Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotentiMSC, also called an induced multipotent progenitor celliMPC) or unipotent(iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.

Three techniques are widely recognized:[1]

In 1895 Thomas Morgan removed one of a frog's two blastomeres and found that amphibians are able to form whole embryos from the remaining part. This meant that the cells can change their differentiation pathway. In 1924 Spemann and Mangold demonstrated the key importance of cellcell inductions during animal development.[20] The reversible transformation of cells of one differentiated cell type to another is called metaplasia.[21] This transition can be a part of the normal maturation process, or caused by an inducement.

One example is the transformation of iris cells to lens cells in the process of maturation and transformation of retinal pigment epithelium cells into the neural retina during regeneration in adult newt eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In Drosophila imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.[22]

The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the embryo. They showed that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, in vivo or in vitro, uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo.[23]

Some types of mature, specialized adult cells can naturally revert to stem cells. For example, "chief" cells express the stem cell marker Troy. While they normally produce digestive fluids for the stomach, they can revert into stem cells to make temporary repairs to stomach injuries, such as a cut or damage from infection. Moreover, they can make this transition even in the absence of noticeable injuries and are capable of replenishing entire gastric units, in essence serving as quiescent "reserve" stem cells.[24] Differentiated airway epithelial cells can revert into stable and functional stem cells in vivo.[25]

After injury, mature terminally differentiated kidney cells dedifferentiate into more primordial versions of themselves and then differentiate into the cell types needing replacement in the damaged tissue[26] Macrophages can self-renew by local proliferation of mature differentiated cells.[27][28] In newts, muscle tissue is regenerated from specialized muscle cells that dedifferentiate and forget the type of cell they had been. This capacity to regenerate does not decline with age and may be linked to their ability to make new stem cells from muscle cells on demand.[29]

A variety of nontumorigenic stem cells display the ability to generate multiple cell types. For instance, multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant adult human stem cells that can self-renew. They form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency and can differentiate into endodermal, ectodermal and mesodermal cells both in vitro and in vivo.[30][31][32][33][34]

Other well-documented examples of transdifferentiation and their significance in development and regeneration were described in detail.[35][36]

Induced totipotent cells can be obtained by reprogramming somatic cells with somatic-cell nuclear transfer (SCNT). The process involves sucking out the nucleus of a somatic (body) cell and injecting it into an oocyte that has had its nucleus removed[3][5][37][38]

Using an approach based on the protocol outlined by Tachibana et al.,[3] hESCs can be generated by SCNT using dermal fibroblasts nuclei from both a middle-aged 35-year-old male and an elderly, 75-year-old male, suggesting that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells.[39] Such reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. Unfortunately, the cells generated by this technology, potentially are not completely protected from the immune system of the patient (donor of nuclei), because they have the same mitochondrial DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for autologous stem cell transplantation therapy, as for the present, it is not clear whether it can induce an immune response of the patient upon treatment.

Induced androgenetic haploid embryonic stem cells can be used instead of sperm for cloning. These cells, synchronized in M phase and injected into the oocyte can produce viable offspring.[40]

These developments, together with data on the possibility of unlimited oocytes from mitotically active reproductive stem cells,[41] offer the possibility of industrial production of transgenic farm animals. Repeated recloning of viable mice through a SCNT method that includes a histone deacetylase inhibitor, trichostatin, added to the cell culture medium,[42] show that it may be possible to reclone animals indefinitely with no visible accumulation of reprogramming or genomic errors[43] However, research into technologies to develop sperm and egg cells from stem cells raises bioethical issues.[44]

Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes.[3][45] For example, the technology could prevent inherited mitochondrial disease from passing to future generations. Mitochondrial genetic material is passed from mother to child. Mutations can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia and other neurological diseases. The nucleus from one human egg has been transferred to another, including its mitochondria, creating a cell that could be regarded as having two mothers. The eggs were then fertilised and the resulting embryonic stem cells carried the swapped mitochondrial DNA.[46] As evidence that the technique is safe author of this method points to the existence of the healthy monkeys that are now more than four years old and are the product of mitochondrial transplants across different genetic backgrounds.[47]

In late-generation telomerase-deficient (Terc/) mice, SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming.[48]

Other cloning and totipotent transformation achievements have been described.[49]

Recently some researchers succeeded to get the totipotent cells without the aid of SCNT. Totipotent cells were obtained using the epigenetic factors such as oocyte germinal isoform of histone.[50] Reprogramming in vivo, by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features. Intraperitoneal injection of such in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic (trophectodermal) markers.[51]

iPSc were first obtained in the form of transplantable teratocarcinoma induced by grafts taken from mouse embryos.[52] Teratocarcinoma formed from somatic cells.[53]Genetically mosaic mice were obtained from malignant teratocarcinoma cells, confirming the cells' pluripotency.[54][55][56] It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent embryonic stem cell in an undifferentiated state, by supplying the culture medium with various factors.[57] In the 1980s, it became clear that transplanting pluripotent/embryonic stem cells into the body of adult mammals, usually leads to the formation of teratomas, which can then turn into a malignant tumor teratocarcinoma.[58] However, putting teratocarcinoma cells into the embryo at the blastocyst stage, caused them to become incorporated in the inner cell mass and often produced a normal chimeric (i.e. composed of cells from different organisms) animal.[59][60][61] This indicated that the cause of the teratoma is a dissonance - mutual miscommunication between young donor cells and surrounding adult cells (the recipient's so-called "niche").

In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic fibroblasts into pluripotent stem cells via the ectopic expression of four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved that the overexpression of a small number of factors can push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.[7]

Reprogramming mechanisms are thus linked, rather than independent and are centered on a small number of genes.[62] IPSC properties are very similar to ESCs.[63] iPSCs have been shown to support the development of all-iPSC mice using a tetraploid (4n) embryo,[64] the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to produce all-iPSC mice because of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster.[18]

An important advantage of iPSC over ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it became possible to obtain iPSC from adult and even elderly patients.[9][65][66]

Reprogramming somatic cells to iPSC leads to rejuvenation. It was found that reprogramming leads to telomere lengthening and subsequent shortening after their differentiation back into fibroblast-like derivatives.[67] Thus, reprogramming leads to the restoration of embryonic telomere length,[68] and hence increases the potential number of cell divisions otherwise limited by the Hayflick limit.[69]

However, because of the dissonance between rejuvenated cells and the surrounding niche of the recipient's older cells, the injection of his own iPSC usually leads to an immune response,[70] which can be used for medical purposes,[71] or the formation of tumors such as teratoma.[72] The reason has been hypothesized to be that some cells differentiated from ESC and iPSC in vivo continue to synthesize embryonic protein isoforms.[73] So, the immune system might detect and attack cells that are not cooperating properly.

A small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via cytochrome c release across the mitochondrial outer membrane) in human pluripotent stem cells, but not in differentiated cells. Shortly after differentiation, daughter cells became resistant to death. When MitoBloCK-6 was introduced to differentiated cell lines, the cells remained healthy. The key to their survival, was hypothesized to be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines has the potential to reduce the risk of teratomas and other problems in regenerative medicine.[74]

In 2012 other small molecules (selective cytotoxic inhibitors of human pluripotent stem cellshPSCs) were identified that prevented human pluripotent stem cells from forming teratomas in mice. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule the undifferentiated cells can be selectively removed from culture.[75][76] An efficient strategy to selectively eliminate pluripotent cells with teratoma potential is targeting pluripotent stem cell-specific antiapoptotic factor(s) (i.e., survivin or Bcl10). A single treatment with chemical survivin inhibitors (e.g., quercetin or YM155) can induce selective and complete cell death of undifferentiated hPSCs and is claimed to be sufficient to prevent teratoma formation after transplantation.[77] However, it is unlikely that any kind of preliminary clearance,[78] is able to secure the replanting iPSC or ESC. After the selective removal of pluripotent cells, they re-emerge quickly by reverting differentiated cells into stem cells, which leads to tumors.[79] This may be due to the disorder of let-7 regulation of its target Nr6a1 (also known as Germ cell nuclear factor - GCNF), an embryonic transcriptional repressor of pluripotency genes that regulates gene expression in adult fibroblasts following micro-RNA miRNA loss.[80]

Teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0,1-5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of Nanog as well as a propensity for increased glucose and cholesterol metabolism.[81] These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.[82] In connection with the above safety problems, the use iPSC for cell therapy is still limited.[83] However, they can be used for a variety of other purposes - including the modeling of disease,[84] screening (selective selection) of drugs, toxicity testing of various drugs.[85]

It is interesting to note that the tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation[86] At the same time, full reprogramming of adult cells in vivo within tissues by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs.[51] Furthermore, partial reprogramming of cells toward pluripotency in vivo in mice demonstrates that incomplete reprogramming entails epigenetic changes (failed repression of Polycomb targets and altered DNA methylation) in cells that drive cancer development.[87]

Determining the unique set of cellular factors that is needed to be manipulated for each cell conversion is a long and costly process that involved much trial and error. As a result, this first step of identifying the key set of cellular factors for cell conversion is the major obstacle researchers face in the field of cell reprogramming. An international team of researchers have developed an algorithm, called Mogrify(1), that can predict the optimal set of cellular factors required to convert one human cell type to another. When tested, Mogrify was able to accurately predict the set of cellular factors required for previously published cell conversions correctly. To further validate Mogrify's predictive ability, the team conducted two novel cell conversions in the laboratory using human cells and these were successful in both attempts solely using the predictions of Mogrify.[89][90][91] Mogrify has been made available online for other researchers and scientists.

By using solely small molecules, Deng Hongkui and colleagues demonstrated that endogenous "master genes" are enough for cell fate reprogramming. They induced a pluripotent state in adult cells from mice using seven small-molecule compounds.[17] The effectiveness of the method is quite high: it was able to convert 0.02% of the adult tissue cells into iPSCs, which is comparable to the gene insertion conversion rate. The authors note that the mice generated from CiPSCs were "100% viable and apparently healthy for up to 6 months". So, this chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.[92][93]

In 2015th year a robust chemical reprogramming system was established with a yield up to 1,000-fold greater than that of the previously reported protocol. So, chemical reprogramming became a promising approach to manipulate cell fates.[94]

The fact that human iPSCs capable of forming teratomas not only in humans but also in some animal body, in particular in mice or pigs, allowed to develop a method for differentiation of iPSCs in vivo. For this purpose, iPSCs with an agent for inducing differentiation into target cells are injected to genetically modified pig or mouse that has suppressed immune system activation on human cells. The formed teratoma is cut out and used for the isolation of the necessary differentiated human cells[95] by means of monoclonal antibody to tissue-specific markers on the surface of these cells. This method has been successfully used for the production of functional myeloid, erythroid and lymphoid human cells suitable for transplantation (yet only to mice).[96] Mice engrafted with human iPSC teratoma-derived hematopoietic cells produced human B and T cells capable of functional immune responses. These results offer hope that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation and drug screening applications. Using MitoBloCK-6[74] and/or PluriSIn # 1 the differentiated progenitor cells can be further purified from teratoma forming pluripotent cells. The fact, that the differentiation takes place even in the teratoma niche, offers hope that the resulting cells are sufficiently stable to stimuli able to cause their transition back to the dedifferentiated (pluripotent) state and therefore safe. A similar in vivo differentiation system, yielding engraftable hematopoietic stem cells from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis, was described by Suzuki et al.[97] They noted that neither leukemia nor tumors were observed in recipients after intravenous injection of iPSC-derived hematopoietic stem cells into irradiated recipients. Moreover, this injection resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Such system provides a useful tool for practical application of iPSCs in the treatment of hematologic and immunologic diseases.[98]

For further development of this method animal in which is grown the human cell graft, for example mouse, must have so modified genome that all its cells express and have on its surface human SIRP.[99] To prevent rejection after transplantation to the patient of the allogenic organ or tissue, grown from the pluripotent stem cells in vivo in the animal, these cells should express two molecules: CTLA4-Ig, which disrupts T cell costimulatory pathways and PD-L1, which activates T cell inhibitory pathway.[100]

See also: US 20130058900 patent.

In the near-future, clinical trials designed to demonstrate the safety of the use of iPSCs for cell therapy of the people with age-related macular degeneration, a disease causing blindness through retina damaging, will begin. There are several articles describing methods for producing retinal cells from iPSCs[101][102] and how to use them for cell therapy.[103][104] Reports of iPSC-derived retinal pigmented epithelium transplantation showed enhanced visual-guided behaviors of experimental animals for 6 weeks after transplantation.[105] However, clinical trials have been successful: ten patients suffering from retinitis pigmentosa have had their eyesight restoredincluding a woman who had only 17 percent of her vision left.[106]

Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis or chronic obstructive pulmonary disease and asthma are leading causes of morbidity and mortality worldwide with a considerable human, societal and financial burden. So there is an urgent need for effective cell therapy and lung tissue engineering.[107][108] Several protocols have been developed for generation of the most cell types of the respiratory system, which may be useful for deriving patient-specific therapeutic cells.[109][110][111][112][113]

Some lines of iPSCs have the potentiality to differentiate into male germ cells and oocyte-like cells in an appropriate niche (by culturing in retinoic acid and porcine follicular fluid differentiation medium or seminiferous tubule transplantation). Moreover, iPSC transplantation make a contribution to repairing the testis of infertile mice, demonstrating the potentiality of gamete derivation from iPSCs in vivo and in vitro.[114]

The risk of cancer and tumors creates the need to develop methods for safer cell lines suitable for clinical use. An alternative approach is so-called "direct reprogramming" - transdifferentiation of cells without passing through the pluripotent state.[115][116][117][118][119][120] The basis for this approach was that 5-azacytidine - a DNA demethylation reagent - can cause the formation of myogenic, chondrogenic and adipogeni clones in the immortal cell line of mouse embryonic fibroblasts[121] and that the activation of a single gene, later named MyoD1, is sufficient for such reprogramming.[122] Compared with iPSC whose reprogramming requires at least two weeks, the formation of induced progenitor cells sometimes occurs within a few days and the efficiency of reprogramming is usually many times higher. This reprogramming does not always require cell division.[123] The cells resulting from such reprogramming are more suitable for cell therapy because they do not form teratomas.[120] For example, Chandrakanthan et al., & Pimanda describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor (platelet-derived growth factorAB (PDGF-AB)) and 5-Azacytidine. These authors claim that: "Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors" and so "has significant scope for application in tissue regeneration."[124][125][126]

Originally only early embryonic cells could be coaxed into changing their identity. Mature cells are resistant to changing their identity once they've committed to a specific kind. However, brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult roundworm Caenorhabditis elegans with no requirement for a dedifferentiated intermediate.[127]

The cell fate can be effectively manipulated by epigenome editing. In particular, by directly activating of specific endogenous gene expression with CRISPR-mediated activator. When dCas9 (that has been modified so that it no longer cuts DNA, but still can be guided to specific sequences and to bind to them) is combined with transcription activators, it can precisely manipulate endogenous gene expression. Using this method, Wei et al., enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus directly converted mouse embryonic stem cells into two extraembryonic lineages, i.e., typical trophoblast stem cells and extraembryonic endoderm cells.[128] An analogous approach was used to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes to convert mouse embryonic fibroblasts to induced neuronal cells.[129] Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Another way of reprogramming is the simulation of the processes that occur during amphibian limb regeneration. In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates into limb tissue. However, sequential small molecule treatment of the muscle fiber with myoseverin, reversine (the aurora B kinase inhibitor) and some other chemicals: BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) causes the formation of new muscle cell types as well as other cell types such as precursors to fat, bone and nervous system cells.[130]

The researchers discovered that GCSF-mimicking antibody can activate a growth-stimulating receptor on marrow cells in a way that induces marrow stem cells that normally develop into white blood cells to become neural progenitor cells. The technique[131] enables researchers to search large libraries of antibodies and quickly select the ones with a desired biological effect.[132]

Schlegel and Liu[133] demonstrated that the combination of feeder cells[134][135][136] and a Rho kinase inhibitor (Y-27632) [137][138] induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. This process occurs without the need for transduction of exogenous viral or cellular genes. These cells have been termed "Conditionally Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results from reprogramming of the entire cell population. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible and removal of Y-27632 and feeders allows the cells to differentiate normally.[133][139][140] CRC technology can generate 2106 cells in 5 to 6 days from needle biopsies and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.[133][141][142]

The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.[133][141][142] Using CRC technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.[143] Engleman's group[144] describes a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance using CRC system. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host. While initial studies revealed that co-culturing epithelial cells with Swiss 3T3 cells J2 was essential for CRC induction, with transwell culture plates, physical contact between feeders and epithelial cells is not required for inducing CRCs and more importantly that irradiation of the feeder cells is required for this induction. Consistent with the transwell experiments, conditioned medium induces and maintains CRCs, which is accompanied by a concomitant increase of cellular telomerase activity. The activity of the conditioned medium correlates directly with radiation-induced feeder cell apoptosis. Thus, conditional reprogramming of epithelial cells is mediated by a combination of Y-27632 and a soluble factor(s) released by apoptotic feeder cells.[145]

Riegel et al.[146] demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neuinduced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs). Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+ and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.

A different approach to CRC is to inhibit CD47a membrane protein that is the thrombospondin-1 receptor. Loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells in vitro and in vivo. Thrombospondin-1 is a key environmental signal that inhibits stem cell self-renewal via CD47. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.[147] In vivo blockade of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation due to increased proliferative capacity of bone marrow-derived cells and radioprotection of radiosensitive gastrointestinal tissues.[148]

Differentiated macrophages can self-renew in tissues and expand long-term in culture.[27] Under certain conditions macrophages can divide without losing features they have acquired while specializing into immune cells - which is usually not possible with differentiated cells. The macrophages achieve this by activating a gene network similar to one found in embryonic stem cells. Single-cell analysis revealed that, in vivo, proliferating macrophages can derepress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. This happened when concentrations of two transcription factors named MafB and c-Maf were naturally low or were inhibited for a short time. Genetic manipulations that turned off MafB and c-Maf in the macrophages caused the cells to start a self-renewal program. The similar network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers.[28]

Hence macrophages isolated from MafB- and c-Maf-double deficient mice divide indefinitely; the self-renewal depends on c-Myc and Klf4.[149]

Indirect lineage conversion is a reprogramming methodology in which somatic cells transition through a plastic intermediate state of partially reprogrammed cells (pre-iPSC), induced by brief exposure to reprogramming factors, followed by differentiation in a specially developed chemical environment (artificial niche).[150]

This method could be both more efficient and safer, since it does not seem to produce tumors or other undesirable genetic changes and results in much greater yield than other methods. However, the safety of these cells remains questionable. Since lineage conversion from pre-iPSC relies on the use of iPSC reprogramming conditions, a fraction of the cells could acquire pluripotent properties if they do not stop the de-differentation process in vitro or due to further de-differentiation in vivo.[151]

A common feature of pluripotent stem cells is the specific nature of protein glycosylation of their outer membrane. That distinguishes them from most nonpluripotent cells, although not white blood cells.[152] The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60 and Tra 1-81.[153] Suila Heli et al.[154] speculate that in human stem cells extracellular O-GlcNAc and extracellular O-LacNAc, play a crucial role in the fine tuning of Notch signaling pathway - a highly conserved cell signaling system, that regulates cell fate specification, differentiation, leftright asymmetry, apoptosis, somitogenesis, angiogenesis and plays a key role in stem cell proliferation (reviewed by Perdigoto and Bardin[155] and Jafar-Nejad et al.[156])

Changes in outer membrane protein glycosylation are markers of cell states connected in some way with pluripotency and differentiation.[157] The glycosylation change is apparently not just the result of the initialization of gene expression, but perform as an important gene regulator involved in the acquisition and maintenance of the undifferentiated state.[158]

For example, activation of glycoprotein ACA,[159] linking glycosylphosphatidylinositol on the surface of the progenitor cells in human peripheral blood, induces increased expression of genes Wnt, Notch-1, BMI1 and HOXB4 through a signaling cascade PI3K/Akt/mTor/PTEN and promotes the formation of a self-renewing population of hematopoietic stem cells.[160]

Furthermore, dedifferentiation of progenitor cells induced by ACA-dependent signaling pathway leads to ACA-induced pluripotent stem cells, capable of differentiating in vitro into cells of all three germ layers.[161] The study of lectins' ability to maintain a culture of pluripotent human stem cells has led to the discovery of lectin Erythrina crista-galli (ECA), which can serve as a simple and highly effective matrix for the cultivation of human pluripotent stem cells.[162]

