A Possible Connection between Mild Allergic Airway Responses and Cardiovascular Risk Featured in Toxicological Sciences  Newswise

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A heart surgeon, Professor Massimo Caputo from the Bristol Heart Institute has stated he "saved the life" of a baby by carrying out a "world-first" operation using stem cells from placentas.

Professor Massimo Caputo used pioneering stem cell injections to correct baby Finley's heart defect and says he now hopes to develop the technology so children born with congenital cardiac disease won't need much surgical operations.

Finley was born with the main arteries in his heart positioned the wrong way round and at just four days old had his first open-heart surgery at Bristol Royal Hospital for Children

Unfortunately the surgery did not solve the issue and his heart function deteriorated significantly, with the left side of the heart suffering from a severe lack of blood flow.

His mother, Melissa, from Corsham, in Wiltshire, said: "We were prepared from the start that the odds of him surviving were not good.

"After 12 hours, Finley finally came out of surgery but he needed a heart and lung bypass machine to keep alive, and his heart function had deteriorated significantly."

After weeks in intensive care it looked like there was no way to treat Finley's condition and he was reliant on drugs to keep his heart going.

But a new procedure was tried, involving stem cells from a placenta bank.

Prof Caputo injected the cells directly into Finley's heart in the hope they would help damaged blood vessels grow.

The so-called "allogeneic" cells were grown by scientists at the Royal Free Hospital in London, and millions of them were injected into Finley's heart muscle.

Allogeneic cells have the ability to grow into tissue that is not rejected and in Finley's case, have regenerated damaged heart muscle.

"We weaned him from all the drugs he was on, we weaned him from ventilation," said Prof Caputo.

"He was discharged from ITU and is now a happy growing little boy."

Finley is now aged two years.

Using a bio-printer, a stem cell scaffold is made to repair abnormalities to valves in blood vessels, and to mend holes between the two main pumping chambers of the heart.

In cardiac surgery, artificial tissue is normally used on babies for cardiac repairs, but it can fail and it does not grow with the heart, so as the children grow, they require more operations.

A child might therefore have to go through the same heart operation multiple times throughout its childhood but Prof Caputo and his team say the stem cell technology could save the UK government an estimated 30,000 for every operation no longer needed.

Dr Stephen Minger, an expert in stem cell biology and director of SLM Blue Skies Innovations Ltd said;

"Most studies that I am aware of in adults with heart dysfunction or failure show only minimal therapeutic benefit with stem cell infusion.

"I'm happy that the clinical team will go on to do a standard clinical trial which should tell us if this was a 'one-off' success and also give us some better understanding of mechanisms behind this."

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An organoid model of colorectal circulating tumor cells with stem cell ...

Cardiac Stem Cells Comments Off on An organoid model of colorectal circulating tumor cells with stem cell … | December 25th, 2022

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

Duchenne muscular dystrophy (DMD) is a genetic disease affecting approximately 1 in 3500 male live births [1]. It results in progressive degeneration of skeletal muscle causing complete paralysis, respiratory and cardiac complications, and ultimately death. Normal symptoms include the delay of motor milestones including the ability to sit and stand independently. DMD is caused by an absence of functional dystrophin protein and skeletal muscle stem cells, as well as the exhaustion of satellite cells following many rounds of muscle degeneration and regeneration [2]. The dystrophin gene is primarily responsible for connecting and maintaining the stability of the cytoskeleton of muscle fibers during contraction and relaxation. Despite the low frequency of occurrence, this disease is incurable and will cause debilitation of the muscle and eventual death in 20 to 30 year olds with recessive X-linked form of muscular dystrophy. Although there are no current treatments developed for DMD, there are several experimental therapies such as stem cell therapies.

Skeletal muscle is known to be a regenerative tissue in the body. This muscle regeneration is mediated by muscle satellite cells, a stem cell population for skeletal muscle [3, 4]. Although satellite cells exhibit some multipotential differentiation capabilities [5], their primary differentiation fate is skeletal muscle cells in normal muscle regeneration. Ex vivo expanded satellite cell-derived myoblasts can be integrated into muscle fibers following injection into damaged muscle, acting as a proof-of-concept of myoblast-mediated cell therapy for muscular dystrophies [69]. However, severe limitations exist in relation to human therapy. The number of available satellite cells or myoblasts from human biopsies is limited. In addition, the poor cell survival and low contribution of transplanted cells have hindered practical application in patients [6, 8, 9]. Human-induced pluripotent stem cells (hiPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell- (ESC-) like state by being forced to express genes and factors important for maintaining the defining properties of ESCs. hiPSCs can be generated from a wide variety of somatic cells [10, 11]. They have the ability to self-renew and successfully turn into any type of cells. With their ability to capture genetic diversity of DMD in an accessible culture system, hiPSCs represent an attractive source for generating myogenic cells for drug screening.

The ESC/iPSC differentiation follows the steps of embryonic development. The origin of skeletal muscle precursor cells comes from the mesodermal lineage, which give rise to skeletal muscle, cardiac muscle, bone, and blood cells. Mesoderm subsequently undergoes unsegmented presomitic mesoderm followed by segmented compartments termed somites from anterior to caudal direction. Dermomyotome is an epithelial cell layer making up the dorsal part of the somite underneath the ectoderm. Dermomyotome expresses Pax3 and Pax7 and gives rise to dermis, skeletal muscle cells, endothelial cells, and vascular smooth muscle [12]. Dermomyotome also serves as a tissue for secreted signaling molecules to the neural tube, notochord, and sclerotome [13, 14]. Upon signals from the neural tube and notochord, the dorsomedial lip of dermomyotome initiates and expresses skeletal muscle-specific transcription factors such as MyoD and Myf5 to differentiate into myogenic cells termed myoblasts. Myoblasts then migrate beneath the dermomyotome to form myotome. Eventually, these myoblasts fuse with each other to form embryonic muscle fibers. ESCs/iPSCs mimic these steps toward differentiation of skeletal muscle cells. Many studies utilize methods of overexpression of muscle-related transcription factors such as MyoD or Pax3 [15], or the addition of small molecules which activate or inhibit myogenic signaling during development. Several studies show that iPSCs retain a bias to form their cell type of origin due to an epigenetic memory [1619], although other papers indicate that such epigenetic memory is erased during the reprogramming processes [2022]. Therefore, this phenomenon is not completely understood at the moment. In light of these developments, we have recently established mouse myoblast-derived iPSCs capable of unlimited expansion [23]. Our data demonstrates that these iPSCs show higher myogenic differentiation potential compared to fibroblast-derived iPSCs. Thus, myogenic precursor cells generated from human myoblast-derived iPSCs expanded ex vivo should provide an attractive cell source for DMD therapy. However, since DMD is a systemic muscle disease, systemic delivery of myoblasts needs to be established for efficient cell-based therapy.

During developmental myogenesis, presomitic mesoderm is first formed by Mesogenin1 upregulation, which is a master regulator of presomitic mesoderm [24]. Then, the paired box transcription factor Pax3 gene begins to be expressed from presomitic mesoderm to dermomyotome [25]. Following Pax3 expression, Pax7 is also expressed in the dermomyotome [26], and then Myf5 and MyoD, skeletal muscle-specific transcription factor genes, begin to be expressed in the dorsomedial lip of the dermomyotome in order to give rise to myoblasts which migrate beneath the dermomyotome to form the myotome. Subsequently, Mrf4 and Myogenin, other skeletal muscle-specific transcription factor genes, followed by skeletal muscle structural genes such as myosin heavy chain (MyHC), are expressed in the myotome for myogenic terminal differentiation (Figure 1) [27, 28]. Pax3 directly and indirectly regulates Myf5 expression in order to induce myotomal cells. Dorsal neural tube-derived Wnt proteins and floor plate cells in neural tube and notochord-derived sonic hedgehog (Shh) positively regulate myotome formation [13, 29]. Neural crest cells migrating from dorsal neural tubes are also involved in myotome formation: Migrating neural crest cells come across the dorsomedial lip of the dermomyotome, and neural crest cell-expressing Delta1 is transiently able to activate Notch1 in the dermomyotome, resulting in conversion of Pax3/7(+) myogenic progenitor cells into MyoD/Myf5(+) myotomal myoblasts [30, 31]. By contrast, bone morphogenetic proteins (BMPs) secreted from lateral plate mesoderm are a negative regulator for the myotome formation by maintaining Pax3/Pax7(+) myogenic progenitor cells [29, 32]. Pax3 also regulates cell migration of myogenic progenitor cells from ventrolateral lip of dermomyotome to the limb bud [33]. Pax3 mutant mice lack limb muscle but trunk muscle development is relatively normal [34]. Pax3/Pax7 double knockout mice display failed generation of myogenic cells, suggesting that Pax3 and Pax7 are critical for proper embryonic myogenesis [35]. Therefore, both Pax3 and Pax7 are also considered master transcription factors for the specification of myogenic progenitor cells. Importantly, MyoD was identified as the first master transcription factor for myogenic specification since MyoD is directly able to reprogram nonmuscle cell type to myogenic lineage when overexpressed [3638]. In addition, genetic ablation of MyoD family gene(s) via a homologous gene recombination technique causes severe myogenic developmental or regeneration defects [3945]. Finally, genetic ablation of combinatory MyoD family genes demonstrates that MyoD/:Myf5/:MRF4/ mice do not form any skeletal muscle during embryogenesis, indicating the essential roles in skeletal muscle development of MyoD family genes [28, 46]. It was proven that Pax3 also possesses myogenic specification capability since ectopic expression of Pax3 is sufficient to induce myogenic programs in both paraxial and lateral plate mesoderm as well as in the neural tube during chicken embryogenesis [47]. In addition, genetic ablation of Pax3 and Myf5 display complete defects of body skeletal muscle formation during mouse embryogenesis [48]. Finally, overexpression of Pax7 can convert CD45(+)Sca-1(+) hematopoietic cells into skeletal muscle cells [49]. From these notions, overexpression of myogenic master transcription factors such as MyoD or Pax3 has become the major strategy for myogenic induction in nonmuscle cells, including ES/iPSCs.

The overexpression of MyoD approach to induce myogenic cells from mESCs was first described by Dekel et al. in 1992. This has been a standard approach for the myogenic induction from pluripotent stem cells (Table 1). Ozasa et al. first utilized Tet-Off systems for MyoD overexpression in mESCs and showed desmin(+) and MyHC(+) myotubes in vitro [50]. Warren et al. transfected synthetic MyoD mRNA in to hiPSCs for 3 days, which resulted in myogenic differentiation (around 40%) with expression of myogenin and MyHC [51]. Tanaka et al. utilized a PiggyBac transposon system to overexpress MyoD in hiPSCs. The PiggyBac transposon system allows cDNAs to stably integrate into the genome for efficient gene expression. After integration, around 70 to 90% of myogenic cells were induced in hiPSC cultures within 5 days [52]. This study also utilized Miyoshi myopathy patient-derived hiPSCs for the MyoD-mediated myogenic differentiation. Miyoshi myopathy is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in dysferlin gene. The patient-derived hiPSC-myogenic cells will be able to provide the opportunity for therapeutic drug screening. Abujarour et al. also established a model of patient-derived skeletal muscle cells which express NCAM, myogenin, and MyHC by doxycycline-inducible overexpression of MyoD in DMD patient-derived hiPSCs [53]. Interestingly, MyoD-induced iPSCs also showed suppression of pluripotent genes such as Nanog and a transient increase in the gene expression levels of T (Brachyury T), Pax3, and Pax7, which belong to paraxial mesodermal/myogenic progenitor genes, upstream genes of myogenesis. It is possible that low levels of MyoD activity in hiPSCs may initially suppress their pluripotent state while failing to induce myogenic programs, which may result in transient paraxial mesodermal induction. Supporting this idea, BAF60C, a SWI/SNF component that is involved in chromatin remodeling and binds to MyoD, is required to induce full myogenic program in MyoD-overexpressing hESCs [54]. Overexpression of MyoD alone in hESC can only induce some paraxial mesodermal genes such as Brachyury T, mesogenin, and Mesp1 but not myogenic genes. Co-overexpression of MyoD and BAF60C was now able to induce myogenic program but not paraxial mesodermal gene expression, indicating that there are different epigenetic landscapes between pluripotent ESCs/iPSCs and differentiating ESC/iPSCs in which MyoD is more accessible to DNA targets than those in pluripotent cells. The authors then argued that without specific chromatin modifiers, only committed cells give rise to myogenic cells by MyoD. These results strongly indicate that nuclear landscapes are important for cell homogeneity for the specific cell differentiation in ESC/iPSC cultures. Similar observations were seen in overexpression of MyoD in P19 embryonal carcinoma stem cells, which can induce paraxial mesodermal genes including Meox1, Pax3, Pax7, Six1, and Eya2 followed by muscle-specific genes. However, these MyoD-induced paraxial mesodermal genes were mediated by direct MyoD binding to their regulatory regions, which was proven by chromatin immunoprecipitation (ChIP) assays, indicating the novel role for MyoD in paraxial mesodermal cell induction [55].

hESCs/iPSCs have been differentiated into myofibers by overexpression of MyoD, and this method is considered an excellent in vitro model for human skeletal muscle diseases for muscle functional tests, therapeutic drug screening, and genetic corrections such as exon skipping and DNA editing. Shoji et al. have shown that DMD patient-derived iPSCs were used for myogenic differentiation via PiggyBac-mediated MyoD overexpression. These myogenic cells were treated with morpholinos for exon-skipping strategies for dystrophin gene correction and showed muscle functional improvement [56]. Li et al. have shown that patient-derived hiPSC gene correction by TALEN and CRISPR-Cas9 systems, and these genetically corrected hiPSCs were used for myogenic differentiation via overexpression of MyoD [57]. This work also revealed that the TALEN and CRISPR-Cas9-mediated exon 44 knock-in approach in the dystrophin gene has high efficiency in gene-editing methods for DMD patient-derived cells in which the exon 44 is missing in the genome.