Cell adhesion protein E-cadherin is indispensable for a robust pluripotent phenotype.[163] During reprogramming for iPS cell generation, N-cadherin can replace function of E-cadherin.[164] These functions of cadherins are not directly related to adhesion because sphere morphology helps maintaining the "stemness" of stem cells.[165] Moreover, sphere formation, due to forced growth of cells on a low attachment surface, sometimes induces reprogramming. For example, neural progenitor cells can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Physical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, "decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)a subunit of H3 methyltranferaseby microgrooved surfaces lead to increased histone H3 acetylation and methylation". Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state.[166]

Substrate rigidity is an important biophysical cue influencing neural induction and subtype specification. For example, soft substrates promote neuroepithelial conversion while inhibiting neural crest differentiation of hESCs in a BMP4-dependent manner. Mechanistic studies revealed a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility.[167]

Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine leukemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, accompanied by an increase in cellsubstratum adhesion and cell spreading. Restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biosubstrates, or by manipulating the cytoskeleton allowed the cells to remain in an undifferentiated and pluripotent state. The effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or siRNA does not promote differentiation.[168] Possible mechanisms of stem cell fate predetermination by physical interactions with the extracellular matrix have been described.[169][170]

A new method has been developed that turns cells into stem cells faster and more efficiently by 'squeezing' them using 3D microenvironment stiffness and density of the surrounding gel. The technique can be applied to a large number of cells to produce stem cells for medical purposes on an industrial scale.[171][172]

Cells involved in the reprogramming process change morphologically as the process proceeds. This results in physical difference in adhesive forces among cells. Substantial differences in 'adhesive signature' between pluripotent stem cells, partially reprogrammed cells, differentiated progeny and somatic cells allowed to develop separation process for isolation of pluripotent stem cells in microfluidic devices,[173] which is:

Stem cells possess mechanical memory (they remember past physical signals)with the Hippo signaling pathway factors:[174] Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) acting as an intracellular mechanical rheostatthat stores information from past physical environments and influences the cells' fate.[175][176]

Stroke and many neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis need cell replacement therapy. The successful use of converted neural cells (cNs) in transplantations open a new avenue to treat such diseases.[177] Nevertheless, induced neurons (iNs), directly converted from fibroblasts are terminally committed and exhibit very limited proliferative ability that may not provide enough autologous donor cells for transplantation.[178] Self-renewing induced neural stem cells (iNSCs) provide additional advantages over iNs for both basic research and clinical applications.[118][119][120][179][180]

For example, under specific growth conditions, mouse fibroblasts can be reprogrammed with a single factor, Sox2, to form iNSCs that self-renew in culture and after transplantation can survive and integrate without forming tumors in mouse brains.[181] INSCs can be derived from adult human fibroblasts by non-viral techniques, thus offering a safe method for autologous transplantation or for the development of cell-based disease models.[180]

Neural chemically induced progenitor cells (ciNPCs) can be generated from mouse tail-tip fibroblasts and human urinary somatic cells without introducing exogenous factors, but - by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, RepSox, an inhibitor of TGF beta signaling pathways), under a physiological hypoxic condition.[182] Alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase and TGF- pathways (where: sodium butyrate (NaB) or Trichostatin A (TSA) could replace VPA, Lithium chloride (LiCl) or lithium carbonate (Li2CO3) could substitute CHIR99021, or Repsox may be replaced with SB-431542 or Tranilast) show similar efficacies for ciNPC induction.[182] Zhang, et al.,[183] also report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components.

Multiple methods of direct transformation of somatic cells into induced neural stem cells have been described.[184]

Proof of principle experiments demonstrate that it is possible to convert transplanted human fibroblasts and human astrocytes directly in the brain that are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes (Ascl1, Brn2a and Myt1l) are activated after transplantation using a drug.[185]

Astrocytesthe most common neuroglial brain cells, which contribute to scar formation in response to injurycan be directly reprogrammed in vivo to become functional neurons that formed networks in mice without the need of cell transplantation.[186] The researchers followed the mice for nearly a year to look for signs of tumor formation and reported finding none. The same researchers have turned scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons in the injured adult spinal cord.[187]

Without myelin to insulate neurons, nerve signals quickly lose power. Diseases that attack myelin, such as multiple sclerosis, result in nerve signals that cannot propagate to nerve endings and as a consequence lead to cognitive, motor and sensory problems. Transplantation of oligodendrocyte precursor cells (OPCs), which can successfully create myelin sheaths around nerve cells, is a promising potential therapeutic response. Direct lineage conversion of mouse and rat fibroblasts into oligodendroglial cells provides a potential source of OPCs. Conversion by forced expression of both eight[188] or of the three[189] transcription factors Sox10, Olig2 and Zfp536, may provide such cells.

Cell-based in vivo therapies may provide a transformative approach to augment vascular and muscle growth and to prevent non-contractile scar formation by delivering transcription factors[115] or microRNAs[14] to the heart.[190] Cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of cardiac core transcription factors ( GATA4, MEF2C, TBX5 and for improved reprogramming plus ESRRG, MESP1, Myocardin and ZFPM2) after coronary ligation.[115][191] These results implicated therapies that can directly remuscularize the heart without cell transplantation. However, the efficiency of such reprogramming turned out to be very low and the phenotype of received cardiomyocyte-like cells does not resemble those of a mature normal cardiomyocyte. Furthermore, transplantation of cardiac transcription factors into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes.[192]

Meanwhile, advances in the methods of obtaining cardiac myocytes in vitro occurred.[193][194] Efficient cardiac differentiation of human iPS cells gave rise to progenitors that were retained within infarcted rat hearts and reduced remodeling of the heart after ischemic damage.[195]

The team of scientists, who were led by Sheng Ding, used a cocktail of nine chemicals (9C) for transdifferentiation of human skin cells into beating heart cells. With this method, more than 97% of the cells began beating, a characteristic of fully developed, healthy heart cells. The chemically induced cardiomyocyte-like cells (ciCMs) uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to ciCMs and developed into healthy-looking heart muscle cells within the organ.[196] This chemical reprogramming approach, after further optimization, may offer an easy way to provide the cues that induce heart muscle to regenerate locally.[197]

In another study, ischemic cardiomyopathy in the murine infarction model was targeted by iPS cell transplantation. It synchronized failing ventricles, offering a regenerative strategy to achieve resynchronization and protection from decompensation by dint of improved left ventricular conduction and contractility, reduced scarring and reversal of structural remodelling.[198] One protocol generated populations of up to 98% cardiomyocytes from hPSCs simply by modulating the canonical Wnt signaling pathway at defined time points in during differentiation, using readily accessible small molecule compounds.[199]

Discovery of the mechanisms controlling the formation of cardiomyocytes led to the development of the drug ITD-1, which effectively clears the cell surface from TGF- receptor type II and selectively inhibits intracellular TGF- signaling. It thus selectively enhances the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells.[200]

One project seeded decellularized mouse hearts with human iPSC-derived multipotential cardiovascular progenitor cells. The introduced cells migrated, proliferated and differentiated in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the hearts. In addition, the heart's extracellular matrix (the substrate of heart scaffold) signalled the human cells into becoming the specialised cells needed for proper heart function. After 20 days of perfusion with growth factors, the engineered heart tissues started to beat again and were responsive to drugs.[201]

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. Mice exposed in vivo, to three cardiac transcription factors GMT (Gata4, Mef2c, Tbx5) and the small-molecules: SB-431542 (the transforming growth factor (TGF)- inhibitor), and XAV939 (the WNT inhibitor) for 2 weeks after myocardial infarction showed significantly improved reprogramming (reprogramming efficiency increased eight-fold) and cardiac function compared to those exposed to only GMT.[202]

See also: review[203]

The elderly often suffer from progressive muscle weakness and regenerative failure owing in part to elevated activity of the p38 and p38 mitogen-activated kinase pathway in senescent skeletal muscle stem cells. Subjecting such stem cells to transient inhibition of p38 and p38 in conjunction with culture on soft hydrogel substrates rapidly expands and rejuvenates them that result in the return of their strength.[204]

In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions.[205]

Myogenic progenitors for potential use in disease modeling or cell-based therapies targeting skeletal muscle could also be generated directly from induced pluripotent stem cells using free-floating spherical culture (EZ spheres) in a culture medium supplemented with high concentrations (100ng/ml) of fibroblast growth factor-2 (FGF-2) and epidermal growth factor.[206]

Unlike current protocols for deriving hepatocytes from human fibroblasts, Saiyong Zhu et al., (2014)[207] did not generate iPSCs but, using small molecules, cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) were efficiently differentiated. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of human primary adult hepatocytes. iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state.

These results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Cocktail of small molecules, Y-27632, A-83-01 (a TGF kinase/activin receptor like kinase (ALK5) inhibitor), and CHIR99021 (potent inhibitor of GSK-3), can convert rat and mouse mature hepatocytes in vitro into proliferative bipotent cells - CLiPs (chemically induced liver progenitors). CLiPs can differentiate into both mature hepatocytes and biliary epithelial cells that can form functional ductal structures. In long-term culture CLiPs did not lose their proliferative capacity and their hepatic differentiation ability, and can repopulate chronically injured liver tissue.[208]

Complications of Diabetes mellitus such as cardiovascular diseases, retinopathy, neuropathy, nephropathy and peripheral circulatory diseases depend on sugar dysregulation due to lack of insulin from pancreatic beta cells and can be lethal if they are not treated. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs).[209] Unfortunately, human PSC-derived insulin-expressing cells resemble human fetal cells rather than adult cells. In contrast to adult cells, fetal cells seem functionally immature, as indicated by increased basal glucose secretion and lack of glucose stimulation and confirmed by RNA-seq of whose transcripts.[210]

An alternative strategy is the conversion of fibroblasts towards distinct endodermal progenitor cell populations and, using cocktails of signalling factors, successful differentiation of these endodermal progenitor cells into functional beta-like cells both in vitro and in vivo.[211]

Overexpression of the three transcription factors, PDX1 (required for pancreatic bud outgrowth and beta-cell maturation), NGN3 (required for endocrine precursor cell formation) and MAFA (for beta-cell maturation) combination (called PNM) can lead to the transformation of some cell types into a beta cell-like state.[212] An accessible and abundant source of functional insulin-producing cells is intestine. PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into -like cells possibly acceptable for transplantation.[213]

Adult proximal tubule cells were directly transcriptionally reprogrammed to nephron progenitors of the embryonic kidney, using a pool of six genes of instructive transcription factors (SIX1, SIX2, OSR1, Eyes absent homolog 1(EYA1), Homeobox A11 (HOXA11) and Snail homolog 2 (SNAI2)) that activated genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule cell line.[214] The generation of such cells may lead to cellular therapies for adult renal disease. Embryonic kidney organoids placed into adult rat kidneys can undergo onward development and vascular development.[215]

As blood vessels age, they often become abnormal in structure and function, thereby contributing to numerous age-associated diseases including myocardial infarction, ischemic stroke and atherosclerosis of arteries supplying the heart, brain and lower extremities. So, an important goal is to stimulate vascular growth for the collateral circulation to prevent the exacerbation of these diseases. Induced Vascular Progenitor Cells (iVPCs) are useful for cell-based therapy designed to stimulate coronary collateral growth. They were generated by partially reprogramming endothelial cells.[150] The vascular commitment of iVPCs is related to the epigenetic memory of endothelial cells, which engenders them as cellular components of growing blood vessels. That is why, when iVPCs were implanted into myocardium, they engrafted in blood vessels and increased coronary collateral flow better than iPSCs, mesenchymal stem cells, or native endothelial cells.[216]

Ex vivo genetic modification can be an effective strategy to enhance stem cell function. For example, cellular therapy employing genetic modification with Pim-1 kinase (a downstream effector of Akt, which positively regulates neovasculogenesis) of bone marrowderived cells[217] or human cardiac progenitor cells, isolated from failing myocardium[218] results in durability of repair, together with the improvement of functional parameters of myocardial hemodynamic performance.

Stem cells extracted from fat tissue after liposuction may be coaxed into becoming progenitor smooth muscle cells (iPVSMCs) found in arteries and veins.[219]

The 2D culture system of human iPS cells[220] in conjunction with triple marker selection (CD34 (a surface glycophosphoprotein expressed on developmentally early embryonic fibroblasts), NP1 (receptor - neuropilin 1) and KDR (kinase insert domain-containing receptor)) for the isolation of vasculogenic precursor cells from human iPSC, generated endothelial cells that, after transplantation, formed stable, functional mouse blood vessels in vivo, lasting for 280 days.[221]

To treat infarction, it is important to prevent the formation of fibrotic scar tissue. This can be achieved in vivo by transient application of paracrine factors that redirect native heart progenitor stem cell contributions from scar tissue to cardiovascular tissue. For example, in a mouse myocardial infarction model, a single intramyocardial injection of human vascular endothelial growth factor A mRNA (VEGF-A modRNA), modified to escape the body's normal defense system, results in long-term improvement of heart function due to mobilization and redirection of epicardial progenitor cells toward cardiovascular cell types.[222]

RBC transfusion is necessary for many patients. However, to date the supply of RBCs remains labile. In addition, transfusion risks infectious disease transmission. A large supply of safe RBCs generated in vitro would help to address this issue. Ex vivo erythroid cell generation may provide alternative transfusion products to meet present and future clinical requirements.[223][224] Red blood cells (RBC)s generated in vitro from mobilized CD34 positive cells have normal survival when transfused into an autologous recipient.[225] RBC produced in vitro contained exclusively fetal hemoglobin (HbF), which rescues the functionality of these RBCs. In vivo the switch of fetal to adult hemoglobin was observed after infusion of nucleated erythroid precursors derived from iPSCs.[226] Although RBCs do not have nuclei and therefore can not form a tumor, their immediate erythroblasts precursors have nuclei. The terminal maturation of erythroblasts into functional RBCs requires a complex remodeling process that ends with extrusion of the nucleus and the formation of an enucleated RBC.[227] Cell reprogramming often disrupts enucleation. Transfusion of in vitro-generated RBCs or erythroblasts does not sufficiently protect against tumor formation.

The aryl hydrocarbon receptor (AhR) pathway (which has been shown to be involved in the promotion of cancer cell development) plays an important role in normal blood cell development. AhR activation in human hematopoietic progenitor cells (HPs) drives an unprecedented expansion of HPs, megakaryocyte- and erythroid-lineage cells.[228] See also Concise Review:[229][230] The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation.[231] (See also overview.[230][232])

Platelets help prevent hemorrhage in thrombocytopenic patients and patients with thrombocythemia. A significant problem for multitransfused patients is refractoriness to platelet transfusions. Thus, the ability to generate platelet products ex vivo and platelet products lacking HLA antigens in serum-free media would have clinical value. An RNA interference-based mechanism used a lentiviral vector to express short-hairpin RNAi targeting 2-microglobulin transcripts in CD34-positive cells. Generated platelets demonstrated an 85% reduction in class I HLA antigens. These platelets appeared to have normal function in vitro[233]

One clinically-applicable strategy for the derivation of functional platelets from human iPSC involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) through doxycycline-dependent overexpression of BMI1 and BCL-XL. The resulting imMKCLs can be expanded in culture over extended periods (45 months), even after cryopreservation. Halting the overexpression (by the removal of doxycycline from the medium) of c-MYC, BMI1 and BCL-XL in growing imMKCLs led to the production of CD42b+ platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo.[234] Thomas et al., describe a forward programming strategy relying on the concurrent exogenous expression of 3 transcription factors: GATA1, FLI1 and TAL1. The forward programmed megakaryocytes proliferate and differentiate in culture for several months with megakaryocyte purity over 90% reaching up to 2x105 mature megakaryocytes per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as one million starting hPSCs.[235] See also overview[236]

A specialised type of white blood cell, known as cytotoxic T lymphocytes (CTLs), are produced by the immune system and are able to recognise specific markers on the surface of various infectious or tumour cells, causing them to launch an attack to kill the harmful cells. Thence, immunotherapy with functional antigen-specific T cells has potential as a therapeutic strategy for combating many cancers and viral infections.[237] However, cell sources are limited, because they are produced in small numbers naturally and have a short lifespan.

A potentially efficient approach for generating antigen-specific CTLs is to revert mature immune T cells into iPSCs, which possess indefinite proliferative capacity in vitro and after their multiplication to coax them to redifferentiate back into T cells.[238][239][240]

Another method combines iPSC and chimeric antigen receptor (CAR)[241] technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture.[242] This approach of generating therapeutic human T cells may be useful for cancer immunotherapy and other medical applications.

Invariant natural killer T (iNKT) cells have great clinical potential as adjuvants for cancer immunotherapy. iNKT cells act as innate T lymphocytes and serve as a bridge between the innate and acquired immune systems. They augment anti-tumor responses by producing interferon-gamma (IFN-).[243] The approach of collection, reprogramming/dedifferentiation, re-differentiation and injection has been proposed for related tumor treatment.[244]

Dendritic cells (DC) are specialized to control T-cell responses. DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and after that be completely eliminated. DC-like antigen-presenting cells obtained from human induced pluripotent stem cells can serve as a source for vaccination therapy.[245]

CCAAT/enhancer binding protein- (C/EBP) induces transdifferentiation of B cells into macrophages at high efficiencies[246] and enhances reprogramming into iPS cells when co-expressed with transcription factors Oct4, Sox2, Klf4 and Myc.[247] with a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population.[248] Furthermore, C/EBPa can convert selected human B cell lymphoma and leukemia cell lines into macrophage-like cells at high efficiencies, impairing the cells' tumor-forming capacity.[249]

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The spine has different sections. The level of paralysis depends on the location of the injury.The spinal cord is made up of millions of nerve cells that send projections up and down the cord and out into other parts of the body. The information that allows us to sit, run, go to the toilet and breathe travels along these projections, called nerves.

When the spinal cord is injured, the initial trauma causes cell damage and destruction, and triggersa cascade of eventsthat spread around the injury site affecting a number of different types of cells. Axons are crushed and torn, and oligodendrocytes, the nerve cells that make up the insulating myelin sheath around axons, begin to die. Exposed axons degenerate, the connection between neurons is disrupted and the flow of information between the brain and the spinal cord is blocked.

The body cannot replace cells lost when the spinal cord is injured, and its function becomes impaired permanently. Patients may end up with severe movement and sensation disabilities. They will generally be paralyzed and without sensation from the level of the injury downwards.

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Spinal cord injuries result from damage to the vertebrae, ligaments or disks of the spinal column or to the spinal cord itself.

A traumatic spinal cord injury may stem from a sudden, traumatic blow to your spine that fractures, dislocates, crushes, or compresses one or more of your vertebrae. It also may result from a gunshot or knife wound that penetrates and cuts your spinal cord.

Additional damage usually occurs over days or weeks because of bleeding, swelling, inflammation and fluid accumulation in and around your spinal cord.

A nontraumatic spinal cord injury may be caused by arthritis, cancer, inflammation, infections or disk degeneration of the spine.

The central nervous system comprises the brain and spinal cord. The spinal cord, made of soft tissue and surrounded by bones (vertebrae), extends downward from the base of your brain and is made up of nerve cells and groups of nerves called tracts, which go to different parts of your body.

The lower end of your spinal cord stops a little above your waist in the region called the conus medullaris. Below this region is a group of nerve roots called the cauda equina.

Tracts in your spinal cord carry messages between the brain and the rest of the body. Motor tracts carry signals from the brain to control muscle movement. Sensory tracts carry signals from body parts to the brain relating to heat, cold, pressure, pain and the position of your limbs.

Whether the cause is traumatic or nontraumatic, the damage affects the nerve fibers passing through the injured area and may impair part or all of your corresponding muscles and nerves below the injury site.

A chest (thoracic) or lower back (lumbar) injury can affect your torso, legs, bowel and bladder control, and sexual function. In addition, a neck (cervical) injury affects movements of your arms and, possibly, your ability to breathe.

The most common causes of spinal cord injuries in the United States are:

.

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An isolated cardiac muscle cell, beating

Cardiac muscle (heart muscle) is an involuntary, striated muscle that is found in the walls and histological foundation of the heart, specifically the myocardium. Cardiac muscle is one of three major types of muscle, the others being skeletal and smooth muscle. These three types of muscle all form in the process of myogenesis. The cells that constitute cardiac muscle, called cardiomyocytes or myocardiocytes, predominantly contain only one nucleus, although populations with two to four nuclei do exist.[1][2][pageneeded] The myocardium is the muscle tissue of the heart, and forms a thick middle layer between the outer epicardium layer and the inner endocardium layer.

Coordinated contractions of cardiac muscle cells in the heart pump blood out of the atria and ventricles to the blood vessels of the left/body/systemic and right/lungs/pulmonary circulatory systems. This complex mechanism illustrates systole of the heart.

Cardiac muscle cells, unlike most other tissues in the body, rely on an available blood and electrical supply to deliver oxygen and nutrients and remove waste products such as carbon dioxide. The coronary arteries help fulfill this function.

Cardiac muscle has cross striations formed by rotating segments of thick and thin protein filaments. Like skeletal muscle, the primary structural proteins of cardiac muscle are myosin and actin. The actin filaments are thin, causing the lighter appearance of the I bands in striated muscle, whereas the myosin filament is thicker, lending a darker appearance to the alternating A bands as observed with electron microscopy. However, in contrast to skeletal muscle, cardiac muscle cells are typically branch-like instead of linear.

Another histological difference between cardiac muscle and skeletal muscle is that the T-tubules in the cardiac muscle are bigger and wider and track laterally to the Z-discs. There are fewer T-tubules in comparison with skeletal muscle. The diad is a structure in the cardiac myocyte located at the sarcomere Z-line. It is composed of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum. The diad plays an important role in excitation-contraction coupling by juxtaposing an inlet for the action potential near a source of Ca2+ ions. This way, the wave of depolarization can be coupled to calcium-mediated cardiac muscle contraction via the sliding filament mechanism. Cardiac muscle forms these instead of the triads formed between the sarcoplasmic reticulum in skeletal muscle and T-tubules. T-tubules play critical role in excitation-contraction coupling (ECC). Recently, the action potentials of T-tubules were recorded optically by Guixue Bu et al.[3]

The cardiac syncytium is a network of cardiomyocytes connected to each other by intercalated discs that enable the rapid transmission of electrical impulses through the network, enabling the syncytium to act in a coordinated contraction of the myocardium. There is an atrial syncytium and a ventricular syncytium that are connected by cardiac connection fibres.[4] Electrical resistance through intercalated discs is very low, thus allowing free diffusion of ions. The ease of ion movement along cardiac muscle fibers axes is such that action potentials are able to travel from one cardiac muscle cell to the next, facing only slight resistance. Each syncytium obeys the all or none law.[5]

Intercalated discs are complex adhering structures that connect the single cardiomyocytes to an electrochemical syncytium (in contrast to the skeletal muscle, which becomes a multicellular syncytium during mammalian embryonic development). The discs are responsible mainly for force transmission during muscle contraction. Intercalated discs consist of three different types of cell-cell junctions: the actin filament anchoring adherens junctions, the intermediate filament anchoring desmosomes , and gap junctions. They allow action potentials to spread between cardiac cells by permitting the passage of ions between cells, producing depolarization of the heart muscle. However, novel molecular biological and comprehensive studies unequivocally showed that intercalated discs consist for the most part of mixed-type adhering junctions named area composita (pl. areae compositae) representing an amalgamation of typical desmosomal and fascia adhaerens proteins (in contrast to various epithelia).[6][7][8] The authors discuss the high importance of these findings for the understanding of inherited cardiomyopathies (such as arrhythmogenic right ventricular cardiomyopathy).

Under light microscopy, intercalated discs appear as thin, typically dark-staining lines dividing adjacent cardiac muscle cells. The intercalated discs run perpendicular to the direction of muscle fibers. Under electron microscopy, an intercalated disc's path appears more complex. At low magnification, this may appear as a convoluted electron dense structure overlying the location of the obscured Z-line. At high magnification, the intercalated disc's path appears even more convoluted, with both longitudinal and transverse areas appearing in longitudinal section.[9]

In contrast to skeletal muscle, cardiac muscle requires extracellular calcium ions for contraction to occur. Like skeletal muscle, the initiation and upshoot of the action potential in ventricular cardiomyocytes is derived from the entry of sodium ions across the sarcolemma in a regenerative process. However, an inward flux of extracellular calcium ions through L-type calcium channels sustains the depolarization of cardiac muscle cells for a longer duration. The reason for the calcium dependence is due to the mechanism of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum that must occur during normal excitation-contraction (EC) coupling to cause contraction. Once the intracellular concentration of calcium increases, calcium ions bind to the protein troponin, which allows myosin to bind to actin and contraction to occur.

Until recently, it was commonly believed that cardiac muscle cells could not be regenerated. However, a study reported in the April 3, 2009 issue of Science contradicts that belief.[10] Olaf Bergmann and his colleagues at the Karolinska Institute in Stockholm tested samples of heart muscle from people born before 1955 who had very little cardiac muscle around their heart, many showing with disabilities from this abnormality. By using DNA samples from many hearts, the researchers estimated that a 4-year-old renews about 20% of heart muscle cells per year, and about 69 percent of the heart muscle cells of a 50-year-old were generated after he or she was born.

One way that cardiomyocyte regeneration occurs is through the division of pre-existing cardiomyocytes during the normal aging process.[11] The division process of pre-existing cardiomyocytes has also been shown to increase in areas adjacent to sites of myocardial injury. In addition, certain growth factors promote the self-renewal of endogenous cardiomyocytes and cardiac stem cells. For example, insulin-like growth factor 1, hepatocyte growth factor, and high-mobility group protein B1 increase cardiac stem cell migration to the affected area, as well as the proliferation and survival of these cells.[12] Some members of the fibroblast growth factor family also induce cell-cycle re-entry of small cardiomyocytes. Vascular endothelial growth factor also plays an important role in the recruitment of native cardiac cells to an infarct site in addition to its angiogenic effect.

Based on the natural role of stem cells in cardiomyocyte regeneration, researchers and clinicians are increasingly interested in using these cells to induce regeneration of damaged tissue. Various stem cell lineages have been shown to be able to differentiate into cardiomyocytes, including bone marrow stem cells. For example, in one study, researchers transplanted bone marrow cells, which included a population of stem cells, adjacent to an infarct site in a mouse model. Nine days after surgery, the researchers found a new band of regenerating myocardium.[13] However, this regeneration was not observed when the injected population of cells was devoid of stem cells, which strongly suggests that it was the stem cell population that contributed to the myocardium regeneration. Other clinical trials have shown that autologous bone marrow cell transplants delivered via the infarct-related artery decreases the infarct area compared to patients not given the cell therapy.[14]

Occlusion (blockage) of the coronary arteries by atherosclerosis and/or thrombosis can lead to myocardial infarction (heart attack), where part of the myocardium is injured due to ischemia (not receiving enough oxygen). This occurs because coronary arteries are functional end arteries - i.e. there is almost no overlap in the areas supplied by different arteries (anastomoses) so that if one fails, others cannot adequately perfuse the region, unlike in other tissues.

Certain viruses lead to myocarditis (inflammation of the myocardium). Cardiomyopathies are inherent diseases of the myocardium, many of which are caused by genetic mutations.

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Durham, NC A new study appearing in the current issue of STEM CELLS Translational Medicine indicates that stem cells harvested from fat (adipose) are more potent than those collected from bone marrow in helping to modulate the bodys immune system.

The finding could have significant implications in developing new stem-cell-based therapies, as adipose tissue-derived stem cells (AT-SCs) are far more plentiful in the body than those found in bone marrow and can be collected from waste material from liposuction procedures. Stem cells are considered potential therapies for a range of conditions, from enhancing skin graft survival to treating inflammatory bowel disease.

Researchers at the Leiden University Medical Centers Department of Immunohematology and Blood Transfusion in Leiden, The Netherlands, led by Helene Roelofs, Ph.D., conducted the study. They were seeking an alternative to bone marrow for stem cell therapies because of the low number of stem cells available in marrow and also because harvesting them involves an invasive procedure.

Adipose tissue is an interesting alternative since it contains approximately a 500-fold higher frequency of stem cells and tissue collection is simple, Dr. Roelofs said.

Moreover, Dr. Sara M. Melief added, 400,000 liposuctions a year are performed in the U.S. alone, where the aspirated adipose tissue is regarded as waste and could be collected without any additional burden or risk for the donor.

For the study, the team used stem cells collected from the bone marrow and fat tissue of age-matched donors. They compared the cells ability to regulate the immune system in vitro and found that the two performed similarly, although it took a smaller dose for the AT-SCs to achieve the same effect on the immune cells.

When it came to secreting cytokines the cell signaling molecules that regulate the immune system the AT-SCs also outperformed the bone marrow-derived cells.

This all adds up to make AT-SC a good alternative to bone marrow stem cells for developing new therapies, Dr. Roelofs concluded.

Cells from bone marrow and from fat were equivalent in terms of their potential to differentiate into multiple cell types, said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of Wake Forest Institute for Regenerative Medicine. The fact that the cells from fat tissue seem to be more potent at suppressing the immune system suggest their promise in clinical therapies.

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Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood.[1][2] It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin).[1][2] It is a medical procedure in the field of hematology, most often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia.[2] In these cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation. Infection and graft-versus-host disease are major complications of allogeneic HSCT.[2]

Hematopoietic stem cell transplantation remains a dangerous procedure with many possible complications; it is reserved for patients with life-threatening diseases. As survival following the procedure has increased, its use has expanded beyond cancer, such as autoimmune diseases.[3][4]

Indications for stem cell transplantation are as follows:

Many recipients of HSCTs are multiple myeloma[5] or leukemia patients[6] who would not benefit from prolonged treatment with, or are already resistant to, chemotherapy. Candidates for HSCTs include pediatric cases where the patient has an inborn defect such as severe combined immunodeficiency or congenital neutropenia with defective stem cells, and also children or adults with aplastic anemia[7] who have lost their stem cells after birth. Other conditions[8] treated with stem cell transplants include sickle-cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing's sarcoma, desmoplastic small round cell tumor, chronic granulomatous disease and Hodgkin's disease. More recently non-myeloablative, "mini transplant(microtransplantation)," procedures have been developed that require smaller doses of preparative chemo and radiation. This has allowed HSCT to be conducted in the elderly and other patients who would otherwise be considered too weak to withstand a conventional treatment regimen.

In 2006 a total of 50,417 first hematopoietic stem cell transplants were reported as taking place worldwide, according to a global survey of 1327 centers in 71 countries conducted by the Worldwide Network for Blood and Marrow Transplantation. Of these, 28,901 (57 percent) were autologous and 21,516 (43 percent) were allogeneic (11,928 from family donors and 9,588 from unrelated donors). The main indications for transplant were lymphoproliferative disorders (54.5 percent) and leukemias (33.8 percent), and the majority took place in either Europe (48 percent) or the Americas (36 percent).[9]

In 2014, according to the World Marrow Donor Association, stem cell products provided for unrelated transplantation worldwide had increased to 20,604 (4,149 bone marrow donations, 12,506 peripheral blood stem cell donations, and 3,949 cord blood units).[10]

Autologous HSCT requires the extraction (apheresis) of haematopoietic stem cells (HSC) from the patient and storage of the harvested cells in a freezer. The patient is then treated with high-dose chemotherapy with or without radiotherapy with the intention of eradicating the patient's malignant cell population at the cost of partial or complete bone marrow ablation (destruction of patient's bone marrow's ability to grow new blood cells). The patient's own stored stem cells are then transfused into his/her bloodstream, where they replace destroyed tissue and resume the patient's normal blood cell production. Autologous transplants have the advantage of lower risk of infection during the immune-compromised portion of the treatment since the recovery of immune function is rapid. Also, the incidence of patients experiencing rejection (and graft-versus-host disease is impossible) is very rare due to the donor and recipient being the same individual. These advantages have established autologous HSCT as one of the standard second-line treatments for such diseases as lymphoma.[11]

However, for other cancers such as acute myeloid leukemia, the reduced mortality of the autogenous relative to allogeneic HSCT may be outweighed by an increased likelihood of cancer relapse and related mortality, and therefore the allogeneic treatment may be preferred for those conditions.[12] Researchers have conducted small studies using non-myeloablative hematopoietic stem cell transplantation as a possible treatment for type I (insulin dependent) diabetes in children and adults. Results have been promising; however, as of 2009[update] it was premature to speculate whether these experiments will lead to effective treatments for diabetes.[13]

Allogeneics HSCT involves two people: the (healthy) donor and the (patient) recipient. Allogeneic HSC donors must have a tissue (HLA) type that matches the recipient. Matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. Even if there is a good match at these critical alleles, the recipient will require immunosuppressive medications to mitigate graft-versus-host disease. Allogeneic transplant donors may be related (usually a closely HLA matched sibling), syngeneic (a monozygotic or 'identical' twin of the patient - necessarily extremely rare since few patients have an identical twin, but offering a source of perfectly HLA matched stem cells) or unrelated (donor who is not related and found to have very close degree of HLA matching). Unrelated donors may be found through a registry of bone marrow donors such as the National Marrow Donor Program. People who would like to be tested for a specific family member or friend without joining any of the bone marrow registry data banks may contact a private HLA testing laboratory and be tested with a mouth swab to see if they are a potential match.[14] A "savior sibling" may be intentionally selected by preimplantation genetic diagnosis in order to match a child both regarding HLA type and being free of any obvious inheritable disorder. Allogeneic transplants are also performed using umbilical cord blood as the source of stem cells. In general, by transfusing healthy stem cells to the recipient's bloodstream to reform a healthy immune system, allogeneic HSCTs appear to improve chances for cure or long-term remission once the immediate transplant-related complications are resolved.[15][16][17]

A compatible donor is found by doing additional HLA-testing from the blood of potential donors. The HLA genes fall in two categories (Type I and Type II). In general, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host disease. In addition a genetic mismatch as small as a single DNA base pair is significant so perfect matches require knowledge of the exact DNA sequence of these genes for both donor and recipient. Leading transplant centers currently perform testing for all five of these HLA genes before declaring that a donor and recipient are HLA-identical.

Race and ethnicity are known to play a major role in donor recruitment drives, as members of the same ethnic group are more likely to have matching genes, including the genes for HLA.[18]

As of 2013[update], there were at least two commercialized allogeneic cell therapies, Prochymal and Cartistem.[19]

To limit the risks of transplanted stem cell rejection or of severe graft-versus-host disease in allogeneic HSCT, the donor should preferably have the same human leukocyte antigens (HLA) as the recipient. About 25 to 30 percent of allogeneic HSCT recipients have an HLA-identical sibling. Even so-called "perfect matches" may have mismatched minor alleles that contribute to graft-versus-host disease.

In the case of a bone marrow transplant, the HSC are removed from a large bone of the donor, typically the pelvis, through a large needle that reaches the center of the bone. The technique is referred to as a bone marrow harvest and is performed under general anesthesia.

Peripheral blood stem cells[20] are now the most common source of stem cells for HSCT. They are collected from the blood through a process known as apheresis. The donor's blood is withdrawn through a sterile needle in one arm and passed through a machine that removes white blood cells. The red blood cells are returned to the donor. The peripheral stem cell yield is boosted with daily subcutaneous injections of Granulocyte-colony stimulating factor, serving to mobilize stem cells from the donor's bone marrow into the peripheral circulation.

It is also possible to extract stem cells from amniotic fluid for both autologous or heterologous use at the time of childbirth.

Umbilical cord blood is obtained when a mother donates her infant's umbilical cord and placenta after birth. Cord blood has a higher concentration of HSC than is normally found in adult blood. However, the small quantity of blood obtained from an Umbilical Cord (typically about 50 mL) makes it more suitable for transplantation into small children than into adults. Newer techniques using ex-vivo expansion of cord blood units or the use of two cord blood units from different donors allow cord blood transplants to be used in adults.

Cord blood can be harvested from the Umbilical Cord of a child being born after preimplantation genetic diagnosis (PGD) for human leucocyte antigen (HLA) matching (see PGD for HLA matching) in order to donate to an ill sibling requiring HSCT.

Unlike other organs, bone marrow cells can be frozen (cryopreserved) for prolonged periods without damaging too many cells. This is a necessity with autologous HSC because the cells must be harvested from the recipient months in advance of the transplant treatment. In the case of allogeneic transplants, fresh HSC are preferred in order to avoid cell loss that might occur during the freezing and thawing process. Allogeneic cord blood is stored frozen at a cord blood bank because it is only obtainable at the time of childbirth. To cryopreserve HSC, a preservative, DMSO, must be added, and the cells must be cooled very slowly in a controlled-rate freezer to prevent osmotic cellular injury during ice crystal formation. HSC may be stored for years in a cryofreezer, which typically uses liquid nitrogen.

The chemotherapy or irradiation given immediately prior to a transplant is called the conditioning regimen, the purpose of which is to help eradicate the patient's disease prior to the infusion of HSC and to suppress immune reactions. The bone marrow can be ablated (destroyed) with dose-levels that cause minimal injury to other tissues. In allogeneic transplants a combination of cyclophosphamide with total body irradiation is conventionally employed. This treatment also has an immunosuppressive effect that prevents rejection of the HSC by the recipient's immune system. The post-transplant prognosis often includes acute and chronic graft-versus-host disease that may be life-threatening. However, in certain leukemias this can coincide with protection against cancer relapse owing to the graft versus tumor effect.[21]Autologous transplants may also use similar conditioning regimens, but many other chemotherapy combinations can be used depending on the type of disease.

A newer treatment approach, non-myeloablative allogeneic transplantation, also termed reduced-intensity conditioning (RIC), uses doses of chemotherapy and radiation too low to eradicate all the bone marrow cells of the recipient.[22]:320321 Instead, non-myeloablative transplants run lower risks of serious infections and transplant-related mortality while relying upon the graft versus tumor effect to resist the inherent increased risk of cancer relapse.[23][24] Also significantly, while requiring high doses of immunosuppressive agents in the early stages of treatment, these doses are less than for conventional transplants.[25] This leads to a state of mixed chimerism early after transplant where both recipient and donor HSC coexist in the bone marrow space.

Decreasing doses of immunosuppressive therapy then allows donor T-cells to eradicate the remaining recipient HSC and to induce the graft versus tumor effect. This effect is often accompanied by mild graft-versus-host disease, the appearance of which is often a surrogate marker for the emergence of the desirable graft versus tumor effect, and also serves as a signal to establish an appropriate dosage level for sustained treatment with low levels of immunosuppressive agents.

Because of their gentler conditioning regimens, these transplants are associated with a lower risk of transplant-related mortality and therefore allow patients who are considered too high-risk for conventional allogeneic HSCT to undergo potentially curative therapy for their disease. The optimal conditioning strategy for each disease and recipient has not been fully established, but RIC can be used in elderly patients unfit for myeloablative regimens, for whom a higher risk of cancer relapse may be acceptable.[22][24]

After several weeks of growth in the bone marrow, expansion of HSC and their progeny is sufficient to normalize the blood cell counts and re-initiate the immune system. The offspring of donor-derived hematopoietic stem cells have been documented to populate many different organs of the recipient, including the heart, liver, and muscle, and these cells had been suggested to have the abilities of regenerating injured tissue in these organs. However, recent research has shown that such lineage infidelity does not occur as a normal phenomenon[citation needed].

HSCT is associated with a high treatment-related mortality in the recipient (1 percent or higher)[citation needed], which limits its use to conditions that are themselves life-threatening. Major complications are veno-occlusive disease, mucositis, infections (sepsis), graft-versus-host disease and the development of new malignancies.

Bone marrow transplantation usually requires that the recipient's own bone marrow be destroyed ("myeloablation"). Prior to "engraftment" patients may go for several weeks without appreciable numbers of white blood cells to help fight infection. This puts a patient at high risk of infections, sepsis and septic shock, despite prophylactic antibiotics. However, antiviral medications, such as acyclovir and valacyclovir, are quite effective in prevention of HSCT-related outbreak of herpetic infection in seropositive patients.[26] The immunosuppressive agents employed in allogeneic transplants for the prevention or treatment of graft-versus-host disease further increase the risk of opportunistic infection. Immunosuppressive drugs are given for a minimum of 6-months after a transplantation, or much longer if required for the treatment of graft-versus-host disease. Transplant patients lose their acquired immunity, for example immunity to childhood diseases such as measles or polio. For this reason transplant patients must be re-vaccinated with childhood vaccines once they are off immunosuppressive medications.

Severe liver injury can result from hepatic veno-occlusive disease (VOD). Elevated levels of bilirubin, hepatomegaly and fluid retention are clinical hallmarks of this condition. There is now a greater appreciation of the generalized cellular injury and obstruction in hepatic vein sinuses, and hepatic VOD has lately been referred to as sinusoidal obstruction syndrome (SOS). Severe cases of SOS are associated with a high mortality rate. Anticoagulants or defibrotide may be effective in reducing the severity of VOD but may also increase bleeding complications. Ursodiol has been shown to help prevent VOD, presumably by facilitating the flow of bile.