Along this line of the strategy, Darabi et al. first performed overexpression of Pax3 gene, which can be activated by treatment with doxycycline in mESCs, and showed efficient induction of MyoD/Myf5(+) skeletal myoblasts in EB cultures [15]. Upon removing doxycycline, these myogenic cells underwent MyHC(+) myotubes. However, teratoma formation was observed after EB cell transplantation into cardiotoxin-injured regenerating skeletal muscle in Rag2/:C/ immunodeficient mice [15]. This indicates that myogenic cell cultures induced by Pax3 in mESCs still contain some undifferentiated cells which gave rise to teratomas. To overcome this problem, the same authors separated paraxial mesodermal cells from Pax3-induced EB cells by FACS using antibodies against cell surface markers as PDGFR(+)Flk-1() cell populations. After cell sorting, isolated Pax3-induced paraxial mesodermal cells were successfully engrafted and contributed to regenerating muscle in mdx:Rag2/:C/ DMD model immunodeficient mice without any teratoma formations. Darabi et al. also showed successful myogenic induction in mESCs and hES/iPSCs by overexpression of Pax7 [58, 59]. Pax3 and Pax7 are not only expressed in myogenic progenitor cells. They are also expressed in neural tube and neural crest cell-derived cells including a part of cardiac cell types in developmental stage, suggesting that further purification to skeletal muscle cell lineage is crucial for therapeutic applications for muscle diseases including DMD.

Taken together, overexpression of myogenic master transcription factors such as MyoD or Pax3/Pax7 is an excellent strategy for myogenic induction in hESCs and hiPSCs, which can be utilized for in vitro muscle disease models for their functional test and drug screening. However, for the safe stem cell therapy, it is essential to maintain the good cellular and genetic qualities of hESC/hiPSC-derived myogenic cells before transplantation. Therefore, random integration sites of overexpression vectors for myogenic master transcription factors and inappropriate expression control of these transgenes may diminish the safety of using these induced myogenic cells for therapeutic stem cell transplantation.

Stepwise induction protocols utilizing small molecules and growth factors have been established as alternative myogenic induction approaches and a more applicable method for therapeutic situations. As described above, during embryonic myogenesis, somites and dermomyotomes receive secreted signals such as Wnts, Notch ligands, Shh, FGF, BMP, and retinoic acid (RA) with morphogen gradients from surrounding tissues in order to induce the formation of myogenic cells (Figure 2). The canonical Wnt signaling pathway has been shown to play essential roles in the development of myogenesis. In mouse embryogenesis, Wnt1 and Wnt3a secreted from the dorsal neural tube can promote myogenic differentiation of dorsomedial dermomyotome via activation of Myf5 [31, 32, 60]. Wnt3a is able to stabilize -catenin which associates with TCF/LEF transcription factors that bind to the enhancer region of Myf5 during myogenesis [61]. Other Wnt proteins, Wnt6 and Wnt7a, which emerge from the surface ectoderm, induce MyoD [62]. BMP functions as an inhibitor of myogenesis by suppression of some myogenic gene expressions. In the lateral mesoderm, BMP4 is able to increase Pax3 expression which delays Myf5 expression in order to maintain an undifferentiated myogenic progenitor state [63]. Therefore, Wnts and BMPs regulate myogenic development by antagonizing each other for myogenic transcription factor gene expression [64, 65]. Wnt also induces Noggin expression to antagonize BMP signals in the dorsomedial lip of the dermomyotome [66]. In this region, MyoD expression level is increased, which causes myotome formation. Notch signaling plays essential roles for cell-cell communication to specify the different cells in developmental stages. During myotome formation, Notch is expressed in dermomyotome, and Notch1 and Notch2 are expressed in dorsomedial lip of dermomyotome. Delta1, a Notch ligand, is expressed in neural crest cells which transiently interact with myogenic progenitor cells in dorsomedial lip of dermomyotome via Notch1 and 2. This contact induces expression of the Myf5 or MyoD gene in the myogenic progenitor cells followed by myotome formation. The loss of function of Delta1 in the neural crest displays delaying skeletal muscle formation [67]. Knockdown of Notch genes or use of a dominant-negative form of mastermind, a Notch transcriptional coactivator, clearly shows dramatically decrease of Myf5 and MyHC(+) myogenic cells. Interestingly, induction of Notch intracellular domain (NICD), a constitutive active form of Notch, can promote myogenesis, while continuous expression of NICD prevents terminal differentiation. Taken together, transient and timely activation of Notch is crucial for myotome formation from dermomyotome [30].

Current studies for myogenic differentiation of ESCs/iPSCs have utilized supplementation with some growth factors and small molecules, which would mimic the myogenic development described above in combination with embryoid body (EB) aggregation and FACS separation of mesodermal cells (Table 2). To induce paraxial mesoderm cells from mESCs, Sakurai et al. utilized BMP4 in serum-free cultures [68]. Three days after treatment with BMP4, mESCs could be differentiated into primitive streak mesodermal-like cells, but the continuous treatment with BMP4 turned the ESCs into osteogenic cells. Therefore, they used LiCl after treatment with BMP4 to enhance Wnt signaling, which is able to induce myogenic differentiation. After treatment with LiCl, PDGFR(+) E-cadherin() paraxial mesodermal cells were sorted by FACS. These sorted cells were cultured with IGF, HGF, and FGF for two weeks in order to induce myogenic differentiation. Hwang et al. have shown that treatment with Wnt3a efficiently promotes skeletal muscle differentiation of hESCs [69]. hESCs were cultured to form EB for 9 days followed by differentiation of EBs for additional 7 days, and then PDGFR(+) cells were sorted by FACS. These PDGFR(+) cells were cultured with Wnt3a for additional 14 days. Consequently, these Wnt3a-treated cells display significantly increased myogenic transcription factors and structural proteins at both mRNA and protein levels. An interesting approach to identify key molecules that induce myogenic cells was reported by Xu et al. [70]. They utilized reporter systems in zebrafish embryos to display myogenic progenitor cell induction and myogenic differentiation in order to identify small compounds for myogenic induction. Myf5-GFP marks myogenic progenitor cells, while myosin light polypeptide 2 (mylz2)-mCherry marks terminally differentiated muscle cells. They found that a mixed cocktail containing GSK3 inhibitor, bFGF, and forskolin has the potential to induce robust myogenic induction in hiPSCs. GSK3 inhibitors act as a canonical Wnt signaling activator via stabilizing -catenin protein, which is crucial for inducing mesodermal cells. Forskolin activates adenylyl cyclase, which then stimulates cAMP signaling. cAMP response element-binding protein (CREB) is able to stimulate cell proliferation of primary myoblasts in vitro, suggesting that the forskolin-cAMP-CREB pathway may help myogenic cell expansion [71], However the precise mechanisms for CREB-mediated myogenic cell expansion remain unclear. The adenylyl cyclase signaling cascade leads to CREB activation [71]. During embryogenesis, phosphorylated CREB has been found at dorsal somite and dermomyotome. CREB gene knockout mice display significantly decreased Myf5 and MyoD expressions in myotomes. While activation of Wnt1 or Wnt7a promotes Pax3, Myf5, and MyoD expressions, inhibition of CREB eliminates these Wnt-mediated myogenic gene expressions without altering the Wnt canonical pathway, suggesting that CREB-induced myogenic activation may be mediated through noncanonical Wnt pathways. Several groups also utilized GSK3 inhibitors for inducing mesodermal cells from ESCs and iPSCs [72, 73]. These mesodermal cell-like cells were expanded by treatment with bFGF, and then ITS (insulin/transferrin/selenite) or N2 medium were used to induce myogenic differentiation. Finally, bFGF is a stimulator for myogenic cell proliferation. Caron et al. demonstrated that hESCs treated with GSK3 inhibitor, ascorbic acid, Alk5 inhibitor, dexamethasone, EGF, and insulin generated around 80% of Pax3(+) myogenic precursor cells in 10 days [74]. Treatment with SB431542, an inhibitor of Alk4, 5, and 7, PDGF, bFGF, oncostatin, and IGF was able to induce these Pax3(+) myogenic precursor cells into around 5060% of MyoD(+) myoblasts in an additional 8 days. For the final step, treatment with insulin, necrosulfonamide, an inhibitor of necrosis, oncostatin, and ascorbic acid was able to induce these myoblasts into myotubes in an additional 8 days. Importantly, the same authors utilized ESCs from human facioscapulohumeral muscular dystrophy (FSHD) to demonstrate the myogenic characterization after myogenic induction by using the protocol described above. Hosoyama et al. have shown that hESCs/iPSCs with high concentrations of bFGF and EGF in combination with cell aggregation, termed EZ spheres, efficiently give rise to myogenic cells [75]. After 6-week culture, around 4050% of cells expressed Pax7, MyoD, or myogenin. However, the authors also showed that EZ spheres included around 30% of Tuj1(+) neural cells. Therefore, the authors discussed the utilization of molecules for activation of mesodermal and myogenic signaling pathways such as BMPs and Wnts.

Taken together, it is likely that the induced cell populations from ESCs/iPSCs may contain other cell types such as neural cells or cardiac cells because neural cells share similar transcription factor gene expression with myogenic cells such as Pax3, and cardiac cells also develop from mesodermal cells. To overcome this limitation, Chal et al. treated ESCs/iPSCs with BMP4 inhibitor, which prevents ESCs/iPSCs from differentiating into lateral mesodermal cells [76, 77]. To identify what genes are involved in myogenic differentiation in vivo, they performed a microarray analysis which compared samples of dissected fragments in mouse embryos, which are able to separate tail bud, presomitic mesoderm, and somite regions. From microarray data, the authors focused on Mesogenin1 (Msgn1) and Pax3 genes. Importantly, they utilized three lineage tracing reporters, Msgn1-repV (Mesogenin1-Venus) marking posterior somitic mesoderm, Pax3-GFP marking anterior somitic mesoderm and myogenic cells, and Myog-repV (Myogenin-Venus) marking differentiated myocytes, allowing the authors to readily detect different differentiation stages during ESC/iPSC cultures. Treatment with GSK3 inhibitors and then BMP inhibitors in ESC cultures induced Msgn1(+) somitic mesoderm with 45 to 65% efficiencies, Pax3(+) anterior somitic mesoderm with 30 to 50% efficiencies, and myogenin(+) myogenic cells with 25 to 30% efficiencies. Furthermore, the authors examined differentiation of mdx ESCs into skeletal muscle cells and revealed abnormal branching myofibers. Current protocols were also published and described more details for hiPSC differentiation [77].

Some nonmuscle cell populations such as mesoangioblasts have the potential to differentiate into skeletal muscle [6]. Mesoangioblasts were originally isolated from embryonic mouse dorsal aorta as vessel-associated pericyte-like cells, which have the ability to differentiate into a myogenic lineage in vitro and in vivo [6, 78]. Mesoangioblasts possess an advantage for the clinical cell-based treatment because they can be injected through an intra-arterial route to systemically deliver cells, which is crucial for therapeutic cell transplantation for muscular dystrophies [79]. Tedesco et al. successfully generated human iPSC-derived mesoangioblast-like stem/progenitor cells called HIDEMs by stepwise protocols without FACS sorting [80, 81]. They displayed similar gene expression profiles as embryonic mesoangioblasts. However, HIDEMs do not spontaneously differentiate into skeletal muscle cells, and thus, the authors utilized overexpression of MyoD to differentiate into skeletal muscle cells. Similar to mesoangioblasts, HIDEM-derived myogenic cells could be delivered to injured muscle via intramuscular and intra-arterial routes. Furthermore, HIDEMs have been generated from hiPSCs derived from limb-girdle muscular dystrophy (LGMD) type 2D patients and used for gene correction and cell transplantation experiments for the potential therapeutic application.