The injury of the mucosal lining of the mouth and throat is a common regimen-related toxicity following ablative HSCT regimens. It is usually not life-threatening but is very painful, and prevents eating and drinking. Mucositis is treated with pain medications plus intravenous infusions to prevent dehydration and malnutrition.

Graft-versus-host disease (GVHD) is an inflammatory disease that is unique to allogeneic transplantation. It is an attack of the "new" bone marrow's immune cells against the recipient's tissues. This can occur even if the donor and recipient are HLA-identical because the immune system can still recognize other differences between their tissues. It is aptly named graft-versus-host disease because bone marrow transplantation is the only transplant procedure in which the transplanted cells must accept the body rather than the body accepting the new cells.[27]

Acute graft-versus-host disease typically occurs in the first 3 months after transplantation and may involve the skin, intestine, or the liver. High-dose corticosteroids such as prednisone are a standard treatment; however this immuno-suppressive treatment often leads to deadly infections. Chronic graft-versus-host disease may also develop after allogeneic transplant. It is the major source of late treatment-related complications, although it less often results in death. In addition to inflammation, chronic graft-versus-host disease may lead to the development of fibrosis, or scar tissue, similar to scleroderma; it may cause functional disability and require prolonged immunosuppressive therapy. Graft-versus-host disease is usually mediated by T cells, which react to foreign peptides presented on the MHC of the host.[citation needed]

Graft versus tumor effect (GVT) or "graft versus leukemia" effect is the beneficial aspect of the Graft-versus-Host phenomenon. For example, HSCT patients with either acute, or in particular chronic, graft-versus-host disease after an allogeneic transplant tend to have a lower risk of cancer relapse.[28][29] This is due to a therapeutic immune reaction of the grafted donor T lymphocytes against the diseased bone marrow of the recipient. This lower rate of relapse accounts for the increased success rate of allogeneic transplants, compared to transplants from identical twins, and indicates that allogeneic HSCT is a form of immunotherapy. GVT is the major benefit of transplants that do not employ the highest immuno-suppressive regimens.

Graft versus tumor is mainly beneficial in diseases with slow progress, e.g. chronic leukemia, low-grade lymphoma, and some cases multiple myeloma. However, it is less effective in rapidly growing acute leukemias.[30]

If cancer relapses after HSCT, another transplant can be performed, infusing the patient with a greater quantity of donor white blood cells (Donor lymphocyte infusion).[30]

Patients after HSCT are at a higher risk for oral carcinoma. Post-HSCT oral cancer may have more aggressive behavior with poorer prognosis, when compared to oral cancer in non-HSCT patients.[31]

Prognosis in HSCT varies widely dependent upon disease type, stage, stem cell source, HLA-matched status (for allogeneic HSCT) and conditioning regimen. A transplant offers a chance for cure or long-term remission if the inherent complications of graft versus host disease, immuno-suppressive treatments and the spectrum of opportunistic infections can be survived.[15][16] In recent years, survival rates have been gradually improving across almost all populations and sub-populations receiving transplants.[32]

Mortality for allogeneic stem cell transplantation can be estimated using the prediction model created by Sorror et al.,[33] using the Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI). The HCT-CI was derived and validated by investigators at the Fred Hutchinson Cancer Research Center (Seattle, WA). The HCT-CI modifies and adds to a well-validated comorbidity index, the Charlson Comorbidity Index (CCI) (Charlson et al.[34]) The CCI was previously applied to patients undergoing allogeneic HCT but appears to provide less survival prediction and discrimination than the HCT-CI scoring system.

The risks of a complication depend on patient characteristics, health care providers and the apheresis procedure, and the colony-stimulating factor used (G-CSF). G-CSF drugs include filgrastim (Neupogen, Neulasta), and lenograstim (Graslopin).

Filgrastim is typically dosed in the 10 microgram/kg level for 45 days during the harvesting of stem cells. The documented adverse effects of filgrastim include splenic rupture (indicated by left upper abdominal or shoulder pain, risk 1 in 40000), Adult respiratory distress syndrome (ARDS), alveolar hemorrage, and allergic reactions (usually expressed in first 30 minutes, risk 1 in 300).[35][36][37] In addition, platelet and hemoglobin levels dip post-procedure, not returning to normal until one month.[37]

The question of whether geriatrics (patients over 65) react the same as patients under 65 has not been sufficiently examined. Coagulation issues and inflammation of atherosclerotic plaques are known to occur as a result of G-CSF injection. G-CSF has also been described to induce genetic changes in mononuclear cells of normal donors.[36] There is evidence that myelodysplasia (MDS) or acute myeloid leukaemia (AML) can be induced by GCSF in susceptible individuals.[38]

Blood was drawn peripherally in a majority of patients, but a central line to jugular/subclavian/femoral veins may be used in 16 percent of women and 4 percent of men. Adverse reactions during apheresis were experienced in 20 percent of women and 8 percent of men, these adverse events primarily consisted of numbness/tingling, multiple line attempts, and nausea.[37]

A study involving 2408 donors (1860 years) indicated that bone pain (primarily back and hips) as a result of filgrastim treatment is observed in 80 percent of donors by day 4 post-injection.[37] This pain responded to acetaminophen or ibuprofen in 65 percent of donors and was characterized as mild to moderate in 80 percent of donors and severe in 10 percent.[37] Bone pain receded post-donation to 26 percent of patients 2 days post-donation, 6 percent of patients one week post-donation, and <2 percent 1 year post-donation. Donation is not recommended for those with a history of back pain.[37] Other symptoms observed in more than 40 percent of donors include myalgia, headache, fatigue, and insomnia.[37] These symptoms all returned to baseline 1 month post-donation, except for some cases of persistent fatigue in 3 percent of donors.[37]

In one metastudy that incorporated data from 377 donors, 44 percent of patients reported having adverse side effects after peripheral blood HSCT.[38] Side effects included pain prior to the collection procedure as a result of GCSF injections, post-procedural generalized skeletal pain, fatigue and reduced energy.[38]

A study that surveyed 2408 donors found that serious adverse events (requiring prolonged hospitalization) occurred in 15 donors (at a rate of 0.6 percent), although none of these events were fatal.[37] Donors were not observed to have higher than normal rates of cancer with up to 48 years of follow up.[37] One study based on a survey of medical teams covered approximately 24,000 peripheral blood HSCT cases between 1993 and 2005, and found a serious cardiovascular adverse reaction rate of about 1 in 1500.[36] This study reported a cardiovascular-related fatality risk within the first 30 days HSCT of about 2 in 10000. For this same group, severe cardiovascular events were observed with a rate of about 1 in 1500. The most common severe adverse reactions were pulmonary edema/deep vein thrombosis, splenic rupture, and myocardial infarction. Haematological malignancy induction was comparable to that observed in the general population, with only 15 reported cases within 4 years.[36]

Georges Math, a French oncologist, performed the first European bone marrow transplant in November 1958 on five Yugoslavian nuclear workers whose own marrow had been damaged by irradiation caused by a criticality accident at the Vina Nuclear Institute, but all of these transplants were rejected.[39][40][41][42][43] Math later pioneered the use of bone marrow transplants in the treatment of leukemia.[43]

Stem cell transplantation was pioneered using bone-marrow-derived stem cells by a team at the Fred Hutchinson Cancer Research Center from the 1950s through the 1970s led by E. Donnall Thomas, whose work was later recognized with a Nobel Prize in Physiology or Medicine. Thomas' work showed that bone marrow cells infused intravenously could repopulate the bone marrow and produce new blood cells. His work also reduced the likelihood of developing a life-threatening complication called graft-versus-host disease.[44]

The first physician to perform a successful human bone marrow transplant on a disease other than cancer was Robert A. Good at the University of Minnesota in 1968.[45] In 1975, John Kersey, M.D., also of the University of Minnesota, performed the first successful bone marrow transplant to cure lymphoma. His patient, a 16-year-old-boy, is today the longest-living lymphoma transplant survivor.[46]

At the end of 2012, 20.2 million people had registered their willingness to be a bone marrow donor with one of the 67 registries from 49 countries participating in Bone Marrow Donors Worldwide. 17.9 million of these registered donors had been ABDR typed, allowing easy matching. A further 561,000 cord blood units had been received by one of 46 cord blood banks from 30 countries participating. The highest total number of bone marrow donors registered were those from the USA (8.0 million), and the highest number per capita were those from Cyprus (15.4 percent of the population).[47]

Within the United States, racial minority groups are the least likely to be registered and therefore the least likely to find a potentially life-saving match. In 1990, only six African-Americans were able to find a bone marrow match, and all six had common European genetic signatures.[48]

Africans are more genetically diverse than people of European descent, which means that more registrations are needed to find a match. Bone marrow and cord blood banks exist in South Africa, and a new program is beginning in Nigeria.[48] Many people belonging to different races are requested to donate as there is a shortage of donors in African, Mixed race, Latino, Aboriginal, and many other communities.

In 2007, a team of doctors in Berlin, Germany, including Gero Htter, performed a stem cell transplant for leukemia patient Timothy Ray Brown, who was also HIV-positive.[49] From 60 matching donors, they selected a [CCR5]-32 homozygous individual with two genetic copies of a rare variant of a cell surface receptor. This genetic trait confers resistance to HIV infection by blocking attachment of HIV to the cell. Roughly one in 1000 people of European ancestry have this inherited mutation, but it is rarer in other populations.[50][51] The transplant was repeated a year later after a leukemia relapse. Over three years after the initial transplant, and despite discontinuing antiretroviral therapy, researchers cannot detect HIV in the transplant recipient's blood or in various biopsies of his tissues.[52] Levels of HIV-specific antibodies have also declined, leading to speculation that the patient may have been functionally cured of HIV. However, scientists emphasise that this is an unusual case.[53] Potentially fatal transplant complications (the "Berlin patient" suffered from graft-versus-host disease and leukoencephalopathy) mean that the procedure could not be performed in others with HIV, even if sufficient numbers of suitable donors were found.[54][55]

In 2012, Daniel Kuritzkes reported results of two stem cell transplants in patients with HIV. They did not, however, use donors with the 32 deletion. After their transplant procedures, both were put on antiretroviral therapies, during which neither showed traces of HIV in their blood plasma and purified CD4 T cells using a sensitive culture method (less than 3 copies/mL). However, the virus was once again detected in both patients some time after the discontinuation of therapy.[56]

Since McAllister's 1997 report on a patient with multiple sclerosis (MS) who received a bone marrow transplant for CML,[57] over 600 reports have been published describing HSCTs performed primarily for MS.[58] These have been shown to "reduce or eliminate ongoing clinical relapses, halt further progression, and reduce the burden of disability in some patients" that have aggressive highly active MS, "in the absence of chronic treatment with disease-modifying agents".[58]

Clincs performing HSCT includes Northwestern University and Karolinska University Hospital.

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In August, 2011 an all natural rejuvenating serum that uses your own adult stem cells to decrease wrinkles and increase moisture retention and elasticity was launched in the United States, and subsequently in Australia. This is a mocha based fusion of the world's most restorative ingredients and a blend of six cytokines that stimulate the proliferation and migration of the skin's stem cells by more than 225%.

There are a number of stem cell based serums and skin care products that have appeared on the marketplace over the past few years, and they constitute a novel frontier in skin care. Although many of them are nothing more than simple skin care products with misleading or spurious stem cell claims, a few are legitimate products. The legitimate ones are all based on the use of compounds called cytokines, which are growth factors supporting the functions of stem cells in the skin. Some of them contain an extract from apple stem cells, whose effectiveness really remains to be proven there is an obvious difference between human skin and an apple! Others contained cytokines from human stem cells. The latter are obviously the premium products.

One of the questions the developers of this product asked was: Of all the natural compounds and herbal extracts known to benefit the skin, which do so by supporting the natural role of stem cells in the skin? Are there natural compounds that can support the intrinsic ability of the skin to renew itself? They studied a broad array of plants and herbal extracts for their effect on the proliferation and differentiation of human skin stem cells grown in vitro, and they discovered a handful of natural compounds that have an effect on the very stem cells of your skin. By supporting the natural role of your skins stem cells, you support the process of rejuvenation of your skin from within.......the way nature intended. These compounds include AFA, the same product from which stem cell nutrition is derived.

AFA alone increased the proliferation of skin stem cells by nearly 100% in the study. Other natural ingredients include: Aloe vera (which increased skin stem cell proliferation by 87%) and a proprietary fucoidan that increased proliferation by 55%. When blended together, the effect of these plants on skin stem cell proliferation was further synergistically increased by ingredients like vanilla, maqui berry, cacao, old mans weed and others. All these ingredients taken together constitute the Stem Cell Complex unique to this product with a Stem Cell Index exceeding 250%

Hyaluronic acid is part of the infrastructure (skeleton of the skin) and is one of the main components forming the matrix of the skin. One of the main roles of hyaluronic acid is to retain moisture in the skin. Good hydration is the hallmark of young skin, and it comes from the presence of hyaluronic acid. Recently a group of scientists discovered that as we age, although we continue to produce hyaluronic acid, its structure is less and less branched. The highly branched hyaluronic acid in young skin allows for greater retention of water in the skin. Since these branches are formed of a derivative of glucosamine, scientists discovered that the best results are obtained when this derivative of glucosamine is applied on the skin, instead of hyaluronic acid itself. This product is the first in the US to contain that very derivative of glucosamine, produced by fermentation.

An all-natural formula Of all the stem cell based skin care products, this is the only one that is truly natural ......even though many make the claim. In essence, all skin care products are oils blended with water extracts of various plants. Since oil and water do not mix, it is necessary to use compounds called emulsifiers that can dissolve in both water and in lipids, thereby helping to create an emulsion. There are very few natural emulsifiers and none that are known to be effective at making a cold emulsion which is essential to the preservation of all the delicate actives found in herbal extracts. This is the only skin care product made cold with an all-natural emulsification system. Products like glycerin are relatively natural and can be used as emulsifiers; however, they are known for their drying effect on the skin. There is no glycerin here. Once produced, natural skin care products are essentially food for bacteria, so they need to be preserved. And this is the biggest challenge, as there are virtually no natural preservatives commercially available. Although the best products claim to have none of the dangerous carcinogenic parabens, they have other compounds just as dangerous such as phenoxyethanol and various forms of benzoic acid, all known to be irritants to the skin. The developers asked the question, Where in nature can we find natural antibacterial compounds? They harvested several flowers known to grow in very moist areas while blooming for weeks, unaffected by bacterial or fungal growth, and they extracted their antibacterial power. To this they added a proprietary process called SoniPure that inactivates bacteria by the use of sound waves a breakthrough innovative process. So this skin serum is 100% stable without delivering harmful compounds to your skin.

The developers intention was to create a product to restructure the skin from within in order to increase water retention and skin elasticity, which in turn would naturally reduce wrinkles and fine lines and this is exactly what was demonstrated in an independent clinical trial. It was shown to increase water retention by 30% and skin elasticity by 10% and to reduce wrinkles by an average of 25% in 28 days. Some people saw significant benefits after only 7 days, while others report wrinkle reduction by as much as 75%. In all participants, wrinkle reduction was already statistically significant after 7 days. So you can easily see how both the developmental process and the resulting formula ensure that this product is undeniably second-to-none in stem cell based skin care.

In healthy individuals, skin youthfulness is maintained by epidermal stem cells which self-renew and generate daughter cells that become new skin. Therefore, part of skin aging is caused by impaired adult stem cell mobilization from the bone marrow and the reduced number of adult stem cells able to respond to repair signals. This means that, if we increase the number of circulating adult stem cells, we can affect the epidermal stem cells. Research also shows that topical application of cytokines stimulates the migration and proliferation of skin stem cells.

In much the same way as stem cell nutrition works with adult stem cells to deliver inner wellness, the rejuvenating skin serum applies the benefits of adult stem cell science to the bodys largest organ, the skin, to achieve and maintain outer vibrance! Taking care of this organ the skin, which exposed to the elements on a continual basis is essential. The rejuvenating skin serum assists in our daily process at the skin level, by a proprietary blend of over two dozen natural ingredients found during years of searching worldwide. Each natural ingredient has been selected for its nutrient-rich attributes that fight the appearance of aging, regenerating cells, decreasing fine lines and wrinkles, increasing moisture retention and increasing skin elasticity. In addition, some of the ingredients have natural sun-protecting components.

After using stem cell serum on one side of face only for only 10 days

Your skin's response to an increase in circulating adult stem cells. The most evident visual response in people's facial skin a few weeks after taking stem cell nutrition is that - it glows. People notice a smoothness and improvement in color of their skin. Skin may also show improvements in age related and hormonal pigmentation, decreased bruising and increased elasticity and tone.

Before and after using stem cell serum

This product is second to none, and early clinical tests have demonstrated the following dramatic results: Decreased fine line & coarse wrinkles 25% in 28 days Increased moisture retention 30% in 28 days Increased elasticity 10% in 28 days

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2015 World Stem Cell Summit & RegMed Capital Conference in Atlanta, GA from December 10 12 (Atlanta, GA) - The 11thWorld Stem Cell Summit & RegMed Capital Conference #WSCS15 brings together over 1,200 scientists, clinicians,

11th Annual Stem Cell Action Awards to be Presented at World Stem Cell Summit #WSCS15, December 10, in Atlanta Five distinguished honorees have been selected for the 2015 Stem Cell Action Awards. For 11 years,

Regenerative medicine through stem cell technology is a source of hope for many suffering from ailments including Alzheimer's disease, diabetes, spinal cord injury and cancer. While new therapeutic options are continually in development, progress has,

New Organ, a collective initiative for biomedical engineering and regenerative medicine, announced today that two new teams have joined the field competing for the New Organ Liver Prize, a global competition sponsored by the Methuselah

Regenerative medicine and stem cell research are presenting possibilities that were unimaginable 15 years ago. Bernard Siegel, Founder of the World Stem Cell Summit, discusses its position at the heart of this community, bringing together

The World Stem Cell Summit honored five champions of stem cell research Thursday evening. They are: Philanthropists Denny Sanford and Malin Burnham; stem cell researcher/blogger/patient advocate Paul Knoepfler; medical journal publisher Mary Ann Liebert, and

Stem cell research has already yielded historic breakthroughs against incurable diseases, a panel of top stem cell researchers said at a public forum Tuesday evening. And that's just the start, said the researchers, who described

(December 5, 2012 -- West Palm Beach, Florida) The winners of the World Stem Cell Summit Poster Prize were announced this morning by Alan Jakimo, Senior Counsel, Sidley Austin.The World Stem Cell Summit Poster Forum

Register now to secure the best registration price for the 8th annualWorld Stem Cell Summit. Our lowest rates are only offered through Friday, October 19, so why wait?The diverse three day program includes: 65 hours

Build on the momentum. Capitalize on your Summit experience. Distill and incorporate what emerged, and further explore what may be ahead. Follow-up with colleagues. And make plans now to continue the dialogue at the 2013

Time magazine last month touted recent advances in stem cell research as one of the world's "10 best shots for tackling our worst problems." But Minnesota and Oklahoma are hellbent on blocking progress. A bill

Rhona Allison is senior director of life sciences for Scottish Enterprise, an economic development group in Scotland. She is responsible for setting and delivering Scottish Enterprise's life sciences strategy to support the economic growth and

The Circuit Court of Appeals for the District of Columbia decided Tuesday afternoon to allow federally funded human embryonic stem cell research to continue, while the federal government appeals a lower court injunction that barred

A U.S. judge blocked the federal government from funding research involving human embryonic stem cells, a surprise blow to one of the most promising yet controversial areas of current scientific research. The ruling, which could

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Spinal Cord Stem Cells Comments Off on World Stem Cell Summit | November 21st, 2016

The Inaugural ISCT-ASBMT Cell Therapy Training Course One Scholars Experience

Beth Sage MBBS, PhD UCL Respiratory University College London, London, UK

Having been lucky enough to be selected as an international scholar for the inaugural Cell Therapy Training Course I was looking forward to leaving behind the rather disappointing British summer and heading towards the much warmer Houston fall. Having googled my destination and accommodation I boarded the plane with great excitement and hopes of enjoying the Texan heat whilst exploring the cosmopolitan offerings of Americas fourth largest city oh and learning something about cell therapy!