Myogenic precursor cells derived from ESCs/iPSCs by various methods may contain nonmuscle cells. Therefore, further purification is mandatory for therapeutic applications. Barberi et al. isolated CD73(+) multipotent mesenchymal precursor cells from hESCs by FACS, and these cells underwent differentiation into fat, cartilage, bone, and skeletal muscle cells [82]. Barberi et al. also demonstrated that hESCs cultured on OP9 stroma cells generated around 5% of CD73(+) adult mesenchymal stem cell-like cells [83]. After FACS, these CD73(+) mesenchymal stem cell-like cells were cultured with ITS medium for 4 weeks and then gave rise to NCAM(+) myogenic cells. After FACS sorting, these NCAM(+) myogenic cells were purified by FACS and transplanted into immunodeficient mice to show their myogenic contribution to regenerating muscle.

It has been shown that many genes are associated with myogenesis. In addition, exhaustive analysis, such as microarray, RNA-seq, and single cell RNA-seq supplies much gene information in many different stages. Chal et al. showed key signaling factors by microarray from presomitic somite, somite, and tail bud cells [76]. They found that initial Wnt signaling has important roles for somite differentiation. Furthermore, mapping differentiated hESCs by single cell RNA-seq analysis is useful to characterize each differentiated stage [84].

As shown above, cell sorting of mesodermal progenitor cells, mesenchymal precursor cells, or myogenic cells is a powerful tool to obtain pure myogenic populations from differentiated pluripotent cells. Sakurai et al. have been able to induce PDGFR(+)Flk-1() mesodermal progenitor cells by FACS followed by myogenic differentiation [85]. Chang et al. and Mizuno et al. have been able to sort SMC-2.6(+) myogenic cells from mouse ESCs/iPSCs [86, 87]. These SMC-2.6(+) myogenic cells were successfully engrafted into mouse regenerating skeletal muscle. However, this SMC-2.6 antibody only recognizes mouse myogenic cells but not human myogenic cells [86, 88]. Therefore, Borchin et al. have shown that hiPSC-derived myogenic cells differentiated into c-met(+)CXCR4(+)ACHR(+) cells, displaying that over 95% of sorted cells are Pax7(+) myogenic cells [72]. Taken together, current myogenic induction protocols utilizing small molecules and growth factors, with or without myogenic transcription factors, have been largely improved in the last 5 years. It is crucial to standardize the induction protocols in the near future to obtain sufficient myogenic cell conversion from pluripotent stem cells.

Recent work demonstrated that cells inherit a stable genetic program partly through various epigenetic marks, such as DNA methylation and histone modifications. This cellular memory needs to be erased during genetic reprogramming, and the cellular program reverted to that of an earlier developmental stage [16, 22, 89]. However, iPSCs retaining an epigenetic memory of their origin can readily differentiate into their original tissues [1619, 90100]. This phenomenon becomes a double-edged sword for the reprogramming process since the retention of epigenetic memory may reduce the quality of pluripotency while increasing the differentiation efficiency into their original tissues. DNA methylation levels are relatively low in the pluripotent stem cells compared to the high levels of DNA methylation seen in somatic cells [101]. Global DNA demethylation is required for the reprogramming process [102]. In the context of these observations, recent work demonstrates that activation-induced cytidine deaminase AID/AICDA contributing to the DNA demethylation can stabilize stem-cell phenotypes by removing epigenetic memory of pluripotent genes. This directly deaminates 5-methylcytosine in concert with base-excision repair to exchange cytosine in genomic DNA [103]. MicroRNA-155 has been identified as a key player for the retention of epigenetic memory during in vitro differentiation of hematopoietic progenitor cell-derived iPSCs toward hematopoietic progenitors [104]. iPSCs that maintained high levels of miR-155 expression tend to differentiate into the original somatic population more efficiently.

Recently, we generated murine skeletal muscle cell-derived iPSCs (myoblast-derived iPSCs) [23] and compared the efficiency of differentiation of myogenic progenitor cells between myoblast-derived iPSCs and fibroblast-derived iPSCs. After EB cultures, more satellite cell/myogenic progenitor cell differentiation occurred in myoblast-derived iPSCs than that in fibroblast-derived-iPSCs (unpublished observation and Figure 3), suggesting that myoblast-derived iPSCs are potential myogenic and satellite cell sources for DMD and other muscular dystrophy therapies (Figure 4). We also noticed that MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce iPSCs (Figure 3) [23]. During overexpression of Oct4, Oct4 first binds to the Oct4 consensus sequence located in two MyoD enhancers (a core enhancer and distal regulatory region) [105107] preceding occupancy at the promoter in myoblasts in order to suppress MyoD gene expression. Interestingly, Oct4 binding to the MyoD core enhancer allows for establishment of a bivalent state in MyoD promoter as a poised state, marked by active (H3K4me3) and repressive (H3K27me3) modifications in fibroblasts, one of the characteristics of stem cells (Figure 3) [23, 108]. It should be investigated whether the similar bivalent state is also established in Oct4-expressing myoblasts during reprogramming process from myoblasts to pluripotent stem cells. It remains to be elucidated whether Oct4-mediated myogenic repression only relies on repression of MyoD expression or is just a general phenomenon of functional antagonism between Oct4 and MyoD on activation of muscle genes. Nevertheless, myoblast-derived iPSCs will enable us to produce an unlimited number of myogenic cells, including satellite cells that could form the basis of novel treatments for DMD and other muscular dystrophies (Figure 4).

There are pros and cons of transgene-free small molecule-mediated myogenic induction protocols. In the transgene-mediated induction protocols, integration of the transgene in the host genome may lead to risk for insertional mutagenesis. To circumvent this issue, there is an obvious advantage for transgene-free induction protocols. Some key molecules such as Wnt, FGF, and BMP have used signaling pathways to induce myogenic differentiation of ES/iPSCs. However, these molecules are also involved in induction of other types of cell lineages, which makes it difficult for ES/iPSCs to induce pure myogenic cell populations in vitro. By contrast, transgene-mediated myogenic induction is able to dictate desired specific cell lineages. In any case, it is necessary to intensively investigate these myogenic induction protocols for the efficient and safe stem cell therapy for patients.

For skeletal muscle diseases, patient-derived hiPSCs, which possess the ability to differentiate into myogenic progenitor cells followed by myotubes, can be a useful tool for drug screening and personalized medicine in clinical practice. However, there are still limitations for utilizing hiPSC-derived myogenic cells for regenerative medicine. For cell-based transplantation therapies such as a clinical situation, animal-free defined medium is essential for stem cell culture and skeletal muscle cell differentiation. Therefore, such animal-free defined medium needs to be established for optimal myogenic differentiation from hiPSCs. Gene correction in DMD patient iPSCs by TALENs and CRISPR-Cas9 systems are promising therapeutic approaches for stem cell transplantation. However, there are still problems for DNA-editing-mediated stem cell therapy such as safety and efficacy. Since iPSC-derived differentiated myotubes do not proliferate, they are not suited for cell transplantation. Therefore, a proper culture method needs to be established for hiPSCs in order to maintain cells in proliferating the myogenic precursor cell stage in vitro in order to expand cells to large quantities of transplantable cells for DMD and other muscular dystrophies. For other issues, it is essential to establish methods to separate ES/iPSC-derived pure skeletal muscle precursor cells from other cell types for safe stem cell therapy that excludes tumorigenic risks of contamination with undifferentiated cells. In the near future, these obstacles will be taken away for more efficient and safe stem cell therapy for DMD and other muscular dystrophies.

The authors declare that they have no conflicts of interest.

This work was supported by the NIH R01 (1R01AR062142) and NIH R21 (1R21AR070319). The authors thank Conor Burke-Smith and Neeladri Chowdhury for critical reading.

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Skeletal Muscle Cell Induction from Pluripotent Stem Cells

Cardiac Stem Cells Comments Off on Skeletal Muscle Cell Induction from Pluripotent Stem Cells | December 1st, 2022

Specific location in the body containing stem cells

Stem-cell niche refers to a microenvironment, within the specific anatomic location where stem cells are found, which interacts with stem cells to regulate cell fate.[1] The word 'niche' can be in reference to the in vivo or in vitro stem-cell microenvironment. During embryonic development, various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem-cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding micro-environment actively signals to stem cells to promote either self-renewal or differentiation to form new tissues. Several factors are important to regulate stem-cell characteristics within the niche: cellcell interactions between stem cells, as well as interactions between stem cells and neighbouring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, and the physicochemical nature of the environment including the pH, ionic strength (e.g. Ca2+ concentration) and metabolites, like ATP, are also important.[2] The stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood.

Scientists are studying the various components of the niche and trying to replicate the in vivo niche conditions in vitro.[2] This is because for regenerative therapies, cell proliferation and differentiation must be controlled in flasks or plates, so that sufficient quantity of the proper cell type are produced prior to being introduced back into the patient for therapy.

Human embryonic stem cells are often grown in fibroblastic growth factor-2 containing, fetal bovine serum supplemented media. They are grown on a feeder layer of cells, which is believed to be supportive in maintaining the pluripotent characteristics of embryonic stem cells. However, even these conditions may not truly mimic in vivo niche conditions.

Adult stem cells remain in an undifferentiated state throughout adult life. However, when they are cultured in vitro, they often undergo an 'aging' process in which their morphology is changed and their proliferative capacity is decreased. It is believed that correct culturing conditions of adult stem cells needs to be improved so that adult stem cells can maintain their stemness over time.[citation needed]

A Nature Insight review defines niche as follows:

"Stem-cell populations are established in 'niches' specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics ... The simple location of stem cells is not sufficient to define a niche. The niche must have both anatomic and functional dimensions."[3]

Though the concept of stem cell niche was prevailing in vertebrates, the first characterization of stem cell niche in vivo was worked out in Drosophila germinal development.

By continuous intravital imaging in mice, researchers were able to explore the structure of the stem cell niche and to obtain the fate of individual stem cells (SCs) and their progeny over time in vivo. In particular in intestinal crypt,[4] two distinct groups of SCs have been identified: the "border stem cells" located in the upper part of the niche at the interface with transit amplifying cells (TAs), and "central stem cells" located at the crypt base. The proliferative potential of the two groups was unequal and correlated with the cells' location (central or border). It was also shown that the two SC compartments acted in accord to maintain a constant cell population and a steady cellular turnover. A similar dependence of self-renewal potential on proximity to the niche border was reported in the context of hair follicle, in an in vivo live-imaging study.[5]

This bi-compartmental structure of stem cell niche has been mathematically modeled to obtain the optimal architecture that leads to the maximum delay in double-hit mutant production.[6] They found that the bi-compartmental SC architecture minimizes the rate of two-hit mutant production compared to the single SC compartment model. Moreover, the minimum probability of double-hit mutant generation corresponds to purely symmetric division of SCs with a large proliferation rate of border stem cells along with a small, but non-zero, proliferation rate of central stem cells.[citation needed]

Stem cell niches harboring continuously dividing cells, such as those located at the base of the intestinal gland, are maintained at small population size. This presents a challenge to the maintenance of multicellular tissues, as small populations of asexually dividing individuals will accumulate deleterious mutations through genetic drift and succumb to mutational meltdown.[7] Mathematical modeling of the intestinal gland reveals that the small population size within the stem cell niche minimizes the probability of carcinogenesis occurring anywhere, at the expense of gradually accumulated deleterious mutations throughout organismal lifetimea process that contributes to tissue degradation and aging.[8] Therefore, the population size of the stem cell niche represents an evolutionary trade-off between the probability of cancer formation and the rate of aging.