The course, chaired by Dave DiGiusto and John Barrett, was the first of its kind, a joint enterprise between the ISCT and the American Society of Blood and Marrow Transplantation. Following a competitive selection procedure 12 scholars from all over the world were invited to attend a 5 day intensive workshop with the primary objective of giving junior cell therapy researchers an insight into the process of taking their research project from bench to bedside, including processing, clinical trial design, regulatory requirements, commercialization and ethical research. Alongside didactic lecture based teaching there were tours of good manufacturing practice (GMP) facilities, both academic and commercial, and most anticipated by the scholars was the opportunity to participate in small group discussions, led by experts in the field, dissecting and improving the individual cell therapy projects.

To break the ice on the light first day each scholar gave a short presentation on their project. It was immediately clear that there is a great breadth of exciting, novel cell therapy projects under investigation throughout the world, from the use of modified T-cells in hematological malignancies to the development of a tissue-engineered oesophagus using amniotic fluid stem cells. Projects ranged from early pre-clinical to those embarking on a first in man clinical trial and every stage in between, making the session interesting and varied. Having fought the jet-lag, the session ended with an ice-breaker drinks and dinner before retiring to prepare for the days ahead.

Over the next few days we were exposed to a wealth of information with detailed talks on pre-clinical development of different cell therapies from CD34 cells to mesenchymal stromal cells, quality systems development and one of the most useful from my personal perspective, manufacturing and release testing of different products. We were able to visit different manufacturing facilities and to understand the processes involved in the production of a clinical grade therapy. It made us challenge the protocols we were developing in the lab as we gained an insight into how it would scale up into a commercially viable process a 26 day culture process of autologous cells requiring purification and multiple cytokine stimulations is significantly more challenging (and expensive) than allogeneic cells cultured for 14 days with no manipulation and simple media exchanges.

Once the process development sessions were complete, we switched gears to look at how to conduct cell therapy clinical trials, covering issues of producing products including normal donors that are used to treat multiple recipients, the challenges of pooling donor cells, how to run multicenter studies and most importantly (although I can say almost universally never thought about by the scholars) how to deal with a regulatory body audit. This really was a really informative session that opened our eyes to the challenges and complexities of working in the field of cell therapy trials.

Just when we were beginning to feel that our jobs over the next few years would be focused on clinical trial design, process validation and filling in an endless paper chain of regulatory documents we were brought back to where we all started the excitement of the translational science. This was, for me, a really interesting session on the importance of correlative studies not only to assess clinical trial performance but to provide mechanistic insights into the behavior of cells when delivered to patients with disease. As scientists we can design and perform many experiments to predict how manipulated cells will behave but the most important data of all comes from the patients themselves. For me, the importance of testing a novel therapy is not just to see if it works but how it works and, just as importantly, if it doesnt - why.

To end to course, our wise leaders Dave DiGiusto and John Barrett decided to test whether we had been listening, and each scholar had to deliver a detailed presentation on how their project had developed during the course. Each scholar had to address the potential pitfalls and specific challenges they faced in moving towards the clinic. Whilst many of us stayed up into the small hours worrying about aspects we were previously oblivious to, undoubtedly we found this one of the most rewarding moments. Despite the potential difficulties we were now aware of, we also felt better placed to solve them and could see a clearer path ahead.

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IPS Cell Therapy Comments Off on Cellular Therapy Training Course – ISCT | November 21st, 2016

Progress in basic research, starting with the work on neural stem cells in the middle 1990's to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author's group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient's own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment is now being investigated in preclinical research. Among the mixture of rod and cone photoreceptors in the donor cells, the percentage of cone photoreceptors is still low. Donor cells with more. cone photoreceptors will be needed. If that will work well, photoreceptor transplantation will be the first example of neural network reconstruction in the central nervous system. These efforts will reach to variety of retinal cell transplantations in the future.

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IPS Cell Therapy Comments Off on [Retinal Cell Therapy Using iPS Cells]. – ncbi.nlm.nih.gov | November 20th, 2016

A bone is a rigid organ that constitutes part of the vertebral skeleton. Bones support and protect the various organs of the body, produce red and white blood cells, store minerals and also enable mobility as well as support for the body. Bone tissue is a type of dense connective tissue. Bones come in a variety of shapes and sizes and have a complex internal and external structure. They are lightweight yet strong and hard, and serve multiple functions. Mineralized osseous tissue, or bone tissue, is of two types, cortical and cancellous, and gives a bone rigidity and a coral-like three-dimensional internal structure. Other types of tissue found in bones include marrow, endosteum, periosteum, nerves, blood vessels and cartilage.

Bone is an active tissue composed of different types of bone cells. Osteoblasts are involved in the creation and mineralisation of bone; osteocytes and osteoclasts are involved in the reabsorption of bone tissue. The mineralised matrix of bone tissue has an organic component of mainly collagen called ossein and an inorganic component of bone mineral made up of various salts.

In the human body at birth, there are over 270 bones,[1] but many of these fuse together during development, leaving a total of 206 separate bones in the adult,[2] not counting numerous small sesamoid bones. The largest bone in the body is the thigh-bone (femur) and the smallest is the stapes in the middle ear.

Bone is not a uniformly solid material, but is mostly a matrix. The primary tissue of bone, bone tissue (osseous tissue), is relatively hard and lightweight. Its matrix is mostly made up of a composite material incorporating the inorganic mineral calcium phosphate in the chemical arrangement termed calcium hydroxylapatite (this is the bone mineral that gives bones their rigidity) and collagen, an elastic protein which improves fracture resistance.[3] Bone is formed by the hardening of this matrix around entrapped cells. When these cells become entrapped from osteoblasts they become osteocytes.[citation needed]

The hard outer layer of bones is composed of cortical bone also called compact bone. Cortical referring to the outer (cortex) layer. The hard outer layer gives bone its smooth, white, and solid appearance, and accounts for 80% of the total bone mass of an adult human skeleton.[citation needed] However, that proportion may be much lower, especially in marine mammals and marine turtles, or in various Mesozoic marine reptiles, such as ichthyosaurs,[4] among others.[5]

Cortical bone consists of multiple microscopic columns, each called an osteon. Each column is multiple layers of osteoblasts and osteocytes around a central canal called the Haversian canal. Volkmann's canals at right angles connect the osteons together. The columns are metabolically active, and as bone is reabsorbed and created the nature and location of the cells within the osteon will change. Cortical bone is covered by a periosteum on its outer surface, and an endosteum on its inner surface. The endosteum is the boundary between the cortical bone and the cancellous bone.

Filling the interior of the bone is the cancellous bone also known as trabecular or spongy bone tissue. It is an open cell porous network. Thin formations of osteoblasts covered in endosteum create an irregular network of spaces. Within these spaces are bone marrow and hematopoietic stem cells that give rise to platelets, red blood cells and white blood cells. Trabecular marrow is composed of a network of rod- and plate-like elements that make the overall organ lighter and allow room for blood vessels and marrow. Trabecular bone accounts for the remaining 20% of total bone mass but has nearly ten times the surface area of compact bone.[8]

Bone marrow, also known as myeloid tissue, can be found in almost any bone that holds cancellous tissue. In newborns, all such bones are filled exclusively with red marrow, but as the child ages it is mostly replaced by yellow, or fatty marrow. In adults, red marrow is mostly found in the bone marrow of the femur, the ribs, the vertebrae and pelvic bones.[citation needed]

Bone is a metabolically active tissue composed of several types of cells. These cells include osteoblasts, which are involved in the creation and mineralization of bone tissue, osteocytes, and osteoclasts, which are involved in the reabsorption of bone tissue. Osteoblasts and osteocytes are derived from osteoprogenitor cells, but osteoclasts are derived from the same cells that differentiate to form macrophages and monocytes. Within the marrow of the bone there are also hematopoietic stem cells. These cells give rise to other cells, including white blood cells, red blood cells, and platelets.

Bones consist of living cells embedded in a mineralized organic matrix. This matrix consists of organic components, mainly collagen "organic" referring to materials produced as a result of the human body and inorganic components, primarily hydroxyapatite and other salts of calcium and phosphate. Above 30% of the acellular part of bone consists of the organic components, and 70% of salts. The strands of collagen give bone its tensile strength, and the interspersed crystals of hydroxyapatite give bone its compressional strength. These effects are synergistic.

The inorganic composition of bone (bone mineral) is primarily formed from salts of calcium and phosphate, the major salt being hydroxyapatite (Ca10(PO4)6(OH)2). The exact composition of the matrix may change over time and with nutrition, with the ratio of calcium to phosphate varying between 1.3 and 2.0 (per weight), and trace minerals such as magnesium, sodium, potassium and carbonate also being found.

The organic part of matrix is mainly composed of Type I collagen. Collagen composes 9095% of the organic matrix, with remainder of the matrix being a homogenous liquid called ground substance consisting of proteoglycans such as hyaluronic acid and chondroitin sulfate. Collagen consists of strands of repeating units, which give bone tensile strength, and are arranged in an overlapping fashion that prevents shear stress. The function of ground substance is not fully known. Two types of bone can be identified microscopically according to the arrangement of collagen:

Woven bone is produced when osteoblasts produce osteoid rapidly, which occurs initially in all fetal bones, but is later replaced by more resilient lamellar bone. In adults woven bone is created after fractures or in Paget's disease. Woven bone is weaker, with a smaller number of randomly oriented collagen fibers, but forms quickly; it is for this appearance of the fibrous matrix that the bone is termed woven. It is soon replaced by lamellar bone, which is highly organized in concentric sheets with a much lower proportion of osteocytes to surrounding tissue. Lamellar bone, which makes its first appearance in humans in the fetus during the third trimester,[16] is stronger and filled with many collagen fibers parallel to other fibers in the same layer (these parallel columns are called osteons). In cross-section, the fibers run in opposite directions in alternating layers, much like in plywood, assisting in the bone's ability to resist torsion forces. After a fracture, woven bone forms initially and is gradually replaced by lamellar bone during a process known as "bony substitution." Compared to woven bone, lamellar bone formation takes place more slowly. The orderly deposition of collagen fibers restricts the formation of osteoid to about 1 to 2m per day. Lamellar bone also requires a relatively flat surface to lay the collagen fibers in parallel or concentric layers.[citation needed]

The extracellular matrix of bone is laid down by osteoblasts, which secrete both collagen and ground substance. These synthesise collagen within the cell, and then secrete collagen fibrils. The collagen fibres rapidly polymerise to form collagen strands. At this stage they are not yet mineralised, and are called "osteoid". Around the strands calcium and phosphate precipitate on the surface of these strands, within a days to weeks becoming crystals of hydroxyapatite.

In order to mineralise the bone, the osteoblasts secrete vesicles containing alkaline phosphatase. This cleaves the phosphate groups and acts as the foci for calcium and phosphate deposition. The vesicles then rupture and act as a centre for crystals to grow on. More particularly, bone mineral is formed from globular and plate structures.[17][18]

There are five types of bones in the human body: long, short, flat, irregular, and sesamoid.[19]

In the study of anatomy, anatomists use a number of anatomical terms to describe the appearance, shape and function of bones. Other anatomical terms are also used to describe the location of bones. Like other anatomical terms, many of these derive from Latin and Greek. Some anatomists still use Latin to refer to bones. The term "osseous", and the prefix "osteo-", referring to things related to bone, are still used commonly today.

Some examples of terms used to describe bones include the term "foramen" to describe a hole through which something passes, and a "canal" or "meatus" to describe a tunnel-like structure. A protrusion from a bone can be called a number of terms, including a "condyle", "crest", "spine", "eminence", "tubercle" or "tuberosity", depending on the protrusion's shape and location. In general, long bones are said to have a "head", "neck", and "body".

When two bones join together, they are said to "articulate". If the two bones have a fibrous connection and are relatively immobile, then the joint is called a "suture".

The formation of bone is called ossification. During the fetal stage of development this occurs by two processes, Intramembranous ossification and endochondral ossification.[citation needed] Intramembranous ossification involves the creation of bone from connective tissue, whereas in the process of endochondral ossification bone is created from cartilage.

Intramembranous ossification mainly occurs during formation of the flat bones of the skull but also the mandible, maxilla, and clavicles; the bone is formed from connective tissue such as mesenchyme tissue rather than from cartilage. The steps in intramembranous ossification are:[citation needed]

Endochondral ossification, on the other hand, occurs in long bones and most of the rest of the bones in the body; it involves an initial hyaline cartilage that continues to grow. The steps in endochondral ossification are:[citation needed]

Endochondral ossification begins with points in the cartilage called "primary ossification centers." They mostly appear during fetal development, though a few short bones begin their primary ossification after birth. They are responsible for the formation of the diaphyses of long bones, short bones and certain parts of irregular bones. Secondary ossification occurs after birth, and forms the epiphyses of long bones and the extremities of irregular and flat bones. The diaphysis and both epiphyses of a long bone are separated by a growing zone of cartilage (the epiphyseal plate). When the child reaches skeletal maturity (18 to 25 years of age), all of the cartilage is replaced by bone, fusing the diaphysis and both epiphyses together (epiphyseal closure).[citation needed] In the upper limbs, only the diaphyses of the long bones and scapula are ossified. The epiphyses, carpal bones, coracoid process, medial border of the scapula, and acromion are still cartilaginous.[21]

The following steps are followed in the conversion of cartilage to bone:

Bones have a variety of functions:

Bones serve a variety of mechanical functions. Together the bones in the body form the skeleton. They provide a frame to keep the body supported, and an attachment point for skeletal muscles, tendons, ligaments and joints, which function together to generate and transfer forces so that individual body parts or the whole body can be manipulated in three-dimensional space (the interaction between bone and muscle is studied in biomechanics).

Bones protect internal organs, such as the skull protecting the brain or the ribs protecting the heart and lungs. Because of the way that bone is formed, bone has a high compressive strength of about 170 MPa (1800 kgf/cm),[3] poor tensile strength of 104121 MPa, and a very low shear stress strength (51.6 MPa).[23][24] This means that bone resists pushing(compressional) stress well, resist pulling(tensional) stress less well, but only poorly resists shear stress (such as due to torsional loads). While bone is essentially brittle, bone does have a significant degree of elasticity, contributed chiefly by collagen. The macroscopic yield strength of cancellous bone has been investigated using high resolution computer models.[25]

Mechanically, bones also have a special role in hearing. The ossicles are three small bones in the middle ear which are involved in sound transduction.

Cancellous bones contain bone marrow. Bone marrow produces blood cells in a process called hematopoiesis.[26] Blood cells that are created in bone marrow include red blood cells, platelets and white blood cells. Progenitor cells such as the hematopoietic stem cell divide in a process called mitosis to produce precursor cells. These include precursors which eventually give rise to white blood cells, and erythroblasts which give rise to red blood cells. Unlike red and white blood cells, created by mitosis, platelets are shed from very large cells called megakaryocytes. This process of progressive differentiation occurs within the bone marrow. After the cells are matured, they enter the circulation. Every day, over 2.5 billion red blood cells and platelets, and 50100 billion granulocytes are produced in this way.

As well as creating cells, bone marrow is also one of the major sites where defective or aged red blood cells are destroyed.

Bone is constantly being created and replaced in a process known as remodeling. This ongoing turnover of bone is a process of resorption followed by replacement of bone with little change in shape. This is accomplished through osteoblasts and osteoclasts. Cells are stimulated by a variety of signals, and together referred to as a remodeling unit. Approximately 10% of the skeletal mass of an adult is remodelled each year.[32] The purpose of remodeling is to regulate calcium homeostasis, repair microdamaged bones from everyday stress, and also to shape and sculpt the skeleton during growth.[citation needed]. Repeated stress, such as weight-bearing exercise or bone healing, results in the bone thickening at the points of maximum stress (Wolff's law). It has been hypothesized that this is a result of bone's piezoelectric properties, which cause bone to generate small electrical potentials under stress.[33]

The action of osteoblasts and osteoclasts are controlled by a number of chemical enzymes that either promote or inhibit the activity of the bone remodeling cells, controlling the rate at which bone is made, destroyed, or changed in shape. The cells also use paracrine signalling to control the activity of each other.[citation needed] For example, the rate at which osteoclasts resorb bone is inhibited by calcitonin and osteoprotegerin. Calcitonin is produced by parafollicular cells in the thyroid gland, and can bind to receptors on osteoclasts to directly inhibit osteoclast activity. Osteoprotegerin is secreted by osteoblasts and is able to bind RANK-L, inhibiting osteoclast stimulation.[34]

Osteoblasts can also be stimulated to increase bone mass through increased secretion of osteoid and by inhibiting the ability of osteoclasts to break down osseous tissue.[citation needed] Increased secretion of osteoid is stimulated by the secretion of growth hormone by the pituitary, thyroid hormone and the sex hormones (estrogens and androgens). These hormones also promote increased secretion of osteoprotegerin.[34] Osteoblasts can also be induced to secrete a number of cytokines that promote reabsorbtion of bone by stimulating osteoclast activity and differentiation from progenitor cells. Vitamin D, parathyroid hormone and stimulation from osteocytes induce osteoblasts to increase secretion of RANK-ligand and interleukin 6, which cytokines then stimulate increased reabsorption of bone by osteoclasts. These same compounds also increase secretion of macrophage colony-stimulating factor by osteoblasts, which promotes the differentiation of progenitor cells into osteoclasts, and decrease secretion of osteoprotegerin.[citation needed]

Bone volume is determined by the rates of bone formation and bone resorption. Recent research has suggested that certain growth factors may work to locally alter bone formation by increasing osteoblast activity. Numerous bone-derived growth factors have been isolated and classified via bone cultures. These factors include insulin-like growth factors I and II, transforming growth factor-beta, fibroblast growth factor, platelet-derived growth factor, and bone morphogenetic proteins.[35] Evidence suggests that bone cells produce growth factors for extracellular storage in the bone matrix. The release of these growth factors from the bone matrix could cause the proliferation of osteoblast precursors. Essentially, bone growth factors may act as potential determinants of local bone formation.[35] Research has suggested that trabecular bone volume in postemenopausal osteoporosis may be determined by the relationship between the total bone forming surface and the percent of surface resorption.[36]

A number of diseases can affect bone, including arthritis, fractures, infections, osteoporosis and tumours. Conditions relating to bone can be managed by a variety of doctors, including rheumatologists for joints, and orthopedic surgeons, who may conduct surgery to fix broken bones. Other doctors, such as rehabilitation specialists may be involved in recovery, radiologists in interpreting the findings on imaging, and pathologists in investigating the cause of the disease, and family doctors may play a role in preventing complications of bone disease such as osteoporosis.

When a doctor sees a patient, a history and exam will be taken. Bones are then often imaged, called radiography. This might include ultrasound X-ray, CT scan, MRI scan and other imaging such as a Bone scan, which may be used to investigate cancer. Other tests such as a blood test for autoimmune markers may be taken, or a synovial fluid aspirate may be taken.

In normal bone, fractures occur when there is significant force applied, or repetitive trauma over a long time. Fractures can also occur when a bone is weakened, such as with osteoporosis, or when there is a structural problem, such as when the bone remodels excessively (such as Paget's disease) or is the site of the growth of cancer. Common fractures include wrist fractures and hip fractures, associated with osteoporosis, vertebral fractures associated with high-energy trauma and cancer, and fractures of long-bones. Not all fractures are painful. When serious, depending on the fractures type and location, complications may include flail chest, compartment syndromes or fat embolism. Compound fractures involve the bone's penetration through the skin.

Fractures and their underlying causes can be investigated by X-rays, CT scans and MRIs. Fractures are described by their location and shape, and several classification systems exist, depending on the location of the fracture. A common long bone fracture in children is a SalterHarris fracture.[39] When fractures are managed, pain relief is often given, and the fractured area is often immobilised. This is to promote bone healing. In addition, surgical measures such as internal fixation may be used. Because of the immobilisation, people with fractures are often advised to undergo rehabilitation.

There are several types of tumour that can affect bone; examples of benign bone tumours include osteoma, osteoid osteoma, osteochondroma, osteoblastoma, enchondroma, giant cell tumor of bone, aneurysmal bone cyst, and fibrous dysplasia of bone.

Cancer can arise in bone tissue, and bones are also a common site for other cancers to spread (metastasise) to. Cancers that arise in bone are called "primary" cancers, although such cancers are rare. Metastases within bone are "secondary" cancers, with the most common being breast cancer, lung cancer, prostate cancer, thyroid cancer, and kidney cancer. Secondary cancers that affect bone can either destroy bone (called a "lytic" cancer) or create bone (a "sclerotic" cancer). Cancers of the bone marrow inside the bone can also affect bone tissue, examples including leukemia and multiple myeloma. Bone may also be affected by cancers in other parts of the body. Cancers in other parts of the body may release parathyroid hormone or parathyroid hormone-related peptide. This increases bone reabsorption, and can lead to bone fractures.