Germline stem cells (GSCs) are found in organisms that continuously produce sperm and eggs until they are sterile. These specialized stem cells reside in the GSC niche, the initial site for gamete production, which is composed of the GSCs, somatic stem cells, and other somatic cells. In particular, the GSC niche is well studied in the genetic model organism Drosophila melanogaster and has provided an extensive understanding of the molecular basis of stem cell regulation.[citation needed]

In Drosophila melanogaster, the GSC niche resides in the anterior-most region of each ovariole, known as the germarium. The GSC niche consists of necessary somatic cells-terminal filament cells, cap cells, escort cells, and other stem cells which function to maintain the GSCs.[9] The GSC niche holds on average 23 GSCs, which are directly attached to somatic cap cells and Escort stem cells, which send maintenance signals directly to the GSCs.[10] GSCs are easily identified through histological staining against vasa protein (to identify germ cells) and 1B1 protein (to outline cell structures and a germline specific fusome structure). Their physical attachment to the cap cells is necessary for their maintenance and activity.[10] A GSC will divide asymmetrically to produce one daughter cystoblast, which then undergoes 4 rounds of incomplete mitosis as it progresses down the ovariole (through the process of oogenesis) eventually emerging as a mature egg chamber; the fusome found in the GSCs functions in cyst formation and may regulate asymmetrical cell divisions of the GSCs.[11] Because of the abundant genetic tools available for use in Drosophila melanogaster and the ease of detecting GSCs through histological stainings, researchers have uncovered several molecular pathways controlling GSC maintenance and activity.[12] [13]

The Bone Morphogenetic Protein (BMP) ligands Decapentaplegic (Dpp) and Glass-bottom-boat (Gbb) ligand are directly signalled to the GSCs, and are essential for GSC maintenance and self-renewal.[14] BMP signalling in the niche functions to directly repress expression of Bag-of-marbles (Bam) in GSCs, which is up-regulated in developing cystoblast cells.[15] Loss of function of dpp in the niche results in de-repression of Bam in GSCs, resulting in rapid differentiation of the GSCs.[10] Along with BMP signalling, cap cells also signal other molecules to GSCs: Yb and Piwi. Both of these molecules are required non-autonomously to the GSCs for proliferation-piwi is also required autonomously in the GSCs for proliferation.[16] In the germarium, BMP signaling has a short-range effect, therefore the physical attachment of GSCs to cap cells is important for maintenance and activity.[citation needed]

The GSCs are physically attached to the cap cells by Drosophila E-cadherin (DE-cadherin) adherens junctions and if this physical attachment is lost GSCs will differentiate and lose their identity as a stem cell.[10] The gene encoding DE-cadherin, shotgun (shg), and a gene encoding Beta-catenin ortholog, armadillo, control this physical attachment.[17] A GTPase molecule, rab11, is involved in cell trafficking of DE-cadherins. Knocking out rab11 in GSCs results in detachment of GSCs from the cap cells and premature differentiation of GSCs.[18] Additionally, zero population growth (zpg), encoding a germline-specific gap junction is required for germ cell differentiation.[19]

Both diet and insulin-like signaling directly control GSC proliferation in Drosophila melanogaster. Increasing levels of Drosophila insulin-like peptide (DILP) through diet results in increased GSC proliferation.[20] Up-regulation of DILPs in aged GSCs and their niche results in increased maintenance and proliferation.[21] It has also been shown that DILPs regulate cap cell quantities and regulate the physical attachment of GSCs to cap cells.[21]

There are two possible mechanisms for stem cell renewal, symmetrical GSC division or de-differentiation of cystoblasts. Normally, GSCs will divide asymmetrically to produce one daughter cystoblast, but it has been proposed that symmetrical division could result in the two daughter cells remaining GSCs.[22][23] If GSCs are ablated to create an empty niche and the cap cells are still present and sending maintenance signals, differentiated cystoblasts can be recruited to the niche and de-differentiate into functional GSCs.[24]

As the Drosophila female ages, the stem cell niche undergoes age-dependent loss of GSC presence and activity. These losses are thought to be caused in part by degradation of the important signaling factors from the niche that maintains GSCs and their activity. Progressive decline in GSC activity contributes to the observed reduction in fecundity of Drosophila melanogaster at old age; this decline in GSC activity can be partially attributed to a reduction of signaling pathway activity in the GSC niche.[25][26] It has been found that there is a reduction in Dpp and Gbb signaling through aging. In addition to a reduction in niche signaling pathway activity, GSCs age cell-autonomously. In addition to studying the decline of signals coming from the niche, GSCs age intrinsically; there is age-dependent reduction of adhesion of GSCs to the cap cells and there is accumulation of Reactive Oxygen species (ROS) resulting in cellular damage which contributes to GSC aging. There is an observed reduction in the number of cap cells and the physical attachment of GSCs to cap cells through aging. Shg is expressed at significantly lower levels in an old GSC niche in comparison to a young one.[26]

Males of Drosophila melanogaster each have two testes long, tubular, coiled structures and at the anterior most tip of each lies the GSC niche. The testis GSC niche is built around a population of non-mitotic hub cells (a.k.a. niche cells), to which two populations of stem cells adhere: the GSCs and the somatic stem cells (SSCs, a.k.a. somatic cyst stem cells/cyst stem cells). Each GSC is enclosed by a pair of SSCs, though each stem cell type is still in contact with the hub cells. In this way, the stem cell niche consists of these three cell types, as not only do the hub cells regulate GSC and SSC behaviour, but the stem cells also regulate the activity of each other. The Drosophila testis GSC niche has proven a valuable model system for examining a wide range of cellular processes and signalling pathways.[27]

The process of spermatogenesis begins when the GSCs divide asymmetrically, producing a GSC that maintains hub contact, and a gonialblast that exits the niche. The SSCs divide with their GSC partner, and their non-mitotic progeny, the somatic cyst cells (SCCs, a.k.a. cyst cells) will enclose the gonialblast. The gonialblast then undergoes four rounds of synchronous, transit-amplifying divisions with incomplete cytokinesis to produce a sixteen-cell spermatogonial cyst. This spermatogonial cyst then differentiates and grows into a spermatocyte, which will eventually undergo meiosis and produce sperm.[27]

The two main molecular signalling pathways regulating stem cell behaviour in the testis GSC niche are the Jak-STAT and BMP signalling pathways. Jak-STAT signalling originates in the hub cells, where the ligand Upd is secreted to the GSCs and SSCs.[28][29] This leads to activation of the Drosophila STAT, Stat92E, a transcription factor which effects GSC adhesion to the hub cells,[30] and SSC self-renewal via Zfh-1.[31] Jak-STAT signalling also influences the activation of BMP signalling, via the ligands Dpp and Gbb. These ligands are secreted into the GSCs from the SSCs and hub cells, activate BMP signalling, and suppress the expression of Bam, a differentiation factor.[32] Outside of the niche, gonialblasts no longer receive BMP ligands, and are free to begin their differentiation program. Other important signalling pathways include the MAPK and Hedgehog, which regulate germline enclosure [33] and somatic cell self-renewal,[34] respectively.

The murine GSC niche in males, also called spermatogonial stem cell (SSC) niche, is located in the basal region of seminiferous tubules in the testes. The seminiferous epithelium is composed of sertoli cells that are in contact with the basement membrane of the tubules, which separates the sertoli cells from the interstitial tissue below. This interstitial tissue comprises Leydig cells, macrophages, mesenchymal cells, capillary networks, and nerves.[35]

During development, primordial germ cells migrate into the seminiferous tubules and downward towards the basement membrane whilst remaining attached to the sertoli cells where they will subsequently differentiate into SSCs, also referred to as Asingle spermatogonia.[35][36] These SSCs can either self-renew or commit to differentiating into spermatozoa upon the proliferation of Asingle into Apaired spermatogonia. The 2 cells of Apaired spermatogonia remain attached by intercellular bridges and subsequently divide into Aaligned spermatogonia, which is made up of 416 connected cells. Aaligned spermatogonia then undergo meiosis I to form spermatocytes and meiosis II to form spermatids which will mature into spermatozoa.[37][38] This differentiation occurs along the longitudinal axis of sertoli cells, from the basement membrane to the apical lumen of the seminiferous tubules. However, sertoli cells form tight junctions that separate SSCs and spermatogonia in contact with the basement membrane from the spermatocytes and spermatids to create a basal and an adluminal compartment, whereby differentiating spermatocytes must traverse the tight junctions.[35][39] These tight junctions form the blood testis barrier (BTB) and have been suggested to play a role in isolating differentiated cells in the adluminal compartment from secreted factors by the interstitial tissue and vasculature neighboring the basal compartment.[35]

The basement membrane of the seminiferous tubule is a modified form of extracellular matrix composed of fibronectin, collagens, and laminin.[35] 1- integrin is expressed on the surface of SSCs and is involved in their adhesion to the laminin component of the basement membrane although other adhesion molecules are likely also implicated in the attachment of SSCs to the basement membrane.[40] E cadherin expression on SSCs in mice, unlike in Drosophila, have been shown to be dispensable as the transplantation of cultured SSCs lacking E-cadherin are able to colonize host seminiferous tubules and undergo spermatogenesis.[41] In addition the blood testis barrier provides architectural support and is composed of tight junction components such as occludins, claudins and zonula occludens (ZOs) which show dynamic expression during spermatogenesis.[42] For example, claudin 11 has been shown to be a necessary component of these tight junctions as mice lacking this gene have a defective blood testis barrier and do not produce mature spermatozoa.[40]

GDNF (Glial cell-derived neurotrophic factor) is known to stimulate self-renewal of SSCs and is secreted by the sertoli cells under the influence of gonadotropin FSH. GDNF is a related member of the TGF superfamily of growth factors and when overexpressed in mice, an increase in undifferentiated spermatogonia was observed which led to the formation of germ tumours.[35][40] In corroboration for its role as a renewal factor, heterozygous knockout male mice for GDNF show decreased spermatogenesis that eventually leads to infertility.[40] In addition the supplementation of GDNF has been shown to extend the expansion of mouse SSCs in culture. However, the GDNF receptor c-RET and co-receptor GFRa1 are not solely expressed on the SSCs but also on Apaired and Aaligned, therefore showing that GDNF is a renewal factor for Asingle to Aaligned in general rather than being specific to the Asingle SSC population. FGF2 (Fibroblast growth factor 2), secreted by sertoli cells, has also been shown to influence the renewal of SSCs and undifferentiated spermatogonia in a similar manner to GDNF.[35]

Although sertoli cells appear to play a major role in renewal, it expresses receptors for testosterone that is secreted by Leydig cells whereas germ cells do not contain this receptor- thus alluding to an important role of Leydig cells upstream in mediating renewal. Leydig cells also produce CSF 1 (Colony stimulating factor 1) for which SSCs strongly express the receptor CSF1R.[37] When CSF 1 was added in culture with GDNF and FGF2 no further increase in proliferation was observed, however, the longer the germ cells remained in culture with CSF-1 the greater the SSC density observed when these germ cells were transplanted into host seminiferous tubules. This showed CSF 1 to be a specific renewal factor that tilts the SSCs towards renewal over differentiation, rather than affecting proliferation of SSCs and spermatogonia. GDNF, FGF 2 and CSF 1 have also been shown to influence self-renewal of stem cells in other mammalian tissues.[35]

Plzf (Promyelocytic leukaemia zinc finger) has also been implicated in regulating SSC self-renewal and is expressed by Asingle, Apaired and Aaligned spermatogonia. Plzf directly inhibits the transcription of a receptor, c-kit, in these early spermatogonia. However, its absence in late spermatogonia permits c-kit expression, which is subsequently activated by its ligand SCF (stem cell factor) secreted by sertoli cells, resulting in further differentiation. Also, the addition of BMP4 and Activin-A have shown to reduce self-renewal of SSCs in culture and increase stem cell differentiation, with BMP4 shown to increase the expression of c-kit.[37]

Prolonged spermatogenesis relies on the maintenance of SSCs, however, this maintenance declines with age and leads to infertility. Mice between 12 and 14 months of age show decreased testis weight, reduced spermatogenesis and SSC content. Although stem cells are regarded as having the potential to infinitely replicate in vitro, factors provided by the niche are crucial in vivo. Indeed, serial transplantation of SSCs from male mice of different ages into young mice 3 months of age, whose endogenous spermatogenesis had been ablated, was used to estimate stem cell content given that each stem cell would generate a colony of spermatogenesis.[35][43] The results of this experiment showed that transplanted SSCs could be maintained far longer than their replicative lifespan for their age. In addition, a study also showed that SSCs from young fertile mice could not be maintained nor undergo spermatogenesis when transplanted into testes of old, infertile mice. Together, these results points towards a deterioration of the SSC niche itself with aging rather than the loss of intrinsic factors in the SSC.[43]

Vertebrate hematopoietic stem cells niche in the bone marrow is formed by cells subendosteal osteoblasts, sinusoidal endothelial cells and bone marrow stromal (also sometimes called reticular) cells which includes a mix of fibroblastoid, monocytic and adipocytic cells (which comprise marrow adipose tissue).[1]

The hair follicle stem cell niche is one of the more closely studied niches thanks to its relative accessibility and role in important diseases such as melanoma. The bulge area at the junction of arrector pili muscle to the hair follicle sheath has been shown to host the skin stem cells which can contribute to all epithelial skin layers. There cells are maintained by signaling in concert with niche cells signals include paracrine (e.g. sonic hedgehog), autocrine and juxtacrine signals.[44] The bulge region of the hair follicle relies on these signals to maintain the stemness of the cells. Fate mapping or cell lineage tracing has shown that Keratin 15 positive stem cells' progeny participate in all epithelial lineages.[45] The follicle undergoes cyclic regeneration in which these stem cells migrate to various regions and differentiate into the appropriate epithelial cell type. Some important signals in the hair follicle stem cell niche produced by the mesenchymal dermal papilla or the bulge include BMP, TGF- and Fibroblast growth factor (FGF) ligands and Wnt inhibitors.[46] While, Wnt signaling pathways and -catenin are important for stem cell maintenance,[47] over-expression of -catenin in hair follicles induces improper hair growth. Therefore, these signals such as Wnt inhibitors produced by surrounding cells are important to maintain and facilitate the stem cell niche.[48]

Intestinal organoids have been used to study intestinal stem cell niches. An intestinal organoid culture can be used to indirectly assess the effect of the manipulation on the stem cells through assessing the organoid's survival and growth. Research using intestinal organoids have demonstrated that the survival of intestinal stem cells is improved by the presence of neurons and fibroblasts,[49] and through the administration of IL-22.[50]

Cardiovascular stem cell niches can be found within the right ventricular free wall, atria and outflow tracks of the heart. They are composed of Isl1+/Flk1+ cardiac progenitor cells (CPCs) that are localized into discrete clusters within a ColIV and laminin extracellular matrix (ECM). ColI and fibronectin are predominantly found outside the CPC clusters within the myocardium. Immunohistochemical staining has been used to demonstrate that differentiating CPCs, which migrate away from the progenitor clusters and into the ColI and fibronectin ECM surrounding the niche, down-regulate Isl1 while up-regulating mature cardiac markers such as troponin C.[51] There is a current controversy over the role of Isl1+ cells in the cardiovascular system. While major publications have identified these cells as CPC's and have found a very large number in the murine and human heart, recent publications have found very few Isl1+ cells in the murine fetal heart and attribute their localization to the sinoatrial node,[52] which is known as an area that contributes to heart pacemaking. The role of these cells and their niche are under intense research and debate.[citation needed]

Neural stem cell niches are divided in two: the Subependymal zone (SEZ) and the Subgranular zone (SGZ).

The SEZ is a thin area beneath the ependymal cell layer that contains three types of neural stem cells: infrequently dividing neural stem cells (NSCs), rapidly dividing transit amplifying precursors (TaPs) and neuroblasts (NBs). The SEZ extracellular matrix (ECM) has significant differences in composition compared to surrounding tissues. Recently, it was described that progenitor cells, NSCs, TaPs and NBs were attached to ECM structures called Fractones.[53] These structures are rich in laminin, collagen and heparan sulfate proteoglycans.[54] Other ECM molecules, such as tenascin-C, MMPs and different proteoglycans are also implicated in the neural stem cell niche.[55]

Cancer tissue is morphologically heterogenous, not only due to the variety of cell types present, endothelial, fibroblast and various immune cells, but cancer cells themselves are not a homogenous population either.[citation needed]

In accordance with the hierarchy model of tumours, the cancer stem cells (CSC) are maintained by biochemical and physical contextual signals emanating from the microenvironment, called the cancer stem cell niche.[56] The CSC niche is very similar to normal stem cells niche (embryonic stem cell (ESC), Adult Stem Cell ASC) in function (maintaining of self-renewal, undifferentiated state and ability to differentiate) and in signalling pathways (Activin/Noda, Akt/PTEN, JAK/STAT, PI3-K, TGF-, Wnt and BMP).[57] It is hypothesized that CSCs arise form aberrant signalling of the microenvironment and participates not only in providing survival signals to CSCs but also in metastasis by induction of epithelial-mesenchymal transition (EMT).[citation needed]

Hypoxic condition in stem cell niches (ESC, ASC or CSC) is necessary for maintaining stem cells in an undifferentiated state and also for minimizing DNA damage via oxidation. The maintaining of the hypoxic state is under control of Hypoxia-Inducible transcription Factors (HIFs).[58] HIFs contribute to tumour progression, cell survival and metastasis by regulation of target genes as VEGF, GLUT-1, ADAM-1, Oct4 and Notch.[57]

Hypoxia plays an important role in the regulation of cancer stem cell niches and EMT through the promotion of HIFs.[59] These HIFs help maintain cancer stem cell niches by regulating important stemness genes such as Oct4, Nanog, SOX2, Klf4, and cMyc.[60][61] HIFs also regulate important tumor suppressor genes such as p53 and genes that promote metastasis.[62][63] Although HIFs increase the survival of cells by decreasing the effects of oxidative stress, they have also been shown to decrease factors such as RAD51 and H2AX that maintain genomic stability.[64] In the hypoxic condition there is an increase of intracellular Reactive Oxygen Species (ROS) which also promote CSCs survival via stress response.[65][66] ROS stabilizes HIF-1 which promotes the Met proto-oncogene, which drives metastasis or motogenic escape in melanoma cells.[67] All of these factors contribute to a cancer stem cell phenotype which is why it is often referred to as a hypoxic stem cell niche. Hypoxic environments are often found in tumors where the cells are dividing faster that angiogenesis can occur. It is important to study hypoxia as an aspect of cancer because hypoxic environments have been shown to be resistant to radiation therapy.[68] Radiation has been shown to increase the amounts of HIF-1.[69] EMT induction by hypoxia though interactions between HIF-1 and ROS is crucial for metastasis in cancers such as melanoma. It has been found that many genes associated with melanoma are regulated by hypoxia such as MXI1, FN1, and NME1.[70]

Epithelialmesenchymal transition is a morphogenetic process, normally occurs in embryogenesis that is "hijacked" by cancer stem cells by detaching from their primary place and migrating to another one. The dissemination is followed by reverse transition so-called Epithelial-Mesenchymal Transition (EMT). This process is regulated by CSCs microenvironment via the same signalling pathways as in embryogenesis using the growth factors (TGF-, PDGF, EGF), cytokine IL-8 and extracellular matrix components. These growth factors' interactions through intracellular signal transducers like -catenin has been shown to induce metastatic potential.[71][72] A characteristic of EMT is loss of the epithelial markers (E-cadherin, cytokeratins, claudin, occluding, desmoglein, desmocolin) and gain of mesenchymal markers (N-cadherin, vimentin, fibronectin).[73]

There is also certain degree of similarity in homing-mobilization of normal stem cells and metastasis-invasion of cancer stem cells. There is an important role of Matrix MetalloProteinases (MMP), the principal extracellular matrix degrading enzymes, thus for example matrix metalloproteinase-2 and 9 are induced to expression and secretion by stromal cells during metastasis of colon cancer via direct contact or paracrine regulation. The next sharing molecule is Stromal cell-Derived Factor-1 (SDF-1).[73][74]

The EMT and cancer progression can be triggered also by chronic inflammation. The main roles have molecules (IL-6, IL-8, TNF-, NFB, TGF-, HIF-1) which can regulate both processes through regulation of downstream signalling that overlapping between EMT and inflammation.[57] The downstream pathways involving in regulation of CSCs are Wnt, SHH, Notch, TGF-, RTKs-EGF, FGF, IGF, HGF.

NFB regulates the EMT, migration and invasion of CSCs through Slug, Snail and Twist. The activation of NFB leads to increase not only in production of IL-6, TNF- and SDF-1 but also in delivery of growth factors.

The source of the cytokine production are lymphocytes (TNF-), Mesenchymal Stem Cells (SDF-1, IL-6, IL8).

Interleukin 6 mediates activation of STAT3. The high level of STAT3 was described in isolated CSCs from liver, bone, cervical and brain cancer. The inhibition of STAT3 results in dramatic reduction in their formation. Generally IL-6 contributes a survival advantage to local stem cells and thus facilitates tumorigenesis.[57]

SDF-1 secreted from Mesenchymal Stem Cells (MSCs) has important role in homing and maintenance of Hematopoietic Stem Cell (HSC) in bone marrow niche but also in homing and dissemination of CSC.[74]

Hypoxia is a main stimulant for angiogenesis, with HIF-1 being the primary mediator. Angiogenesis induced by hypoxic conditions is called an "Angiogenic switch". HIF-1 promotes expression of several angiogenic factors: Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), Placenta-Like Growth Factor (PLGF), Platelet-Derived Growth Factor (PDGF) and Epidermal Growth Factor. But there is evidence that the expression of angiogenic agens by cancer cells can also be HIF-1 independent. It seems that there is an important role of Ras protein, and that intracellular levels of calcium regulate the expression of angiogenic genes in response to hypoxia.[73]

The angiogenic switch downregulates angiogenesis suppressor proteins, such as thrombospondin, angiostatin, endostatin and tumstatin. Angiogenesis is necessary for the primary tumour growth.[citation needed]

During injury, support cells are able to activate a program for repair, recapitulating aspects of development in the area of damage. These areas become permissive for stem cell renewal, migration and differentiation. For instance in the CNS, injury is able to activate a developmental program in astrocytes that allow them to express molecules that support stem cells such as chemokines i.e. SDF-1[75] and morphogens such as sonic hedgehog.[76]

It is evident that biophysio-chemical characteristics of ECM such as composition, shape, topography, stiffness, and mechanical strength can control the stem cell behavior. These ECM factors are equally important when stem cells are grown in vitro. Given a choice between niche cell-stem cell interaction and ECM-stem cell interaction, mimicking ECM is preferred as that can be precisely controlled by scaffold fabrication techniques, processing parameters or post-fabrication modifications. In order to mimic, it is essential to understand natural properties of ECM and their role in stem cell fate processes. Various studies involving different types of scaffolds that regulate stem cells fate by mimicking these ECM properties have been done.[2])

[77]

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Stem-cell niche - Wikipedia

Cardiac Stem Cells Comments Off on Stem-cell niche – Wikipedia | December 1st, 2022

A protein that helps make neurons also works to reprogram scar tissue cells into heart muscle cells, especially in partnership with a second protein, according to a study led by Li Qian, PhD, at the UNC School of Medicine.

CHAPEL HILL, N.C. Scientists at the UNC School of Medicine have made a significant advance in the promising field of cellular reprogramming and organ regeneration, and the discovery could play a major role in future medicines to heal damaged hearts.

In a study published in the journal Cell Stem Cell, scientists at the University of North Carolina at Chapel Hill discovered a more streamlined and efficient method for reprogramming scar tissue cells (fibroblasts) to become healthy heart muscle cells (cardiomyocytes). Fibroblasts produce the fibrous, stiff tissue that contributes to heart failure after a heart attack or because of heart disease. Turning fibroblasts into cardiomyocytes is being investigated as a potential future strategy for treating or even someday curing this common and deadly condition.

Surprisingly, the key to the new cardiomyocyte-making technique turned out to be a gene activity-controlling protein called Ascl1, which is known to be a crucial protein involved in turning fibroblasts into neurons. Researchers had thought Ascl1 was neuron-specific.

Its an outside-the-box finding, and we expect it to be useful in developing future cardiac therapies and potentially other kinds of therapeutic cellular reprogramming, said study senior author Li Qian, PhD, associate professor in the UNC Department of Pathology and Lab Medicine and associate director of the McAllister Heart Institute at UNC School of Medicine.

Scientists over the last 15 years have developed various techniques to reprogram adult cells to become stem cells, then to induce those stem cells to become adult cells of some other type. More recently, scientists have been finding ways to do this reprogramming more directly straight from one mature cell type to another. The hope has been that when these methods are made maximally safe, effective, and efficient, doctors will be able to use a simple injection into patients to reprogram harm-causing cells into beneficial ones.

Reprogramming fibroblasts has long been one of the important goals in the field, Qian said. Fibroblast over-activity underlies many major diseases and conditions including heart failure, chronic obstructive pulmonary disease, liver disease, kidney disease, and the scar-like brain damage that occurs after strokes.

In the new study, Qians team, including co-first-authors Haofei Wang, PhD, a postdoctoral researcher, and MD/PhD student Benjamin Keepers, used three existing techniques to reprogram mouse fibroblasts into cardiomyocytes, liver cells, and neurons. Their aim was to catalogue and compare the changes in cells gene activity patterns and gene-activity regulation factors during these three distinct reprogrammings.

Unexpectedly, the researchers found that the reprogramming of fibroblasts into neurons activated a set of cardiomyocyte genes. Soon they determined that this activation was due to Ascl1, one of the master-programmer transcription factor proteins that had been used to make the neurons.

Since Ascl1 activated cardiomyocyte genes, the researchers added it to the three-transcription-factor cocktail they had been using for making cardiomyocytes, to see what would happen. They were astonished to find that it dramatically increased the efficiency of reprogramming the proportion of successfully reprogrammed cells by more than ten times. In fact, they found that they could now dispense with two of the three factors from their original cocktail, retaining only Ascl1 and another transcription factor called Mef2c.

In further experiments they found evidence that Ascl1 on its own activates both neuron and cardiomyocyte genes, but it shifts away from the pro-neuron role when accompanied by Mef2c. In synergy with Mef2c, Ascl1 switches on a broad set of cardiomyocyte genes.

Ascl1 and Mef2c work together to exert pro-cardiomyocyte effects that neither factor alone exerts, making for a potent reprogramming cocktail, Qian said.

The results show that the major transcription factors used in direct cellular reprogramming arent necessarily exclusive to one targeted cell type.

Perhaps more importantly, they represent another step on the path towards future cell-reprogramming therapies for major disorders. Qian says that she and her team hope to make a two-in-one synthetic protein that contains the effective bits of both Ascl1 and Mef2c, and could be injected into failing hearts to mend them.

Cross-lineage Potential of Ascl1 Uncovered by Comparing Diverse Reprogramming Regulatomes was co-authored by Haofei Wang, Benjamin Keepers, Yunzhe Qian, Yifang Xie, Marazzano Colon, Jiandong Liu, and Li Qian. Funding was provided by the American Heart Association and the National Institutes of Health (T32HL069768, F30HL154659, R35HL155656, R01HL139976, R01HL139880).