Bone tissue that is destroyed or altered as a result of cancers is distorted, weakened, and more prone to fracture. This may lead to compression of the spinal cord, destruction of the marrow resulting in bruising, bleeding and immunosuppression, and is one cause of bone pain. If the cancer is metastatic, then there might be other symptoms depending on the site of the original cancer. Some bone cancers can also be felt.

Cancers of the bone are managed according to their type, their stage, prognosis, and what symptoms they cause. Many primary cancers of bone are treated with radiotherapy. Cancers of bone marrow may be treated with chemotherapy, and other forms of targeted therapy such as immunotherapy may be used.Palliative care, which focuses on maximising a person's quality of life, may play a role in management, particularly if the likelihood of survival within five years is poor.

Osteoporosis is a disease of bone where there is reduced bone mineral density, increasing the likelihood of fractures. Osteoporosis is defined by the World Health Organization in women as a bone mineral density 2.5 standard deviations below peak bone mass, relative to the age and sex-matched average, as measured by Dual energy X-ray absorptiometry, with the term "established osteoporosis" including the presence of a fragility fracture.[43] Osteoporosis is most common in women after menopause, when it is called "postmenopausal osteoporosis", but may develop in men and premenopausal women in the presence of particular hormonal disorders and other chronic diseases or as a result of smoking and medications, specifically glucocorticoids. Osteoporosis usually has no symptoms until a fracture occurs. For this reason, DEXA scans are often done in people with one or more risk factors, who have developed osteoporosis and be at risk of fracture.

Osteoporosis treatment includes advice to stop smoking, decrease alcohol consumption, exercise regularly, and have a healthy diet. Calcium supplements may also be advised, as may Vitamin D. When medication is used, it may include bisphosphonates, Strontium ranelate, and osteoporosis may be one factor considered when commencing Hormone replacement therapy.[44]

The study of bones and teeth is referred to as osteology. It is frequently used in anthropology, archeology and forensic science for a variety of tasks. This can include determining the nutritional, health, age or injury status of the individual the bones were taken from. Preparing fleshed bones for these types of studies can involve the process of maceration.

Typically anthropologists and archeologists study bone tools made by Homo sapiens and Homo neanderthalensis. Bones can serve a number of uses such as projectile points or artistic pigments, and can also be made from external bones such as antlers.

Bird skeletons are very lightweight. Their bones are smaller and thinner, to aid flight. Among mammals, bats come closest to birds in terms of bone density, suggesting that small dense bones are a flight adaptation. Many bird bones have little marrow due to their being hollow.[45]

A bird's beak is primarily made of bone as projections of the mandibles which are covered in keratin.

A deer's antlers are composed of bone which is an unusual example of bone being outside the skin of the animal once the velvet is shed.[46]

The extinct predatory fish Dunkleosteus had sharp edges of hard exposed bone along its jaws.[citation needed]

Many animals possess an exoskeleton that is not made of bone, These include insects and crustaceans.

Bones from slaughtered animals have a number of uses. In prehistoric times, they have been used for making bone tools. They have further been used in bone carving, already important in prehistoric art, and also in modern time as crafting materials for buttons, beads, handles, bobbins, calculation aids, head nuts, dice, poker chips, pick-up sticks, ornaments, etc. A special genre is scrimshaw.

Bone glue can be made by prolonged boiling of ground or cracked bones, followed by filtering and evaporation to thicken the resulting fluid. Historically once important, bone glue and other animal glues today have only a few specialized uses, such as in antiques restoration. Essentially the same process, with further refinement, thickening and drying, is used to make gelatin.

Broth is made by simmering several ingredients for a long time, traditionally including bones.

Ground bones are used as an organic phosphorus-nitrogen fertilizer and as additive in animal feed. Bones, in particular after calcination to bone ash, are used as source of calcium phosphate for the production of bone china and previously also phosphorus chemicals.[citation needed]

Bone char, a porous, black, granular material primarily used for filtration and also as a black pigment, is produced by charring mammal bones.

Oracle bone script was a writing system used in Ancient china based on inscriptions in bones.

To point the bone at someone is considered bad luck in some cultures, such as Australian aborigines, such as by the Kurdaitcha.

Osteopathic medicine is a school of medical thought originally developed based on the idea of the link between the musculoskeletal system and overall health, but now very similar to mainstream medicine. As of 2012[update], over 77,000 physicians in the United States are trained in Osteopathic medicine colleges.[47]

The wishbones of fowl have been used for divination, and are still customarily used in a tradition to determine which one of two people pulling on either prong of the bone may make a wish.

Various cultures throughout history have adopted the custom of shaping an infant's head by the practice of artificial cranial deformation. A widely practised custom in China was that of foot binding to limit the normal growth of the foot.

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Bone Marrow Stem Cells Comments Off on Bone – Wikipedia | November 18th, 2016

BACKGROUND. Low vitamin D status in pregnancy was proposed as a risk factor of preeclampsia.

METHODS. We assessed the effect of vitamin D supplementation (4,400 vs. 400 IU/day), initiated early in pregnancy (1018 weeks), on the development of preeclampsia. The effects of serum vitamin D (25-hydroxyvitamin D [25OHD]) levels on preeclampsia incidence at trial entry and in the third trimester (3238 weeks) were studied. We also conducted a nested case-control study of 157 women to investigate peripheral blood vitamin Dassociated gene expression profiles at 10 to 18 weeks in 47 participants who developed preeclampsia.

RESULTS. Of 881 women randomized, outcome data were available for 816, with 67 (8.2%) developing preeclampsia. There was no significant difference between treatment (N = 408) or control (N = 408) groups in the incidence of preeclampsia (8.08% vs. 8.33%, respectively; relative risk: 0.97; 95% CI, 0.611.53). However, in a cohort analysis and after adjustment for confounders, a significant effect of sufficient vitamin D status (25OHD 30 ng/ml) was observed in both early and late pregnancy compared with insufficient levels (25OHD <30 ng/ml) (adjusted odds ratio, 0.28; 95% CI, 0.100.96). Differential expression of 348 vitamin Dassociated genes (158 upregulated) was found in peripheral blood of women who developed preeclampsia (FDR <0.05 in the Vitamin D Antenatal Asthma Reduction Trial [VDAART]; P < 0.05 in a replication cohort). Functional enrichment and network analyses of this vitamin Dassociated gene set suggests several highly functional modules related to systematic inflammatory and immune responses, including some nodes with a high degree of connectivity.

CONCLUSIONS. Vitamin D supplementation initiated in weeks 1018 of pregnancy did not reduce preeclampsia incidence in the intention-to-treat paradigm. However, vitamin D levels of 30 ng/ml or higher at trial entry and in late pregnancy were associated with a lower risk of preeclampsia. Differentially expressed vitamin Dassociated transcriptomes implicated the emergence of an early pregnancy, distinctive immune response in women who went on to develop preeclampsia.

TRIAL REGISTRATION. ClinicalTrials.gov NCT00920621.

FUNDING. Quebec Breast Cancer Foundation and Genome Canada Innovation Network. This trial was funded by the National Heart, Lung, and Blood Institute. For details see Acknowledgments.

Hooman Mirzakhani, Augusto A. Litonjua, Thomas F. McElrath, George OConnor, Aviva Lee-Parritz, Ronald Iverson, George Macones, Robert C. Strunk, Leonard B. Bacharier, Robert Zeiger, Bruce W. Hollis, Diane E. Handy, Amitabh Sharma, Nancy Laranjo, Vincent Carey, Weilliang Qiu, Marc Santolini, Shikang Liu, Divya Chhabra, Daniel A. Enquobahrie, Michelle A. Williams, Joseph Loscalzo, Scott T. Weiss

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JCI - Welcome

Bone Marrow Stem Cells Comments Off on JCI – Welcome | November 17th, 2016

Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cellsectoderm, endoderm and mesoderm (see induced pluripotent stem cells)but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.

There are three known accessible sources of autologous adult stem cells in humans:

Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one may bank his or her own blood for elective surgical procedures.

Adult stem cells are frequently used in various medical therapies (e.g., bone marrow transplantation). Stem cells can now be artificially grown and transformed (differentiated) into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic stem cells generated through somatic cell nuclear transfer or dedifferentiation have also been proposed as promising candidates for future therapies.[1] Research into stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.[2][3]

The classical definition of a stem cell requires that it possess two properties:

Two mechanisms exist to ensure that a stem cell population is maintained:

Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.[4]

In practice, stem cells are identified by whether they can regenerate tissue. For example, the defining test for bone marrow or hematopoietic stem cells (HSCs) is the ability to transplant the cells and save an individual without HSCs. This demonstrates that the cells can produce new blood cells over a long term. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.

Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, in which single cells are assessed for their ability to differentiate and self-renew.[7][8] Stem cells can also be isolated by their possession of a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells shall behave in a similar manner in vivo. There is considerable debate as to whether some proposed adult cell populations are truly stem cells.[citation needed]

Embryonic stem (ES) cells are the cells of the inner cell mass of a blastocyst, an early-stage embryo.[9] Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

During embryonic development these inner cell mass cells continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as 'neurectoderm', which will become the future central nervous system.[10] Later in development, neurulation causes the neurectoderm to form the neural tube. At the neural tube stage, the anterior portion undergoes encephalization to generate or 'pattern' the basic form of the brain. At this stage of development, the principal cell type of the CNS is considered a neural stem cell. These neural stem cells are pluripotent, as they can generate a large diversity of many different neuron types, each with unique gene expression, morphological, and functional characteristics. The process of generating neurons from stem cells is called neurogenesis. One prominent example of a neural stem cell is the radial glial cell, so named because it has a distinctive bipolar morphology with highly elongated processes spanning the thickness of the neural tube wall, and because historically it shared some glial characteristics, most notably the expression of glial fibrillary acidic protein (GFAP).[11][12] The radial glial cell is the primary neural stem cell of the developing vertebrate CNS, and its cell body resides in the ventricular zone, adjacent to the developing ventricular system. Neural stem cells are committed to the neuronal lineages (neurons, astrocytes, and oligodendrocytes), and thus their potency is restricted.[10]

Nearly all research to date has made use of mouse embryonic stem cells (mES) or human embryonic stem cells (hES) derived from the early inner cell mass. Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin as an extracellular matrix (for support) and require the presence of leukemia inhibitory factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2).[13] Without optimal culture conditions or genetic manipulation,[14] embryonic stem cells will rapidly differentiate.

A human embryonic stem cell is also defined by the expression of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency.[15] The cell surface antigens most commonly used to identify hES cells are the glycolipids stage specific embryonic antigen 3 and 4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. By using human embryonic stem cells to produce specialized cells like nerve cells or heart cells in the lab, scientists can gain access to adult human cells without taking tissue from patients. They can then study these specialized adult cells in detail to try and catch complications of diseases, or to study cells reactions to potentially new drugs. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.[16]

There are currently no approved treatments using embryonic stem cells. The first human trial was approved by the US Food and Drug Administration in January 2009.[17] However, the human trial was not initiated until October 13, 2010 in Atlanta for spinal cord injury research. On November 14, 2011 the company conducting the trial (Geron Corporation) announced that it will discontinue further development of its stem cell programs.[18] ES cells, being pluripotent cells, require specific signals for correct differentiationif injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face.[19] Due to ethical considerations, many nations currently have moratoria or limitations on either human ES cell research or the production of new human ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.

Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer

The primitive stem cells located in the organs of fetuses are referred to as fetal stem cells.[20] There are two types of fetal stem cells:

Adult stem cells, also called somatic (from Greek , "of the body") stem cells, are stem cells which maintain and repair the tissue in which they are found.[22] They can be found in children, as well as adults.[23]

Pluripotent adult stem cells are rare and generally small in number, but they can be found in umbilical cord blood and other tissues.[24] Bone marrow is a rich source of adult stem cells,[25] which have been used in treating several conditions including liver cirrhosis,[26] chronic limb ischemia [27] and endstage heart failure.[28] The quantity of bone marrow stem cells declines with age and is greater in males than females during reproductive years.[29] Much adult stem cell research to date has aimed to characterize their potency and self-renewal capabilities.[30] DNA damage accumulates with age in both stem cells and the cells that comprise the stem cell environment. This accumulation is considered to be responsible, at least in part, for increasing stem cell dysfunction with aging (see DNA damage theory of aging).[31]

Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, dental pulp stem cell, etc.).[32][33]

Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants.[34] Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.[35]

The use of adult stem cells in research and therapy is not as controversial as the use of embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. Additionally, in instances where adult stem cells are obtained from the intended recipient (an autograft), the risk of rejection is essentially non-existent. Consequently, more US government funding is being provided for adult stem cell research.[36]

Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic, endothelial, hepatic and also neuronal lines.[37] Amniotic stem cells are a topic of active research.

Use of stem cells from amniotic fluid overcomes the ethical objections to using human embryos as a source of cells. Roman Catholic teaching forbids the use of embryonic stem cells in experimentation; accordingly, the Vatican newspaper "Osservatore Romano" called amniotic stem cells "the future of medicine".[38]

It is possible to collect amniotic stem cells for donors or for autologuous use: the first US amniotic stem cells bank [39][40] was opened in 2009 in Medford, MA, by Biocell Center Corporation[41][42][43] and collaborates with various hospitals and universities all over the world.[44]

These are not adult stem cells, but rather adult cells (e.g. epithelial cells) reprogrammed to give rise to pluripotent capabilities. Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue.[45][46][47]Shinya Yamanaka and his colleagues at Kyoto University used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4[45] in their experiments on human facial skin cells. Junying Yu, James Thomson, and their colleagues at the University of WisconsinMadison used a different set of factors, Oct4, Sox2, Nanog and Lin28,[45] and carried out their experiments using cells from human foreskin.

As a result of the success of these experiments, Ian Wilmut, who helped create the first cloned animal Dolly the Sheep, has announced that he will abandon somatic cell nuclear transfer as an avenue of research.[48]

Frozen blood samples can be used as a source of induced pluripotent stem cells, opening a new avenue for obtaining the valued cells.[49]

To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation diagram). Symmetric division gives rise to two identical daughter cells both endowed with stem cell properties. Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.[50]

An alternative theory is that stem cells remain undifferentiated due to environmental cues in their particular niche. Stem cells differentiate when they leave that niche or no longer receive those signals. Studies in Drosophila germarium have identified the signals decapentaplegic and adherens junctions that prevent germarium stem cells from differentiating.[51][52]

Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is a form of stem cell therapy that has been used for many years without controversy. No stem cell therapies other than bone marrow transplant are widely used.[53][54]

Stem cell treatments may require immunosuppression because of a requirement for radiation before the transplant to remove the person's previous cells, or because the patient's immune system may target the stem cells. One approach to avoid the second possibility is to use stem cells from the same patient who is being treated.

Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type. It is also difficult to obtain the exact cell type needed, because not all cells in a population differentiate uniformly. Undifferentiated cells can create tissues other than desired types.[55]

Some stem cells form tumors after transplantation;[56] pluripotency is linked to tumor formation especially in embryonic stem cells, fetal proper stem cells, induced pluripotent stem cells. Fetal proper stem cells form tumors despite multipotency.[citation needed]

Some of the fundamental patents covering human embryonic stem cells are owned by the Wisconsin Alumni Research Foundation (WARF) they are patents 5,843,780, 6,200,806, and 7,029,913 invented by James A. Thomson. WARF does not enforce these patents against academic scientists, but does enforce them against companies.[57]

In 2006, a request for the US Patent and Trademark Office (USPTO) to re-examine the three patents was filed by the Public Patent Foundation on behalf of its client, the non-profit patent-watchdog group Consumer Watchdog (formerly the Foundation for Taxpayer and Consumer Rights).[57] In the re-examination process, which involves several rounds of discussion between the USTPO and the parties, the USPTO initially agreed with Consumer Watchdog and rejected all the claims in all three patents,[58] however in response, WARF amended the claims of all three patents to make them more narrow, and in 2008 the USPTO found the amended claims in all three patents to be patentable. The decision on one of the patents (7,029,913) was appealable, while the decisions on the other two were not.[59][60] Consumer Watchdog appealed the granting of the '913 patent to the USTPO's Board of Patent Appeals and Interferences (BPAI) which granted the appeal, and in 2010 the BPAI decided that the amended claims of the '913 patent were not patentable.[61] However, WARF was able to re-open prosecution of the case and did so, amending the claims of the '913 patent again to make them more narrow, and in January 2013 the amended claims were allowed.[62]

In July 2013, Consumer Watchdog announced that it would appeal the decision to allow the claims of the '913 patent to the US Court of Appeals for the Federal Circuit (CAFC), the federal appeals court that hears patent cases.[63] At a hearing in December 2013, the CAFC raised the question of whether Consumer Watchdog had legal standing to appeal; the case could not proceed until that issue was resolved.[64]

Diseases and conditions where stem cell treatment is being investigated include:

Research is underway to develop various sources for stem cells, and to apply stem cell treatments for neurodegenerative diseases and conditions, diabetes, heart disease, and other conditions.[80]

In more recent years, with the ability of scientists to isolate and culture embryonic stem cells, and with scientists' growing ability to create stem cells using somatic cell nuclear transfer and techniques to create induced pluripotent stem cells, controversy has crept in, both related to abortion politics and to human cloning.

Hepatotoxicity and drug-induced liver injury account for a substantial number of failures of new drugs in development and market withdrawal, highlighting the need for screening assays such as stem cell-derived hepatocyte-like cells, that are capable of detecting toxicity early in the drug development process.[81]

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Stem cell - Wikipedia

Bone Marrow Stem Cells Comments Off on Stem cell – Wikipedia | November 16th, 2016

Everyone knows that the heart is a vital organ. We cannot live without our heart. However, when you get right down to it, the heart is just a pump. A complex and important one, yes, but still just a pump. As with all other pumps it can become clogged, break down and need repair. This is why it is critical that we know how the heart works. With a little knowledge about your heart and what is good or bad for it, you can significantly reduce your risk for heart disease.

Heart disease is the leading cause of death in the United States. Almost 2,000 Americans die of heart disease each day. That is one death every 44 seconds. The good news is that the death rate from heart disease has been steadily decreasing. Unfortunately, heart disease still causes sudden death and many people die before even reaching the hospital.

The heart holds a special place in our collective psyche as well. Of course the heart is synonymous with love. It has many other associations, too. Here are just a few examples:

Certainly no other bodily organ elicits this kind of response. When was the last time you had a heavy pancreas?

In this article, we will look at this important organ so that you can understand exactly what makes your heart tick.

Read more:
How Your Heart Works | HowStuffWorks

Bone Marrow Stem Cells Comments Off on How Your Heart Works | HowStuffWorks | November 16th, 2016

Some of the promise of stem cell therapy has been realized. A prime example is bone marrow transplantation. Even here, however, manyproblems remain to be solved.

Challenges facing stem cell therapy include the following:

Adult stem cells Tissue-specific stem cells in adult individuals tend to be rare. Furthermore, while they can regenerate themselves in an animal or person they are generally very difficult to grow and to expand in the laboratory. Because of this, it is difficult to obtain sufficient numbers of many adult stem cell types for study and clinical use. Hematopoietic or blood-forming stem cells in the bone marrow, for example, only make up one in a hundred thousand cells of the bone marrow. They can be isolated, but can only be expanded a very limited amount in the laboratory. Fortunately, large numbers of whole bone marrow cells can be isolated and administered for the treatment for a variety of diseases of the blood. Skin stem cells can be expanded however, and are used to treat burns. For other types of stem cells, such as mesenchymal stem cells, some success has been achieved in expanding the cellsin vitro, but application in animals has been difficult. One major problem is the mode of administration. Bone marrow cells can be infused in the blood stream, and will find their way to the bone marrow. For other stem cells, such as muscle stem cells, mesenchymal stem cells and neural stem cells, the route of administration in humans is more problematic. It is believed, however, that once healthy stem cells find their niche, they will start repairing the tissue. In another approach, attempts are made to differentiate stem cells into functional tissue, which is then transplanted. A final problem is rejection. If stem cells from the patients are used, rejection by the immune system is not a problem. However, with donor stem cells, the immune system of the recipient will reject the cells, unless the immune system is suppressed by drugs. In the case of bone marrow transplantation, another problem arises. The bone marrow contains immune cells from the donor. These will attack the tissues of the recipient, causing the sometimes deadly graft-versus-host disease.

Pluripotent stem cells All embryonic stem cell lines are derived from very early stage embryos, and will therefore be genetically different from any patient. Hence, immune rejection will be major issue. For this reason, iPS cells, which are generated from the cells of the patient through a process of reprogramming, are a major breakthrough, since these will not be rejected. A problem however is that many iPS cell lines are generated by insertion of genes using viruses, carrying the risk of transformation into cancer cells. Furthermore, undifferentiated embryonic stem cells or iPS cells form tumors when transplanted into mice. Therefore, cells derived from embryonic stem cells or iPS cells have to be devoid of the original stem cells to avoid tumor formation. This is a major safety concern.

A second major challenge is differentiation of pluripotent cells into cells or tissues that are functional in an adult patient and that meet the standards that are required for 'transplantation grade' tissues and cells.

A major advantage of pluripotent cells is that they can be grown and expanded indefinitely in the laboratory. Therefore, in contrast to adult stem cells, cell number will be less of a limiting factor. Another advantage is that given their very broad potential, several cell types that are present in an organ might be generated. Sophisticated tissue engineering approaches are therefore being developed to reconstruct organs in the lab.

While results from animal models are promising, the research on stem cells and their applications to treat various human diseases is still at a preliminary stage. As with any medical treatment, a rigorous research and testing process must be followed to ensure long-term efficacy and safety.

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Stem Cell FAQ

IPS Cell Therapy Comments Off on Stem Cell FAQ | November 13th, 2016

1. Introduction

Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indications are restricted because of critical complications including regimen-related toxicities involving conditioning, infection, and graft-versus-host disease.

Studies in recent decades have shown that HSCT can have a long-term effect in the treatment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined immunodeficiency (SCID-X1) resulted in tumorigenesis [4]. In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.

However, as technological advances have increased the safety and efficiency of introducing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various hereditary diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress [8].

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+CD38-c-Mpl- population, CD34+CD38-c-Mpl+ cells show significantly better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-renewal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A signal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC re-entry into the cell cycle, transforming growth factor- (TGF-) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs. Stem cell factor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor - via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc. San Diego, CA, U.S.A. http://www.biolegend.com/cell_markers)

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromosome. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentiation-specific or non-specific promoters or enhancers during the vector design process.

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activity, are widely used for inserting genes in host chromosomes. Although adeno-associated virus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called simply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a gag/pol gene that codes for viral structure proteins, protease and reverse transcriptase, an env gene that codes for the envelope glycoprotein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the gag/pol gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collecting the culture solution.

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG islands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5 LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vector, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disassembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although various combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one advantage of lentiviral vectors in HSC gene therapy.

Both lentiviruses and gammaretroviruses have gag, pol, and env genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (vif, vpr, vpu, nef) and regulatory genes (tat, rev). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, allowing the viral genome to be inserted into the host cell chromosome.

HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B). The viral long terminal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the gag and pol genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human -globin gene. As the transcripts of the gag and pol genes contain cis-repressive sequences, they are expressed only if rev promotes their nuclear export by binding to the RRE. All tat and rev exons have been deleted, and the viral sequences upstream of the gag gene have been replaced. The rev plasmid expresses rev cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5 LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on tat. The 3 LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the integrated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivating (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with modifications from [26]).

Although first-generation lentiviral vectors included modification genes, they were removed in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifications included the deletion of tat, use of multiple vector plasmids, and introduction of self-inactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the packaging plasmid, rev plasmid, SIN vector plasmid and envelope plasmid. To prevent production of wild type HIV-1, tat, a regulatory gene indispensable to viral reproduction was deleted, and the rev gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of tat, it was replaced with the cytomegalovirus (CMV) promoter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replacing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary genes with the integrated gene (Fig. 3) [27].

Mechanism of gene activation induced by vector insertion. The genomic integration site of an MLV-based retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a self-inactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modification from [27]).

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vectors displaying early-acting cytokines such as TPO and SCF. This vector can promote survival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].

Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient targeting of hematopoietic stem cells. Blood. 2005;106(10):3333)

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the development of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular proto-oncogene and mediates transformation. This mechanism does not occur in replication-incompetent vectors. Second, the provirus 3 LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5 LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector [30].

Retroviral mechanisms of oncogenesis. The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes respectively (Reproduced from [30]).

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-Gpseudotyped HIV vectors have an integration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integration sites.

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than retroviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene undergoes differentiation.

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and metabolic diseases. Most are congenital or hereditary diseases. The characteristic clinical features and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.

Clinical applications of hematopoietic stem cell gene therapy.

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two -globin and two -globin chains combined with a heme group. -thalassemia is an autosomal hemoglobin disorder caused by decreased -globin chain synthesis. Although individuals with -thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, -thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfusion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector containing a functional -globin gene has been performed in an HbE/ -thalassemia (E/ 0) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months [33].

The human -globin locus is located in a large 70kb area which also contains some -like globulin genes (, G, A, , ). Gene switching takes place according to the development stage, and the -globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5 side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5 side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

The structure of the lentiviral SIN vector used in gene therapy for -thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal () of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken -globin insulators) were inserted in the U3 region of the HIV 3 LTR. Furthermore, the amino acid at the 87th position of -globin was changed from threonine to glutamine. This altered -globin can be distinguished from normal adult -globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and +-thalassemia patients [33].

Diagram of the human -globin gene in a lentiviral vector. HIV LTR, human immunodeficiency type-1 virus long terminal repeat; +, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive element; p, human -globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color modification from [33])

A clinical study using this vector was performed in two -thalassemia patients. As with autologous bone marrow transplantation, some of the patients marrow cells were cryopreserved as a backup. The lentiviral vector particles containing a functional -globin were introduced into the remaining cells. After the transfected cells were cultured for one week ex vivo, some were also cryopreserved. The patients were conditioned with intravenous busulfan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before transplantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term -globin production; one-third of the patients hemoglobin was produced by the genetically modified cells [33].

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the HMGA2 gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was expressive production of a truncated form of the HMGA2 protein. Since truncated or overexpressed HMGA2 is observed with some blood cancers and non-malignant expansions of blood cells, caution is recommended with this therapy [34].

Gene-therapy procedure for patient with b-thalassemia. a. Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with -thalassemia and maintained them in culture. b, Lentiviral-vector particles containing a functional -globin gene were then introduced into the cells and allowed them to expand further in culture. c. To eradicate the patients remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemotherapy. d. The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two hereditary persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic levels of A-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated -globin gene addition and genetic reactivation of endogenous -globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with -globin deficiency [36]. In addition, a trial of -globin induction with -globin production using mithramycin, an inducer of -globin expression, to remove excess -globin proteins in -thalassemic erythroid progenitor cells was reported [37].

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approximately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the ten-year probability of survival after transplant from an Human leukocyte antigen (HLA) -identical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN) have been cloned. Mutations in FANCB are associated with an X-linked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, FANCA gene abnormalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during ex vivo culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the FANCA and FANCC genes [20, 42-44]. In a phase I trial of FANCA gene therapy, gene transfer was performed with patient bone marrow-derived CD34+ cells and the MSCV retroviral vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi anemia patients due to possible bone marrow insufficiency, but in this study, sufficient target CD34+ cells were obtained from most patients. Two patients had FANCA-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient improvements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clinical study of FANCC gene therapy using a retroviral vector involved four patients. Although functional FANCC gene expression was observed in peripheral blood and bone marrow cells, the results were transient [43].

Engraftment efficiency of FANCA-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakaryocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleukin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with FANCA abnormalities with short transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-L-cysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells mediates efficient phenotypic correction of hematopoietic progenitor cells and CD34- mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In Fancg -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs in vitro [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for FANCC gene transfer in mice was able to successfully introduce the FANCC gene to HSCs. This result provides evidence that targeting the HSCs directly in their native environment enables efficient and long-term correction of bone marrow defects in Fanconi anemia [47].

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of FANCA gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of FANCA. The PGK-FANCA lentiviral vectors with either a wild-type woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3 region have higher levels of FANCA gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and efficiency for Fanconi anemia gene therapy [48].

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a FANCA gene abnormality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed in patients with hematologic disorders [49].

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemophilia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with hemophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually produced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibroblasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vector or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein expression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, partial replacement of the mutated liver cells by healthy cells in hemophilia A mice was challenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia [55].

Although HSCT has been widely performed as curative treatment for primary immunodeficiencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, frequent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lymphocytes continued for two years after the cessation of treatment [1], gene therapy attracted attention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered [56].

SCID-X1 is an X-linked disease caused by deficiency of the common (c) chain in the IL-2 receptor. Because the c chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be achieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. After gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as LIM domain only 2 (LMO2), Bmi1, cyclin D2 (CCND2) are near the gene insertion region, there was a high frequency of T-cell leukemia after treatment. Furthermore, in the patients who developed leukemia, additional chromosomal changes, including activating mutations of Notch1, changes in the T cell receptor region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (CDKN2A) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 positive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus influences the target cells dormant capacity for survival, engraftment, and proliferation.

Although continuous T cell production was founded in many cases, there was little reconstruction of myeloid cells and B cells, and some patients required continuous immunoglobulin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after c chain gene therapy. There is decreased NK cell reconstruction without conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after allergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children [60].

To study whether activation of genes near the region of gene insertion or inserted c chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the human c chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- c chain gene) was performed in mice. When the CD2- c chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in c chain-gene deficient mice. This study demonstrated that when the c c chain gene is expressed externally, SCID-X1 may be treated safely [61].

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or c chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive disease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetriphosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined immunodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycolmodified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase peripheral T cell counts was attempted during the early stages of gene therapy. Although adverse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the ADA gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was discontinued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Epstein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-induced leukemia [25].

As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as LMO2, or proto-oncogenes [66]. In clinical studies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, including heart, skeletal muscle, and kidney [71].

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in gp91phox on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, leading to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis reduces the incidence of infections, the overall annual mortality rate remains high (2%5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flare-ups at infected sites [56].

In the initial trials of CGD gene therapy without any conditioning therapy, p47phox or gp91phox gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte colony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74].

In a German study where gp91phox was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leukocytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as myelodysplastic syndrome 1-ecotropic virus integration site 1 (MDS1/EVI1), PR domain containing protein 16 (PRDM16), SET binding protein 1 (SETBP1). Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to EVI1 activation. One died of severe sepsis 27 months after gene therapy. Although the gene-inserted cells remained expressed in this patient, methylation of the CpG site in the LTR of the viral vector was observed and the expression of the inserted gp91phox gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although gp91phox gene expression was decreased, the activation of EVI1 near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene insertion site will be increased, there remains a concern about tumorigenesis with peripheral stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lentiviral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were studied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce sequence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of gp91phox was targeted into one allele of the "safe harbor" AAVS1 locus in iPSs [79].

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WAS gene result in a wide spectrum of clinical manifestations ranging from relatively mild X-linked thrombocytopenia to the classic WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous in vivo reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are sometimes seen [81].

In one preclinical study introducing the WAS gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigenesis is necessary, as with SCID-X1 and CGD [84, 85].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].

Since a study using human embryonic stem cells (hESCs) and WAS-promoterdriven lentiviral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expression in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs [91].

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-haplo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is successful in most cases.

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of JAK3 into the patients bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was observed for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigenesis remains a concern with JAK gene therapy [92].

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by lethal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human PNP resulted in human PNP expression, improved thymocyte maturation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of PNP-transduced cells and the percentage of engrafted cells decreased [94].

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte integrin CD11/CD18 heterodimer due to mutations in the CD18 gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious bacterial infections immediately after birth.

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the CD18 gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. In vivo animal experiments using a PGK or an elongation factor 1 promoter did not lead to symptom improvement [95-97], but improvement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral vector in one animal study [98].

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumulation into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is divided into seven types: type I (-L-iduronidase deficiency, Hurler syndrome, Sheie syndrome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, -N-acetylglucosaminidase deficiency, -glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase deficiency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syndrome), type VII (-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lysosomes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retardation, varies with MPS type.

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major challenges in the treatment of MPS. Since replaced enzymes easily cannot pass the blood-brain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for example, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, although the underlying mechanism is unknown. There are also trials where AAV vectors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue [99].

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts [101].

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or without mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, infantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is considered in cases with little improvement with enzyme replacement therapy [103].

For Gaucher disease without CNS symptoms, a animal model using an AAV vector to produce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher -glucocerebrosidase activity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelination of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (ABCD1) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be obtained with HSCT [107].

One study has reported the introduction of wild-type ABCD1 using a lentiviral vector into peripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 714% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

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Recent Advances in Hematopoietic Stem Cell Gene Therapy ...

IPS Cell Therapy Comments Off on Recent Advances in Hematopoietic Stem Cell Gene Therapy … | November 13th, 2016

SIGNIFICANCE:

Heart disease is the primary cause of death in the industrialized world. Cardiac failure is dictated by an uncompensated reduction in the number of viable and fully functional cardiomyocytes. While current pharmacological therapies alleviate the symptoms associated with cardiac deterioration, heart transplantation remains the only therapy for advanced heart failure. Therefore, there is a pressing need for novel therapeutic modalities. Cell-based therapies involving cardiac stem cells (CSCs) constitute a promising emerging approach for the replenishment of the lost tissue and the restoration of cardiac contractility.

CSCs reside in the adult heart and govern myocardial homeostasis and repair after injury by producing new cardiomyocytes and vascular structures. In the last decade, different classes of immature cells expressing distinct stem cell markers have been identified and characterized in terms of their growth properties, differentiation potential, and regenerative ability. Phase I clinical trials, employing autologous CSCs in patients with ischemic cardiomyopathy, are being completed with encouraging results.

Accumulating evidence concerning the role of CSCs in heart regeneration imposes a reconsideration of the mechanisms of cardiac aging and the etiology of heart failure. Deciphering the molecular pathways that prevent activation of CSCs in their environment and understanding the processes that affect CSC survival and regenerative function with cardiac pathologies, commonly accompanied by alterations in redox conditions, are of great clinical importance.

Further investigations of CSC biology may be translated into highly effective and novel therapeutic strategies aiming at the enhancement of the endogenous healing capacity of the diseased heart.

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Cardiac stem cells: biology and clinical applications.

Cardiac Stem Cells Comments Off on Cardiac stem cells: biology and clinical applications. | November 7th, 2016

Spinal cord injury (SCI) causes the irreversible loss of spinal cord parenchyma including astroglia, oligodendroglia and neurons. In particular, severe injuries can lead to an almost complete neural cell loss at the lesion site and structural and functional recovery might only be accomplished by appropriate cell and tissue replacement. Stem cells have the capacity to differentiate into all relevant neural cell types necessary to replace degenerated spinal cord tissue and can now be obtained from virtually any stage of development. Within the last two decades, many in vivo studies in small animal models of SCI have demonstrated that stem cell transplantation can promote morphological and, in some cases, functional recovery via various mechanisms including remyelination, axon growth and regeneration, or neuronal replacement. However, only two well-documented neural-stem-cell-based transplantation strategies have moved to phase I clinical trials to date. This review aims to provide an overview about the current status of preclinical and clinical neural stem cell transplantation and discusses future perspectives in the field.

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Neural Stem Cells for Spinal Cord Repair

Spinal Cord Stem Cells Comments Off on Neural Stem Cells for Spinal Cord Repair | November 2nd, 2016

This article is about the medical therapy. For the cell type, see Stem cell.

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition.

Bone marrow transplant is the most widely used stem-cell therapy, but some therapies derived from umbilical cord blood are also in use. Research is underway to develop various sources for stem cells, and to apply stem-cell treatments for neurodegenerative diseases and conditions such as diabetes, heart disease, and other conditions.

Stem-cell therapy has become controversial following developments such as the ability of scientists to isolate and culture embryonic stem cells, to create stem cells using somatic cell nuclear transfer and their use of techniques to create induced pluripotent stem cells. This controversy is often related to abortion politics and to human cloning. Additionally, efforts to market treatments based on transplant of stored umbilical cord blood have been controversial.

For over 30 years, bone marrow has been used to treat cancer patients with conditions such as leukaemia and lymphoma; this is the only form of stem-cell therapy that is widely practiced.[1][2][3] During chemotherapy, most growing cells are killed by the cytotoxic agents. These agents, however, cannot discriminate between the leukaemia or neoplastic cells, and the hematopoietic stem cells within the bone marrow. It is this side effect of conventional chemotherapy strategies that the stem-cell transplant attempts to reverse; a donor's healthy bone marrow reintroduces functional stem cells to replace the cells lost in the host's body during treatment. The transplanted cells also generate an immune response that helps to kill off the cancer cells; this process can go too far, however, leading to graft vs host disease, the most serious side effect of this treatment.[4]

Another stem-cell therapy called Prochymal, was conditionally approved in Canada in 2012 for the management of acute graft-vs-host disease in children who are unresponsive to steroids.[5] It is an allogenic stem therapy based on mesenchymal stem cells (MSCs) derived from the bone marrow of adult donors. MSCs are purified from the marrow, cultured and packaged, with up to 10,000 doses derived from a single donor. The doses are stored frozen until needed.[6]

The FDA has approved five hematopoietic stem-cell products derived from umbilical cord blood, for the treatment of blood and immunological diseases.[7]

In 2014, the European Medicines Agency recommended approval of Holoclar, a treatment involving stem cells, for use in the European Union. Holoclar is used for people with severe limbal stem cell deficiency due to burns in the eye.[8]

In March 2016 GlaxoSmithKline's Strimvelis (GSK2696273) therapy for the treatment ADA-SCID was recommended for EU approval.[9]

Stem cells are being studied for a number of reasons. The molecules and exosomes released from stem cells are also being studied in an effort to make medications.[10]

Research has been conducted on the effects of stem cells on animal models of brain degeneration, such as in Parkinson's, Amyotrophic lateral sclerosis, and Alzheimer's disease.[11][12][13] There have been preliminary studies related to multiple sclerosis.[14][15]

Healthy adult brains contain neural stem cells which divide to maintain general stem-cell numbers, or become progenitor cells. In healthy adult laboratory animals, progenitor cells migrate within the brain and function primarily to maintain neuron populations for olfaction (the sense of smell). Pharmacological activation of endogenous neural stem cells has been reported to induce neuroprotection and behavioral recovery in adult rat models of neurological disorder.[16][17][18]

Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons and oligodendrocytes within the brain. A small clinical trial was underway in Scotland in 2013, in which stem cells were injected into the brains of stroke patients.[19]

Clinical and animal studies have been conducted into the use of stem cells in cases of spinal cord injury.[20][21][22]

The pioneering work[23] by Bodo-Eckehard Strauer has now been discredited by the identification of hundreds of factual contradictions.[24] Among several clinical trials that have reported that adult stem-cell therapy is safe and effective, powerful effects have been reported from only a few laboratories, but this has covered old[25] and recent[26] infarcts as well as heart failure not arising from myocardial infarction.[27] While initial animal studies demonstrated remarkable therapeutic effects,[28][29] later clinical trials achieved only modest, though statistically significant, improvements.[30][31] Possible reasons for this discrepancy are patient age,[32] timing of treatment[33] and the recent occurrence of a myocardial infarction.[34] It appears that these obstacles may be overcome by additional treatments which increase the effectiveness of the treatment[35] or by optimizing the methodology although these too can be controversial. Current studies vary greatly in cell-procuring techniques, cell types, cell-administration timing and procedures, and studied parameters, making it very difficult to make comparisons. Comparative studies are therefore currently needed.