Media contact: Mark Derewicz, 919-923-0959

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Scientists Discover Protein Partners that Could Heal Heart Muscle | Newsroom - UNC Health and UNC School of Medicine

Cardiac Stem Cells Comments Off on Scientists Discover Protein Partners that Could Heal Heart Muscle | Newsroom – UNC Health and UNC School of Medicine | October 13th, 2022

ReportLinker

Abstract: Whats New for 2022?? Global competitiveness and key competitor percentage market shares. Market presence across multiple geographies - Strong/Active/Niche/Trivial.

New York, Oct. 10, 2022 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Induced Pluripotent Stem Cell (iPSC) Industry" - https://www.reportlinker.com/p05798831/?utm_source=GNW

Online interactive peer-to-peer collaborative bespoke updates

Access to our digital archives and MarketGlass Research Platform

Complimentary updates for one yearGlobal Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027- In the changed post COVID-19 business landscape, the global market for Induced Pluripotent Stem Cell ((iPSC) estimated at US$1.4 Billion in the year 2020, is projected to reach a revised size of US$0 Thousand by 2027, growing at a CAGR of -100% over the analysis period 2020-2027. Vascular Cells, one of the segments analyzed in the report, is projected to record a -100% CAGR and reach US$0 Thousand by the end of the analysis period. Taking into account the ongoing post pandemic recovery, growth in the Cardiac Cells segment is readjusted to a revised -100% CAGR for the next 7-year period.- The U.S. Market is Estimated at $629.2 Million, While China is Forecast to Grow at -100% CAGR- The Induced Pluripotent Stem Cell ((iPSC) market in the U.S. is estimated at US$629.2 Million in the year 2020. China, the world`s second largest economy, is forecast to reach a projected market size of US$0 Thousand by the year 2027 trailing a CAGR of -100% over the analysis period 2020 to 2027. Among the other noteworthy geographic markets are Japan and Canada, each forecast to grow at -100% and -100% respectively over the 2020-2027 period. Within Europe, Germany is forecast to grow at approximately -100% CAGR.Neuronal Cells Segment to Record -100% CAGR- In the global Neuronal Cells segment, USA, Canada, Japan, China and Europe will drive the -100% CAGR estimated for this segment. These regional markets accounting for a combined market size of US$188.9 Million in the year 2020 will reach a projected size of US$0 Thousand by the close of the analysis period. China will remain among the fastest growing in this cluster of regional markets.

Select Competitors (Total 51 Featured)Axol Bioscience Ltd.Cynata Therapeutics LimitedEvotec SEFate Therapeutics, Inc.FUJIFILM Cellular Dynamics, Inc.NcardiaPluricell BiotechREPROCELL USA, Inc.Sumitomo Dainippon Pharma Co., Ltd.Takara Bio, Inc.Thermo Fisher Scientific, Inc.ViaCyte, Inc.

Read the full report: https://www.reportlinker.com/p05798831/?utm_source=GNW

I. METHODOLOGY

II. EXECUTIVE SUMMARY

1. MARKET OVERVIEWInfluencer Market InsightsImpact of Covid-19 and a Looming Global RecessionInduced Pluripotent Stem Cells (iPSCs) Market Gains fromIncreasing Use in Research for COVID-19Studies Employing iPSCs in COVID-19 ResearchStem Cells, Application Areas, and the Different Types: A PreludeApplications of Stem CellsTypes of Stem CellsInduced Pluripotent Stem Cell (iPSC): An IntroductionProduction of iPSCsFirst & Second Generation Mouse iPSCsHuman iPSCsKey Properties of iPSCsTranscription Factors Involved in Generation of iPSCsNoteworthy Research & Application Areas for iPSCsInduced Pluripotent Stem Cell ((iPSC) Market: Growth Prospectsand OutlookDrug Development Application to Witness Considerable GrowthTechnical Breakthroughs, Advances & Clinical Trials to SpurGrowth of iPSC MarketNorth America Dominates Global iPSC MarketCompetitionRecent Market ActivitySelect Innovation/AdvancementInduced Pluripotent Stem Cell (iPSC) - Global Key CompetitorsPercentage Market Share in 2022 (E)Competitive Market Presence - Strong/Active/Niche/Trivial forPlayers Worldwide in 2022 (E)

2. FOCUS ON SELECT PLAYERSAxol Bioscience Ltd. (UK)Cynata Therapeutics Limited (Australia)Evotec SE (Germany)Fate Therapeutics, Inc. (USA)FUJIFILM Cellular Dynamics, Inc. (USA)Ncardia (Belgium)Pluricell Biotech (Brazil)REPROCELL USA, Inc. (USA)Sumitomo Dainippon Pharma Co., Ltd. (Japan)Takara Bio, Inc. (Japan)Thermo Fisher Scientific, Inc. (USA)ViaCyte, Inc. (USA)

3. MARKET TRENDS & DRIVERSEffective Research Programs Hold Key in Roll Out of AdvancediPSC TreatmentsInduced Pluripotent Stem Cells: A Giant Leap in the TherapeuticApplicationsResearch Trends in Induced Pluripotent Stem Cell SpaceWorldwide Publication of hESC and hiPSC Research Papers for thePeriod 2008-2010, 2011-2013 and 2014-2016Number of Original Research Papers on hESC and iPSC PublishedWorldwide (2014-2016)Concerns Related to Embryonic Stem Cells Shift the Focus ontoiPSCsRegenerative Medicine: A Promising Application of iPSCsInduced Pluripotent: A Potential Competitor to hESCs?Global Regenerative Medicine Market Size in US$ Billion for2019, 2021, 2023 and 2025Global Stem Cell & Regenerative Medicine Market by Product(in %) for the Year 2019Global Regenerative Medicines Market by Category: Breakdown(in %) for Biomaterials, Stem Cell Therapies and TissueEngineering for 2019Pluripotent Stem Cells Hold Significance for CardiovascularRegenerative MedicineLeading Causes of Mortality Worldwide: Number of Deaths inMillions & % Share of Deaths by Cause for 2017Leading Causes of Mortality for Low-Income and High-IncomeCountriesGrowing Importance of iPSCs in Personalized Drug DiscoveryPersistent Advancements in Genetics Space and Subsequent Growthin Precision Medicine Augur Well for iPSCs MarketGlobal Precision Medicine Market (In US$ Billion) for the Years2018, 2021 & 2024Increasing Prevalence of Chronic Disorders Supports Growth ofiPSCs MarketWorldwide Cancer Incidence: Number of New Cancer CasesDiagnosed for 2012, 2018 & 2040Number of New Cancer Cases Reported (in Thousands) by CancerType: 2018Fatalities by Heart Conditions: Estimated Percentage Breakdownfor Cardiovascular Disease, Ischemic Heart Disease, Stroke,and OthersRising Diabetes Prevalence Presents Opportunity for iPSCsMarket: Number of Adults (20-79) with Diabetes (in Millions)by Region for 2017 and 2045Aging Demographics Add to the Global Burden of ChronicDiseases, Presenting Opportunities for iPSCs MarketExpanding Elderly Population Worldwide: Breakdown of Number ofPeople Aged 65+ Years in Million by Geographic Region for theYears 2019 and 2030Growth in Number of Genomics Projects Propels Market GrowthGenomic Initiatives in Select CountriesNew Gene-Editing Tools Spur Interest and Investments inGenetics, Driving Lucrative Growth Opportunities for iPSCs:Total VC Funding (In US$ Million) in Genetics for the Years2014, 2015, 2016, 2017 and 2018Launch of Numerous iPSCs-Related Clinical Trials Set to BenefitMarket GrowthNumber of Induced Pluripotent Stem Cells based Studies bySelect Condition: As on Oct 31, 2020iPSCs-based Clinical Trial for Heart DiseasesInduced Pluripotent Stem Cells for Stroke Treatment?Off-the-shelf? Stem Cell Treatment for Cancer Enters ClinicalTrialiPSCs for Hematological DisordersMarket Benefits from Growing Funding for iPSCs-Related R&DInitiativesStem Cell Research Funding in the US (in US$ Million) for theYears 2016 through 2021Human iPSC Banks: A Review of Emerging Opportunities and DrawbacksHuman iPSC Banks Worldwide: An OverviewCell Sources and Reprogramming Methods Used by Select iPSC BanksInnovations, Research Studies & Advancements in iPSCsKey iPSC Research Breakthroughs for Regenerative MedicineResearchers Develop Novel Oncogene-Free and Virus-Free iPSCProduction MethodScientists Study Concerns of Genetic Mutations in iPSCsiPSCs Hold Tremendous Potential in Transforming Research EffortsResearchers Highlight Potential Use of iPSCs for DevelopingNovel Cancer VaccinesScientists Use Machine Learning to Improve Reliability of iPSCSelf-OrganizationSTEMCELL Technologies Unveils mTeSR? PlusChallenges and Risks Related to Pluripotent Stem CellsA Glance at Issues Related to Reprogramming of Adult Cells toiPSCsA Note on Legal, Social and Ethical Considerations with iPSCs

4. GLOBAL MARKET PERSPECTIVETable 1: World Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Geographic Region -USA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld Markets - Independent Analysis of Annual Sales in US$Thousand for Years 2020 through 2025 and % CAGR

Table 2: World 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Geographic Region - Percentage Breakdown ofValue Sales for USA, Canada, Japan, China, Europe, Asia-Pacificand Rest of World Markets for Years 2021 & 2025

Table 3: World Recent Past, Current & Future Analysis forVascular Cells by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 4: World 5-Year Perspective for Vascular Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 5: World Recent Past, Current & Future Analysis forCardiac Cells by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 6: World 5-Year Perspective for Cardiac Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 7: World Recent Past, Current & Future Analysis forNeuronal Cells by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 8: World 5-Year Perspective for Neuronal Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 9: World Recent Past, Current & Future Analysis for LiverCells by Geographic Region - USA, Canada, Japan, China, Europe,Asia-Pacific and Rest of World Markets - Independent Analysisof Annual Sales in US$ Thousand for Years 2020 through 2025 and% CAGR

Table 10: World 5-Year Perspective for Liver Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 11: World Recent Past, Current & Future Analysis forImmune Cells by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 12: World 5-Year Perspective for Immune Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 13: World Recent Past, Current & Future Analysis forOther Cell Types by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 14: World 5-Year Perspective for Other Cell Types byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 15: World Recent Past, Current & Future Analysis forCellular Reprogramming by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 16: World 5-Year Perspective for Cellular Reprogrammingby Geographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 17: World Recent Past, Current & Future Analysis for CellCulture by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 18: World 5-Year Perspective for Cell Culture byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 19: World Recent Past, Current & Future Analysis for CellDifferentiation by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 20: World 5-Year Perspective for Cell Differentiation byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 21: World Recent Past, Current & Future Analysis for CellAnalysis by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 22: World 5-Year Perspective for Cell Analysis byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 23: World Recent Past, Current & Future Analysis forCellular Engineering by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 24: World 5-Year Perspective for Cellular Engineering byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 25: World Recent Past, Current & Future Analysis forOther Research Methods by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 26: World 5-Year Perspective for Other Research Methodsby Geographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 27: World Recent Past, Current & Future Analysis for DrugDevelopment & Toxicology Testing by Geographic Region - USA,Canada, Japan, China, Europe, Asia-Pacific and Rest of WorldMarkets - Independent Analysis of Annual Sales in US$ Thousandfor Years 2020 through 2025 and % CAGR

Table 28: World 5-Year Perspective for Drug Development &Toxicology Testing by Geographic Region - Percentage Breakdownof Value Sales for USA, Canada, Japan, China, Europe,Asia-Pacific and Rest of World for Years 2021 & 2025

Table 29: World Recent Past, Current & Future Analysis forAcademic Research by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 30: World 5-Year Perspective for Academic Research byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 31: World Recent Past, Current & Future Analysis forRegenerative Medicine by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 32: World 5-Year Perspective for Regenerative Medicine byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 33: World Recent Past, Current & Future Analysis forOther Applications by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 34: World 5-Year Perspective for Other Applications byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

III. MARKET ANALYSIS

UNITED STATESInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in the United Statesfor 2022 (E)Table 35: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 36: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 37: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 38: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 39: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 40: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

CANADATable 41: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 42: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 43: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 44: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 45: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 46: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

JAPANInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Japan for 2022 (E)Table 47: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 48: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 49: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 50: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 51: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 52: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

CHINAInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in China for 2022 (E)Table 53: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 54: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 55: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 56: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 57: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 58: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

EUROPEInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Europe for 2022 (E)Table 59: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Geographic Region -France, Germany, Italy, UK and Rest of Europe Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 60: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Geographic Region - Percentage Breakdown ofValue Sales for France, Germany, Italy, UK and Rest of EuropeMarkets for Years 2021 & 2025

Table 61: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 62: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 63: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 64: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 65: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 66: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

FRANCEInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in France for 2022 (E)Table 67: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 68: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 69: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 70: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 71: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 72: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

GERMANYInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Germany for 2022 (E)Table 73: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 74: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 75: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 76: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 77: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 78: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

ITALYTable 79: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 80: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 81: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 82: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 83: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 84: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

UNITED KINGDOMInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in the United Kingdomfor 2022 (E)Table 85: UK Recent Past, Current & Future Analysis for InducedPluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells,Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells andOther Cell Types - Independent Analysis of Annual Sales in US$Thousand for the Years 2020 through 2025 and % CAGR

Table 86: UK 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Read the rest here:
Global Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027 - Yahoo Finance

Cardiac Stem Cells Comments Off on Global Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027 – Yahoo Finance | October 13th, 2022

Self-organizing lumps of human brain tissue grown in the laboratory have been successfully transplanted into the nervous systems of newborn rats in a step towards finding new ways to treat neuropsychiatric disorders.