Stem-cell therapy for treatment of myocardial infarction usually makes use of autologous bone-marrow stem cells (a specific type or all), however other types of adult stem cells may be used, such as adipose-derived stem cells.[36] Adult stem cell therapy for treating heart disease was commercially available in at least five continents as of 2007.[citation needed]

Possible mechanisms of recovery include:[11]

It may be possible to have adult bone-marrow cells differentiate into heart muscle cells.[11]

The first successful integration of human embryonic stem cell derived cardiomyocytes in guinea pigs (mouse hearts beat too fast) was reported in August 2012. The contraction strength was measured four weeks after the guinea pigs underwent simulated heart attacks and cell treatment. The cells contracted synchronously with the existing cells, but it is unknown if the positive results were produced mainly from paracrine as opposed to direct electromechanical effects from the human cells. Future work will focus on how to get the cells to engraft more strongly around the scar tissue. Whether treatments from embryonic or adult bone marrow stem cells will prove more effective remains to be seen.[37]

In 2013 the pioneering reports of powerful beneficial effects of autologous bone marrow stem cells on ventricular function were found to contain "hundreds" of discrepancies.[38] Critics report that of 48 reports there seemed to be just five underlying trials, and that in many cases whether they were randomized or merely observational accepter-versus-rejecter, was contradictory between reports of the same trial. One pair of reports of identical baseline characteristics and final results, was presented in two publications as, respectively, a 578 patient randomized trial and as a 391 patient observational study. Other reports required (impossible) negative standard deviations in subsets of patients, or contained fractional patients, negative NYHA classes. Overall there were many more patients published as having receiving stem cells in trials, than the number of stem cells processed in the hospital's laboratory during that time. A university investigation, closed in 2012 without reporting, was reopened in July 2013.[39]

One of the most promising benefits of stem cell therapy is the potential for cardiac tissue regeneration to reverse the tissue loss underlying the development of heart failure after cardiac injury.[40]

Initially, the observed improvements were attributed to a transdifferentiation of BM-MSCs into cardiomyocyte-like cells.[28] Given the apparent inadequacy of unmodified stem cells for heart tissue regeneration, a more promising modern technique involves treating these cells to create cardiac progenitor cells before implantation to the injured area.[41]

The specificity of the human immune-cell repertoire is what allows the human body to defend itself from rapidly adapting antigens. However, the immune system is vulnerable to degradation upon the pathogenesis of disease, and because of the critical role that it plays in overall defense, its degradation is often fatal to the organism as a whole. Diseases of hematopoietic cells are diagnosed and classified via a subspecialty of pathology known as hematopathology. The specificity of the immune cells is what allows recognition of foreign antigens, causing further challenges in the treatment of immune disease. Identical matches between donor and recipient must be made for successful transplantation treatments, but matches are uncommon, even between first-degree relatives. Research using both hematopoietic adult stem cells and embryonic stem cells has provided insight into the possible mechanisms and methods of treatment for many of these ailments.[citation needed]

Fully mature human red blood cells may be generated ex vivo by hematopoietic stem cells (HSCs), which are precursors of red blood cells. In this process, HSCs are grown together with stromal cells, creating an environment that mimics the conditions of bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stem cells to complete terminal differentiation into red blood cells.[42] Further research into this technique should have potential benefits to gene therapy, blood transfusion, and topical medicine.

In 2004, scientists at King's College London discovered a way to cultivate a complete tooth in mice[43] and were able to grow bioengineered teeth stand-alone in the laboratory. Researchers are confident that the tooth regeneration technology can be used to grow live teeth in human patients.

In theory, stem cells taken from the patient could be coaxed in the lab turning into a tooth bud which, when implanted in the gums, will give rise to a new tooth, and would be expected to be grown in a time over three weeks.[44] It will fuse with the jawbone and release chemicals that encourage nerves and blood vessels to connect with it. The process is similar to what happens when humans grow their original adult teeth. Many challenges remain, however, before stem cells could be a choice for the replacement of missing teeth in the future.[45][46]

Research is ongoing in different fields, alligators which are polyphyodonts grow up to 50 times a successional tooth (a small replacement tooth) under each mature functional tooth for replacement once a year.[47]

Heller has reported success in re-growing cochlea hair cells with the use of embryonic stem cells.[48]

Since 2003, researchers have successfully transplanted corneal stem cells into damaged eyes to restore vision. "Sheets of retinal cells used by the team are harvested from aborted fetuses, which some people find objectionable." When these sheets are transplanted over the damaged cornea, the stem cells stimulate renewed repair, eventually restore vision.[49] The latest such development was in June 2005, when researchers at the Queen Victoria Hospital of Sussex, England were able to restore the sight of forty patients using the same technique. The group, led by Sheraz Daya, was able to successfully use adult stem cells obtained from the patient, a relative, or even a cadaver. Further rounds of trials are ongoing.[50]

In April 2005, doctors in the UK transplanted corneal stem cells from an organ donor to the cornea of Deborah Catlyn, a woman who was blinded in one eye when acid was thrown in her eye at a nightclub. The cornea, which is the transparent window of the eye, is a particularly suitable site for transplants. In fact, the first successful human transplant was a cornea transplant. The absence of blood vessels within the cornea makes this area a relatively easy target for transplantation. The majority of corneal transplants carried out today are due to a degenerative disease called keratoconus.

The University Hospital of New Jersey reports that the success rate for growth of new cells from transplanted stem cells varies from 25 percent to 70 percent.[51]

In 2014, researchers demonstrated that stem cells collected as biopsies from donor human corneas can prevent scar formation without provoking a rejection response in mice with corneal damage.[52]

In January 2012, The Lancet published a paper by Steven Schwartz, at UCLA's Jules Stein Eye Institute, reporting two women who had gone legally blind from macular degeneration had dramatic improvements in their vision after retinal injections of human embryonic stem cells.[53]

In June 2015, the Stem Cell Ophthalmology Treatment Study (SCOTS), the largest adult stem cell study in ophthalmology ( http://www.clinicaltrials.gov NCT # 01920867) published initial results on a patient with optic nerve disease who improved from 20/2000 to 20/40 following treatment with bone marrow derived stem cells.[54]

Diabetes patients lose the function of insulin-producing beta cells within the pancreas.[55] In recent experiments, scientists have been able to coax embryonic stem cell to turn into beta cells in the lab. In theory if the beta cell is transplanted successfully, they will be able to replace malfunctioning ones in a diabetic patient.[56]

Human embryonic stem cells may be grown in cell culture and stimulated to form insulin-producing cells that can be transplanted into the patient.

However, clinical success is highly dependent on the development of the following procedures:[11]

Clinical case reports in the treatment orthopaedic conditions have been reported. To date, the focus in the literature for musculoskeletal care appears to be on mesenchymal stem cells. Centeno et al. have published MRI evidence of increased cartilage and meniscus volume in individual human subjects.[57][58] The results of trials that include a large number of subjects, are yet to be published. However, a published safety study conducted in a group of 227 patients over a 3-4-year period shows adequate safety and minimal complications associated with mesenchymal cell transplantation.[59]

Wakitani has also published a small case series of nine defects in five knees involving surgical transplantation of mesenchymal stem cells with coverage of the treated chondral defects.[60]

Stem cells can also be used to stimulate the growth of human tissues. In an adult, wounded tissue is most often replaced by scar tissue, which is characterized in the skin by disorganized collagen structure, loss of hair follicles and irregular vascular structure. In the case of wounded fetal tissue, however, wounded tissue is replaced with normal tissue through the activity of stem cells.[61] A possible method for tissue regeneration in adults is to place adult stem cell "seeds" inside a tissue bed "soil" in a wound bed and allow the stem cells to stimulate differentiation in the tissue bed cells. This method elicits a regenerative response more similar to fetal wound-healing than adult scar tissue formation.[61] Researchers are still investigating different aspects of the "soil" tissue that are conducive to regeneration.[61]

Culture of human embryonic stem cells in mitotically inactivated porcine ovarian fibroblasts (POF) causes differentiation into germ cells (precursor cells of oocytes and spermatozoa), as evidenced by gene expression analysis.[62]

Human embryonic stem cells have been stimulated to form Spermatozoon-like cells, yet still slightly damaged or malformed.[63] It could potentially treat azoospermia.

In 2012, oogonial stem cells were isolated from adult mouse and human ovaries and demonstrated to be capable of forming mature oocytes.[64] These cells have the potential to treat infertility.

Destruction of the immune system by the HIV is driven by the loss of CD4+ T cells in the peripheral blood and lymphoid tissues. Viral entry into CD4+ cells is mediated by the interaction with a cellular chemokine receptor, the most common of which are CCR5 and CXCR4. Because subsequent viral replication requires cellular gene expression processes, activated CD4+ cells are the primary targets of productive HIV infection.[65] Recently scientists have been investigating an alternative approach to treating HIV-1/AIDS, based on the creation of a disease-resistant immune system through transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and progenitor cells (GM-HSPC).[66]

On 23 January 2009, the US Food and Drug Administration gave clearance to Geron Corporation for the initiation of the first clinical trial of an embryonic stem-cell-based therapy on humans. The trial aimed evaluate the drug GRNOPC1, embryonic stem cell-derived oligodendrocyte progenitor cells, on patients with acute spinal cord injury. The trial was discontinued in November 2011 so that the company could focus on therapies in the "current environment of capital scarcity and uncertain economic conditions".[67] In 2013 biotechnology and regenerative medicine company BioTime (NYSEMKT:BTX) acquired Geron's stem cell assets in a stock transaction, with the aim of restarting the clinical trial.[68]

Scientists have reported that MSCs when transfused immediately within few hours post thawing may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth(fresh), so cryopreserved MSCs should be brought back into log phase of cell growth in invitro culture before these are administered for clinical trials or experimental therapies, re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various clinical trials on MSCs have failed which used cryopreserved product immediately post thaw as compared to those clinical trials which used fresh MSCs.[69]

Research currently conducted on horses, dogs, and cats can benefit the development of stem cell treatments in veterinary medicine and can target a wide range of injuries and diseases such as myocardial infarction, stroke, tendon and ligament damage, osteoarthritis, osteochondrosis and muscular dystrophy both in large animals, as well as humans.[70][71][72][73] While investigation of cell-based therapeutics generally reflects human medical needs, the high degree of frequency and severity of certain injuries in racehorses has put veterinary medicine at the forefront of this novel regenerative approach.[74] Companion animals can serve as clinically relevant models that closely mimic human disease.[75][76]

There is widespread controversy over the use of human embryonic stem cells. This controversy primarily targets the techniques used to derive new embryonic stem cell lines, which often requires the destruction of the blastocyst. Opposition to the use of human embryonic stem cells in research is often based on philosophical, moral, or religious objections.[110] There is other stem cell research that does not involve the destruction of a human embryo, and such research involves adult stem cells, amniotic stem cells, and induced pluripotent stem cells.

Stem-cell research and treatment was practiced in the People's Republic of China. The Ministry of Health of the People's Republic of China has permitted the use of stem-cell therapy for conditions beyond those approved of in Western countries. The Western World has scrutinized China for its failed attempts to meet international documentation standards of these trials and procedures.[111]

Since 2008 many universities, centers and doctors tried a diversity of methods; in Lebanon proliferation for stem cell therapy, in-vivo and in-vitro techniques were used, Thus this country is considered the launching place of the Regentime[112] procedure. http://www.researchgate.net/publication/281712114_Treatment_of_Long_Standing_Multiple_Sclerosis_with_Regentime_Stem_Cell_Technique The regenerative medicine also took place in Jordan and Egypt.[citation needed]

Stem-cell treatment is currently being practiced at a clinical level in Mexico. An International Health Department Permit (COFEPRIS) is required. Authorized centers are found in Tijuana, Guadalajara and Cancun. Currently undergoing the approval process is Los Cabos. This permit allows the use of stem cell.[citation needed]

In 2005, South Korean scientists claimed to have generated stem cells that were tailored to match the recipient. Each of the 11 new stem cell lines was developed using somatic cell nuclear transfer (SCNT) technology. The resultant cells were thought to match the genetic material of the recipient, thus suggesting minimal to no cell rejection.[113]

As of 2013, Thailand still considers Hematopoietic stem cell transplants as experimental. Kampon Sriwatanakul began with a clinical trial in October 2013 with 20 patients. 10 are going to receive stem-cell therapy for Type-2 diabetes and the other 10 will receive stem-cell therapy for emphysema. Chotinantakul's research is on Hematopoietic cells and their role for the hematopoietic system function in homeostasis and immune response.[114]

Today, Ukraine is permitted to perform clinical trials of stem-cell treatments (Order of the MH of Ukraine 630 "About carrying out clinical trials of stem cells", 2008) for the treatment of these pathologies: pancreatic necrosis, cirrhosis, hepatitis, burn disease, diabetes, multiple sclerosis, critical lower limb ischemia. The first medical institution granted the right to conduct clinical trials became the "Institute of Cell Therapy"(Kiev).

Other countries where doctors did stem cells research, trials, manipulation, storage, therapy: Brazil, Cyprus, Germany, Italy, Israel, Japan, Pakistan, Philippines, Russia, Switzerland, Turkey, United Kingdom, India, and many others.

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Stem-cell therapy - Wikipedia

Spinal Cord Stem Cells Comments Off on Stem-cell therapy – Wikipedia | November 2nd, 2016

Bone marrow is the flexible tissue in the interior of bones. In humans, red blood cells are produced by cores of bone marrow in the heads of long bones in a process known as hematopoiesis.[2] On average, bone marrow constitutes 4% of the total body mass of humans; in an adult having 65 kilograms of mass (143 lbs), bone marrow typically accounts for approximately 2.6 kilograms (5.7lb). The hematopoietic component of bone marrow produces approximately 500 billion blood cells per day, which use the bone marrow vasculature as a conduit to the body's systemic circulation.[3] Bone marrow is also a key component of the lymphatic system, producing the lymphocytes that support the body's immune system.[4]

Bone marrow transplants can be conducted to treat severe diseases of the bone marrow, including certain forms of cancer such as leukemia. Additionally, bone marrow stem cells have been successfully transformed into functional neural cells,[5] and can also potentially be used to treat illnesses such as inflammatory bowel disease.[6]

The two types of bone marrow are "red marrow" (Latin: medulla ossium rubra), which consists mainly of hematopoietic tissue, and "yellow marrow" (Latin: medulla ossium flava), which is mainly made up of fat cells. Red blood cells, platelets, and most white blood cells arise in red marrow. Both types of bone marrow contain numerous blood vessels and capillaries. At birth, all bone marrow is red. With age, more and more of it is converted to the yellow type; only around half of adult bone marrow is red. Red marrow is found mainly in the flat bones, such as the pelvis, sternum, cranium, ribs, vertebrae and scapulae, and in the cancellous ("spongy") material at the epiphyseal ends of long bones such as the femur and humerus. Yellow marrow is found in the medullary cavity, the hollow interior of the middle portion of short bones. In cases of severe blood loss, the body can convert yellow marrow back to red marrow to increase blood cell production.

The stroma of the bone marrow is all tissue not directly involved in the marrow's primary function of hematopoiesis.[2] Yellow bone marrow makes up the majority of bone marrow stroma, in addition to smaller concentrations of stromal cells located in the red bone marrow. Though not as active as parenchymal red marrow, stroma is indirectly involved in hematopoiesis, since it provides the hematopoietic microenvironment that facilitates hematopoiesis by the parenchymal cells. For instance, they generate colony stimulating factors, which have a significant effect on hematopoiesis. Cell types that constitute the bone marrow stroma include:

In addition, the bone marrow contains hematopoietic stem cells, which give rise to the three classes of blood cells that are found in the circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes).[7]

The bone marrow stroma contains mesenchymal stem cells (MSCs),[7] also known as marrow stromal cells. These are multipotent stem cells that can differentiate into a variety of cell types. MSCs have been shown to differentiate, in vitro or in vivo, into osteoblasts, chondrocytes, myocytes, adipocytes and beta-pancreatic islets cells.

The blood vessels of the bone marrow constitute a barrier, inhibiting immature blood cells from leaving the marrow. Only mature blood cells contain the membrane proteins, such as aquaporin and glycophorin, that are required to attach to and pass the blood vessel endothelium.[9]Hematopoietic stem cells may also cross the bone marrow barrier, and may thus be harvested from blood.

The red bone marrow is a key element of the lymphatic system, being one of the primary lymphoid organs that generate lymphocytes from immature hematopoietic progenitor cells.[4] The bone marrow and thymus constitute the primary lymphoid tissues involved in the production and early selection of lymphocytes. Furthermore, bone marrow performs a valve-like function to prevent the backflow of lymphatic fluid in the lymphatic system.

Biological compartmentalization is evident within the bone marrow, in that certain cell types tend to aggregate in specific areas. For instance, erythrocytes, macrophages, and their precursors tend to gather around blood vessels, while granulocytes gather at the borders of the bone marrow.[7]

Animal bone marrow has been used in cuisine worldwide for millennia, such as the famed Milanese Ossobuco.[citation needed]

The normal bone marrow architecture can be damaged or displaced by aplastic anemia, malignancies such as multiple myeloma, or infections such as tuberculosis, leading to a decrease in the production of blood cells and blood platelets. The bone marrow can also be affected by various forms of leukemia, which attacks its hematologic progenitor cells.[10] Furthermore, exposure to radiation or chemotherapy will kill many of the rapidly dividing cells of the bone marrow, and will therefore result in a depressed immune system. Many of the symptoms of radiation poisoning are due to damage sustained by the bone marrow cells.

To diagnose diseases involving the bone marrow, a bone marrow aspiration is sometimes performed. This typically involves using a hollow needle to acquire a sample of red bone marrow from the crest of the ilium under general or local anesthesia.[11]

On CT and plain film, marrow change can be seen indirectly by assessing change to the adjacent ossified bone. Assessment with MRI is usually more sensitive and specific for pathology, particularly for hematologic malignancies like leukemia and lymphoma. These are difficult to distinguish from the red marrow hyperplasia of hematopoiesis, as can occur with tobacco smoking, chronically anemic disease states like sickle cell anemia or beta thalassemia, medications such as granulocyte colony-stimulating factors, or during recovery from chronic nutritional anemias or therapeutic bone marrow suppression.[12] On MRI, the marrow signal is not supposed to be brighter than the adjacent intervertebral disc on T1 weighted images, either in the coronal or sagittal plane, where they can be assessed immediately adjacent to one another.[13] Fatty marrow change, the inverse of red marrow hyperplasia, can occur with normal aging,[14] though it can also be seen with certain treatments such as radiation therapy. Diffuse marrow T1 hypointensity without contrast enhancement or cortical discontinuity suggests red marrow conversion or myelofibrosis. Falsely normal marrow on T1 can be seen with diffuse multiple myeloma or leukemic infiltration when the water to fat ratio is not sufficiently altered, as may be seen with lower grade tumors or earlier in the disease process.[15]

Bone marrow examination is the pathologic analysis of samples of bone marrow obtained via biopsy and bone marrow aspiration. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells. While much information can be gleaned by testing the blood itself (drawn from a vein by phlebotomy), it is sometimes necessary to examine the source of the blood cells in the bone marrow to obtain more information on hematopoiesis; this is the role of bone marrow aspiration and biopsy.

The ratio between myeloid series and erythroid cells is relevant to bone marrow function, and also to diseases of the bone marrow and peripheral blood, such as leukemia and anemia. The normal myeloid-to-erythroid ratio is around 3:1; this ratio may increase in myelogenous leukemias, decrease in polycythemias, and reverse in cases of thalassemia.[16]

In a bone marrow transplant, hematopoietic stem cells are removed from a person and infused into another person (allogenic) or into the same person at a later time (autologous). If the donor and recipient are compatible, these infused cells will then travel to the bone marrow and initiate blood cell production. Transplantation from one person to another is conducted for the treatment of severe bone marrow diseases, such as congenital defects, autoimmune diseases or malignancies. The patient's own marrow is first killed off with drugs or radiation, and then the new stem cells are introduced. Before radiation therapy or chemotherapy in cases of cancer, some of the patient's hematopoietic stem cells are sometimes harvested and later infused back when the therapy is finished to restore the immune system.[17]

Bone marrow stem cells can be induced to become neural cells to treat neurological illnesses,[5] and can also potentially be used for the treatment of other illnesses, such as inflammatory bowel disease.[6] In 2013, following a clinical trial, scientists proposed that bone marrow transplantation could be used to treat HIV in conjunction with antiretroviral drugs;[18][19] however, it was later found that HIV remained in the bodies of the test subjects.[20]

The stem cells are typically harvested directly from the red marrow in the iliac crest, often under general anesthesia. The procedure is minimally invasive and does not require stitches afterwards. Depending on the donor's health and reaction to the procedure, the actual harvesting can be an outpatient procedure, or can require 12 days of recovery in the hospital.[21]

Another option is to administer certain drugs that stimulate the release of stem cells from the bone marrow into circulating blood.[22] An intravenous catheter is inserted into the donor's arm, and the stem cells are then filtered out of the blood. This procedure is similar to that used in blood or platelet donation. In adults, bone marrow may also be taken from the sternum, while the tibia is often used when taking samples from infants.[11] In newborns, stem cells may be retrieved from the umbilical cord.[23]

The earliest fossilised evidence of bone marrow was discovered in 2014 in Eusthenopteron, a lobe-finned fish which lived during the Devonian period approximately 370 million years ago.[24] Scientists from Uppsala University and the European Synchrotron Radiation Facility used X-ray synchrotron microtomography to study the fossilised interior of the skeleton's humerus, finding organised tubular structures akin to modern vertebrate bone marrow.[24]Eusthenopteron is closely related to the early tetrapods, which ultimately evolved into the land-dwelling mammals and lizards of the present day.[24]

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Bone marrow - Wikipedia

Bone Marrow Stem Cells Comments Off on Bone marrow – Wikipedia | October 25th, 2016