The 3D organoids, developed from stem cells to resemble a simplified model of the human cortex, connected and integrated with the surrounding tissue in each rat's cortex to form a functional part of the rodent's own brain, displaying activity related to sensory perception.

This, according to a team of researchers led by neuroscientist Sergiu Paca of Stanford University, overcomes the limitations of dish-grown organoids, and gives us a new platform for modeling human brain development and disease in a living system.

"Most of the work that my lab has been doing has been motivated by this mission of trying to understand psychiatric disorders at the biological level so that we can actually find effective therapeutics," Paca explained in a press briefing.

"Many of these psychiatric conditions, such as autism and schizophrenia, are likely uniquely human, or at least, they are anchored in unique features of the human brain. And the human brain has certainly not been very accessible, which has precluded the progress we've been making in understanding the biology of these conditions."

In 2008, scientists made a breakthrough: brain cells grown from induced pluripotent stem cells. Mature cells harvested from adult humans were reverse engineered (or induced) to return them to the 'blank' state of stem cells the form cells take before they grow into cells with specializations, such as skin cells or cardiac cells.

These stem cells were then guided to develop into brain cells, which scientists cultivated to form lumps of brain-like tissue called organoids. These models of key areas of brain anatomy, such as the wrinkled outer cortex, could be used to study functions and development of brains up close.

As useful as they are, in vitro cortical organoids have limitations. Because they aren't connected to living systems, they don't complete maturation, depriving researchers of an opportunity to observe how they integrate with other major parts of a brain.

In addition, a brain organoid in a dish can't reveal the behavioral consequences of any defects scientists might identify. Since psychiatric disorders are defined by behavior, this stymies the ability to identify the physiological characteristics of these disorders.

In previous research, scientists have tried to overcome these hurdles by implanting human brain organoids into the brains of adult rats. Because of the developmental mismatch, the transplants didn't take: the developing neurons in the organoid couldn't form a strong connection with the fully developed network of an adult rat brain.

So Paca and his colleagues tried something else: grafting the human brain tissue onto the brains of newborn rats, whose own brains have not yet developed and matured.

Human cortical organoids were cultured in a dish, and then transplanted directly into the somatosensory cortex (the area of the brain responsible for receiving and processing sensory information) of rat pups just a few days old. These rats were then left to grow into adults for another 140 days (rats are fully sexually mature between 6 and 12 weeks).

Then, the scientists studied the rats. They had genetically engineered the organoids to respond to blue light simulation, activating neurons when blue light is shone on them. This stimulation on the human neurons was performed while the rats were being trained to lick a spout to receive water. Later, when the blue light was shone on the organoids, the rats would automatically lick displaying a response not seen in control groups.

This indicated that not only was the organoid functioning as part of the rat brain, it could help drive reward-seeking behavior.

Another group of neurons in the organoid showed activity when the scientist pushed the rats' whiskers evidence that the neurons can respond to sensory stimulation.

Brain cells cultivated from three human patients with a genetic disease called Timothy syndrome were also used for some of the organoids. Timothy syndrome affects the heart, digits and nervous system, and usually results in early death.

After the behavioral tests, the rats were euthanized and their brains extracted and dissected, allowing the researchers to observe the integration of the organoids on a cellular level. They found the organoid neurons grew much larger than any neurons grown in vitro, extending into the rats' brains and forming networks with the native rat neurons.

The neurons in the rats with Timothy syndrome transplants showed less elaborate shapes, and formed different synaptic connections with the surrounding brain tissue compared to control groups. This is a new discovery, and could not have been discovered in a brain organoid in a dish.

Although the platform still has some limitations, the team believes that it has the potential to become a powerful new tool for understanding brain development and disease.

"Overall, this in vivo platform represents a powerful resource to complement in vitro studies of human brain development and disease," the authors write in their paper.

"We anticipate that this platform will allow us to uncover new circuit-level phenotypes in patient-derived cells that have otherwise been elusive and to test novel therapeutic strategies."

The research has been published in Nature.

Originally posted here:
Scientists Spliced Human Brain Tissue Into The Brains of Baby Rats - ScienceAlert

Cardiac Stem Cells Comments Off on Scientists Spliced Human Brain Tissue Into The Brains of Baby Rats – ScienceAlert | October 13th, 2022

Tissue source and processing

Paired sections of tissue, including both artery and plaque, were recovered from the atherosclerotic core (AC) and proximally adjacent (PA) region of three patients with asymptomatic type VII calcified plaques who underwent carotid endarterectomy (Fig. S1a, TableS1). Due to the rich cellular composition of carotid artery and plethora of debris in plaque (i.e., lipid, fibrinogen, etc.), dissociation and generation of single-cell suspensions amenable to single-cell RNA sequencing were difficult. After tissue collection, enzymatic digestion, RBC lysis, and filtration were the initial steps required to generate single cells (see Methods and Fig. S1b). However, despite efficient enzymatic dissociation and significant filtering of our sample, we were still challenged by abundant plaque debris, which ultimately resulted in poor single-cell capture rates. In order to overcome this issue without isolating specific cell types through cell-marker antibody labeling, we devised a strategy to label all cells in the sample with a far-red excitation-emission live/dead cell nuclear stain (DRAQ5). All cells in the sample were stained, with debris being left unstained by the dye. Previous studies have used nuclear staining in library preparation and sequencing experiments to discriminate single versus doublet cells during cell sorting without adverse effects for downstream applications such as single-cell and bulk RNA sequencing17,18,19,20. Subsequently, DRAQ5+ cells were manually gated and sorted from the remainder of the debris using FACS. Cells isolated from the entire filtered sample represented <1% of the total particles in the sample (Figs. S2aS2f). Viability of remaining cells was assessed and was always >80% using this technique for cell separation (see Methods). The cells were then processed for single-cell sequencing.

The analytical approach in this manuscript is depicted in Fig.1a. Generation of single cells from three patient-matched AC and PA samples (batched per patient on a single NextSeq flow cell) yielded 51,981 cells total, with an average of ~13,000 AC cells/patient and ~5000PA cells/patient. Cell number disparities are due to the difference in size of the AC vs PA tissue itself. Given the abundance of AC versus PA cells, down-sampling was performed to equalize the contribution of each sample and condition to the unsupervised discovery of cell types and to mitigate bias due to class imbalance. UMAP-based clustering (see Methods) of this down-sampled dataset reveals 15 distinct cell partitions (Fig. S1c, d), representing 17,100 cells total. In order to assign partitions to major cell types we examined genes expressed in >80% of cells per partition and at a mean expression count >2. A dotplot representing three marker genes selected for each partition is presented in (Fig. S1e). A comparison of VSMC marker genes used in our study with those in the literature15 is provided (Fig. S1f). Cell-type labels assigned to these 15 initial partitions based on these marker genes include: T-lymphocytes (2 partitions), macrophages, VSMCs (2 partitions), ECs (2 partitions), B-lymphocytes, natural killer T-cells, B1-lymphocytes, mast cells, lymphoid progenitors, plasmacytoid dendritic cells, and an unidentified partition (TableS2). Following doublet filtering using a marker-gene exclusion method (see Supplemental Methods), removal of partitions with too few cells for differential gene expression analysis (mast cells, lymphoid progenitors, plasmacytoid dendritic cells, and the unidentified partition), and merging of partitions assigned to the same cell-type, we assessed differential gene expression between AC and PA regions across the 6 remaining major cell types: macrophages, ECs, VSMCs, NKT cells, T- and B-lymphocytes (Fig.1bd, Fig. S4, Supplementary Data16). We performed a number of independent partitioning experiments using various algorithmic variations to confirm the reproducibility of these partitions and cell-label assignments (see Supplemental Methods).

a Schematic diagram of analytical steps from tissue dissociation to key driver analysis. b, c UMAP visualization of 6 major cell types following doublet removal via gene exclusion criteria (see Supplemental Methods), separated by anatomic location (b), and by cell type (c). d Dotplot depicting cell-type marker genes, resulting in the identification of macrophages, ECs, VSMCs, NKT cells, T- and B-Lymphocytes. Dot size depicts the fraction of cells expressing a gene. Dot color depicts the degree of expression of each gene. n=3 for PA and AC groups.

GWAS results have highlighted biological processes in the vessel wall as key drivers of coronary artery disease (CAD)21. Our prior work has demonstrated the vascular wall to be involved in the most impactful common genetic risk factor for CAD22. Our results here also demonstrate extensive differential expression in these cell types across anatomic locations compared to the remaining cell types. Therefore, we chose to focus our efforts on dissecting expression alterations in VSMCs and ECs in order to illuminate pathogenic genomic signatures within these cell-types. As above, each cell type is compared across anatomic location (Fig.2a, e), and the top differentially expressed genes are shown (Fig.3b, f), revealing interesting spatial and expression magnitude differences between AC and PA cells.

a, e UMAP visualization of VSMCs (a) and ECs (e), separated by anatomic location. b, f Volcano plots of the top differentially expressed genes in VSMCs (b) and ECs (f). Dotted lines represented q-value 0.5 and <0.5 corresponding to PA and AC cells, respectively. c, d UMAP visualization of the top 4 upregulated genes in AC VSMCs (c), and PA VSMCs (d). Gray-colored cells indicate 0 expression of designated gene, while color bar gradient indicates lowest (black) to highest (yellow) gene expression level. g, h UMAP visualization of the top 4 upregulated genes in AC ECs (g), and PA ECs (h). Color scheme is similar to the above-described parameters. VSMCs=3674 cells; ECs=2764 cells. n=3 for PA and AC groups.

a, b Normalized enrichment score (NES) ranking of all significant PA and AC Hallmarks generated from GSEA analysis of differentially expressed genes for VSMCs (a) and ECs (b) (FDR q-value<0.05). c Fully clustered on/off heatmap visualization of overlap between leading edge EMT hallmark genes generated by GSEA. Heatmaps are downsampled and represent 448 cells from each cell type and anatomic location (1792 total cells). A dotplot corresponding to gene expression levels for each cell type in the heatmap is included. Dot size depicts the fraction of cells expressing a gene. Dot color depicts the degree of expression of each gene. d Volcano plot of differentially expressed genes between the two groups of VSMCs in (c). Dotted lines represented q-value<0.01 and normalized effect >0.5 and <0.5. e, f Gene co-expression networks generated from VSMC Module 13 (d) and EC Module 1 (e) representing the EMT hallmark from GSEA analysis. Genes are separated by anatomic location (red=AC genes, cyan=PA genes), differential expression (darker shade=higher DE, gray=non-significantly DEGs), correlation with other connected genes (green line=positive correlation, orange line=negative correlation) and strength of correlation (connecting line thickness). Significantly DEGs (q<0.05) with high connectivity scores (>0.3) are denoted by a box instead of a circle. n=3 for PA and AC groups.

VSMCs generate three subclusters in the UMAP plot. A large fraction of PA VSMCs form a PA-specific VSMC subcluster. In contrast, AC VSMCs form 2 separate clusters both of which are intermingled with PA VSMCs. This suggests VSMCs occupy three major cell states, including one completely distinct PA subtype, and two that are predominantly AC VSMCs (Fig.2a). The top four upregulated genes in the AC are sparsely expressed and include SPP1, SFRP5, IBSP, and CRTAC1 (Fig.2b, c), while APOD, PLA2G2A, C3, and MFAP5 are upregulated in many PA VSMCs (Fig.2b, d).

The spatial clustering of upregulated genes in AC VSMCs suggests the presence of separate subpopulations of matrix-secreting VSMCs involved with ECM remodeling (Fig.2c). SPP1 (osteopontin) is a secreted glycoprotein involved in bone remodeling23 and has been implicated in atherosclerosis for inhibiting vascular calcification and inflammation in the plaque milieu24. IBSP (bone sialoprotein) is a significant component of bone, cartilage, and other mineralized tissues25. CRTAC1 is a marker to distinguish chondrocytes from osteoblasts and other mesenchymal stem cells26,27. These findings suggest the presence of cartilaginous and osseous matrix-secreting VSMCs in the AC region. SFRP5, an adipokine that is a direct WNT antagonist, reduces the secretion of inflammatory factors28 and is thought to exert favorable effects on the development of atherosclerosis29. The high expression of SFRP5 in the AC suggests a deceleration of these inflammatory processes in the core of the plaque, and an overall shift in the AC to calcification and matrix remodeling.

Conversely, the upregulated genes in PA VSMCs are more ubiquitously expressed by VSMCs in a PA-specific region of the UMAP plot (Fig.2d). C3 is highly differentially expressed in many PA cells (Fig.2d). Complement activation has long been appreciated for its role in atherosclerosis30, with maturation of plaque shown to be dependent, in part, on C3 opsonization for macrophage recruitment and stimulation of antibody responses31. Its predominance in our PA samples suggests complement activation in atherosclerosis is anatomically driven by VSMCs located adjacent to areas of maximal plaque build-up. PLA2G2A (phospholipase A2 group IIA), also selectively expressed by this group of cells, is pro-atherogenic, modulates LDL oxidation and cellular oxidative stress, and promotes inflammatory cytokine secretion32, further facilitating the inflammatory properties of this group of VSMCs. Full differential expression results for VSMCs are provided (Supplementary Data5).

Overall, we identify increased calcification and ECM remodeling by VSMCs in the AC versus pro-inflammatory signaling by VSMCs in the PA. These differences in biological processes are strongly supported further in the systems analyses below.

In contrast to VSMCs, for ECs we observe a more complete separation of cells into two distinct subgroups (Fig.2e). PA ECs significantly outnumber the AC ECs (2316 vs 448 cells, respectively), possibly due to intimal erosion and loss of endothelial cell layer integrity during advanced disease5,33,34,35 resulting in fewer captured ECs in the AC. Cellular transdifferentiation may also cause a subpopulation of ECs to lose common EC marker expression, resulting in lower numbers of ECs identified in AC compared to the PA counterpart. Histologic assessment of AC plaque collected from our patients supports the assertion of endothelial layer attenuation as the principal reason for lower AC EC capture (Fig. S3b, c). In contrast to VSMCs, there is a skew toward higher magnitude expression changes in AC ECs vs PA ECs. The top four upregulated genes are ITLN1, DKK2, F5, and FN1 in the AC and IL6, MLPH, HLA-DQA1, and ACKR1 in PA ECs (Fig.2g, h).

The upregulated genes in AC ECs again suggest a synthetic profile. ITLN (omentin) is an adipokine enhancing insulin-sensitivity in adipocytes36. Interestingly, circulating plasma omentin levels were shown to negatively correlate with carotid intima-media thickness37, inhibit TNF-induced vascular inflammation in human ECs38, and promote revascularization39, suggesting an anti-inflammatory and intimal repair role in AC ECs. DKK2 further indicates intimal repair as it stimulates angiogenesis in ECs40. The significant upregulation of FN1 (fibronectin) in this group further suggests active ECM remodeling and may serve as a marker for mesenchymal cells and EMT-related processes41.

Similarly to PA VSMCs, the upregulated genes in PA ECs suggest an overall inflammatory profile. Central players in inflammation and antigen presentation are upregulated specifically in PA ECs (Fig.2h). IL6, a key inflammatory cytokine associated with plaque42, is the most upregulated gene. Furthermore, ACKR1, highly upregulated in many PA ECs, binds and internalizes numerous chemokines and facilitates their presentation on the cell surface in order to boost leukocyte recruitment and augment inflammation43. Antigen presentation on ECs via HLA-DQA1 (MHC class II molecule) may support activation and exhaustion of CD4+ T-cells44,45 as previously described. Full differential expression results for ECs are provided (Supplementary Data6).

Overall, we identify two main EC subtypes: synthetic ECs in the AC that appear to participate in intimal repair, revascularization, and ECM modulation, and inflammatory ECs in the PA region that likely facilitate inflammation via antigen/chemokine presentation and recruitment of immune cells, including CD4+ T-cells. These differences in biological processes are strongly supported further in the systems analyses below.

In order to explore the anatomic differences for these cell types further, gene set enrichment analysis (GSEA) was used to asses hallmark processes most significantly altered in VSMCs and ECs (Fig.3a, b). Epithelial to mesenchymal transition (EMT), oxidative phosphorylation, and myogenesis gene upregulation were strongly enriched in both AC VSMCs and ECs, collectively suggesting an increase in cellular metabolic activity and proliferation. In contrast, a distinctly inflammatory profile was seen in PA VSMCs and ECs, with IFN gamma/alpha responses and TNFa signaling via NFkB dominating the enriched processes in these groups of cells. Because EMT and TNFa signaling were both shared and strongly enriched processes in the two cell types, the gene signatures associated with these hallmarks were further scrutinized through generation of heatmaps consisting of leading-edge differentially expressed genes from each hallmark process (EMTFig.3c, TNFa signaling via NFkBFig. S5a).

While overlapping at the hallmark level, separation of cells by cell type as well as anatomic location in the EMT hallmark heatmap suggests the overlapping processes are mediated by distinct gene sets in each cell type. Moreover, analysis of EMT hallmark genes further supports the presence of 2 cellular subtypes of AC VSMCs as they appear to cluster into two distinct groups of cells with dichotomous expression of contractile (MYL9, TPM2, TAGLN, FLNA) versus synthetic/EMT (POSTN, LUM, FBLN2, DCN, PCOLCE2, MGP, COL3A1) gene signatures (Fig.3c, d). These results indicate a group of VSMCs in the AC may perform the contractile functions of the blood vessel wall, while the other group of VSMCs may be involved with CTD and ECM remodeling. Furthermore, cells with an ACTA2+Thy1 gene signature in Fig.3c may be, in part, plaque-stabilizing myofibroblasts (orange line), indicating that these contractile cells may also have a large role in ECM remodeling.

In contrast to distinct subclustering of cells by EMT-related genes, there appears to be a common gradient of genes involved in inflammation and response to inflammation expressed throughout the atherosclerotic tissue, with higher levels of TNF-related inflammatory genes expressed in PA VSMCs and ECs compared to AC cells, indicating a predominance of inflammatory processes occurring in the PA region overall (Fig. S5a). Collectively these genes (EIF1, FOS, JUN, JUNB, ZFP36, PNRC1, KLF2, IER2, CEBPD, NFKBIA, GADD45B, EGR1, PPP1R15A, and SOCS3), in addition to IL6 expression in PA ECs, appear to coordinate the inflammatory response pathways in plaque and its adjacent structures. All cell types analyzed thus far are coordinated along this gradient of inflammation.

To further dissect VSMC and EC anatomical gene expression differences in order to identify candidate key genes driving the significant hallmark processes, we reconstructed gene co-expression networks using a partial correlation-based approach (see Methods), defined modules by clustering, and overlaid differential expression analysis results on these modules to identify those enriched in genes differentially expressed between AC and PA tissues.

Using this strategy, 31 and 39 distinct gene network modules were generated in our VSMC and EC datasets, respectively (see Supplemental Methods, Supplementary Data7, 8). Of these, 8 modules in VSMCs, and 5 modules in ECs were enriched with differentially expressed genes (p-value<0.05, Fishers exact test, see Methods). Furthermore, differentially expressed EMT-related hallmark genes overlapped significantly and specifically with a single VSMC and EC module. Differentially expressed TNFa signaling via NFkB-related hallmark genes also overlapped significantly with one VSMCs and EC module (p-value<0.05, Fishers exact test). No other hallmark processes overlapped with generated network modules.

The EMT gene signature generated from GSEA analysis of network modules and the robust upregulation of genes found in matrix-secreting cells in this cohort suggests the possibility of CTD occurring and/or completing in the atherosclerotic core. Therefore, in order to further characterize genes which may stimulate CTD in AC VSMCs and ECs we examined gene co-expression networks in conjunction with differential expression data from the modules enriched with EMT hallmark genes. In VSMCs we identified 9 genes (SPP1, IBSP, POSTN, MMP11, COL15A1, FN1, COL4A1, SMOC1, TIMP1) whose expression was significantly upregulated in AC cells and with strong network connectivity (see Methods). Among these genes we identify POSTN, SPP1, and IBSP as possible key gene drivers of CTD processes in AC VSMCs due to their strong central connectivity and high degree of differential expression in the network module (Fig.3e). POSTN (periostin) is expressed by osteoblasts and other connective tissue cell types involved with ECM maturation46 and stabilization during EMT in non-cardiac lineages47,48. POSTN, SPP1, and IBSP are highly interconnected in our network and likely serve as drivers of CTD by modulating other correlated genes such as TIMP1, VCAN, TPST2, SMOC1, MMP11, FN1, and COL4A1 (Fig.3e), all genes which are involved with cellular differentiation49 and extracellular matrix remodeling50,51.

In our EC network we identified 18 genes (ITLN1, FN1, OMD, S100A4, SCX, PRELP, GDF7, TMP2, SERPINE2, SLPI, HEY2, IGFBP3, FOXC2, RARRES2, PTGDS, TAGLN, LINC01235, and COL6A2) whose expression was significantly upregulated in AC cells and with strong network connectivity. Among these genes, we identify ITLN1, S100A4, and SCX as possible gene drivers of CTD in ECs associated with the AC (Fig.3f). ITLN1 (omentin) is highly upregulated in ECs associated with the atherosclerotic core, and network data indicate it is strongly correlated with genes involved with cellular proliferation and ECM modulation. ITLN is also strongly correlated to OGN (osteoglycin) which induces ectopic bone formation52, indicating that ITLN1 may modulate ECs with osteoblast-like features in the atherosclerotic core. SCX (scleraxin), a transcription factor that plays a critical role in mesoderm formation, and the development of chondrocyte lineages53, as well as regulating gene expression involved with ECM synthesis and breakdown in tenocytes54, is co-expressed with IL11RA, an interleukin receptor implicated in chondrogenesis55, as well as with a variety of genes involved with cellular development and modulation of ECM structures. Thus, SCX may modulate chondrocyte-like ECs in the AC. S100A4 is a calcium-binding protein that is highly expressed in smooth muscle cells of human coronary arteries during intimal thickening56, and silencing this gene in endothelial cells prevents endothelial tube formation and tumor angiogenesis in mice57. Co-expression with HEY2, a transcription factor involved with NOTCH signaling and critical for vascular development58, may indicate an important role in repair via re-endothelialization of plaque-denuded artery.

Next, genes critical to stimulating TNFa signaling via NFkB in PA VSMCs and ECs were evaluated. In the VSMC module we identified 14 genes (APOLD1, MT1A, ZFP36, EGR1, JUNB, FOSB, JUN, FOS, RERGL, MT1M, DNAJB1, CCNH, HSPA1B, and HSPA1A) whose expression was significantly upregulated in PA cells and with strong network connectivity. Among these genes we identify immediate-early (IE) genes ZFP36, EGR1, JUNB, FOSB, and FOS as critical response genes in this hallmark process. Importantly, the paired-sample study design in which AC and PA samples from the same patient are processed identically at the same time ensures that these IE genes preferentially upregulated in the PA region are critical for the inflammatory response and not an artifact of tissue processing stressors.

In the EC module we identified two genes (IER2 and FOS) whose expression was significantly increased in PA EC cells (Fig. S5e), and are highly correlated with other critical transcription factors in our network, including FOSB, JUNB, EGR1, and ZFP36, further supporting this group of genes importance in the TNFa signaling hallmark (Fig. S5d).

Finally, in order to identify and characterize refined subpopulations from each anatomic region, we selected the 7 VSMC and 5 EC differentially expressed modules described above and biclustered cells and genes (Fig.4a, d). The likely biological functions of these subpopulations were then inferred based on the genes differentially expressed and subsequent gene ontology enrichment analysis across these subpopulations. A continuous gene expression model, based on the fraction of AC cells per subpopulation, and subsequent gene ontology enrichment analysis was used to evaluate these cell subtype differences (Fig.4b, c, e, f).

a, d Biclustered heatmap visualization of all significant genes (q<0.05) from VSMC (a) and EC (d) modules enriched with differentially expressed genes. a 1224 VSMCs from each anatomic location (2448 cells total). Large color bar denotes PA (cyan) and AC (orange) VSMCs. Small color bar above denotes distinct cell subpopulations (blue, forest green, lime green, brown, purple, magenta, red). d 448 ECs from each anatomic location (896 cells total) in. Large color bar denotes PA (blue) and AC (red) ECs. Small color bar above denotes distinct cell subpopulations (cyan, green, magenta). A dotplot corresponding to gene expression levels for each cell subpopulation on the heatmap is included. Colored dots next to specific genes correspond to critical genes related to the designated cell subpopulation. Continuous gene expression based gene ontology enrichment analysis of biological function performed based on the fraction of AC cells per subpopulation of VSMCs (b, c) and ECs (e, f). n=3 for PA and AC groups.

We identified four cell subpopulations of VSMCs with some overlapping features in our analysis (Fig.4a). The four subpopulations appear to form a continuum of cell states, starting with a population that consists exclusively of PA VSMCs (Fig.4a, green bar), characterized by genes involved in recruitment of inflammatory mediators, with early signs of CTD. Specifically, C3 (opsonization and macrophage recruitment; normalized effect=6.5, q=1.74e07) is highly differentially expressed in this subpopulation and likely augments PA inflammation and macrophage recruitment. This group of VSMCs also shows evidence of early migratory and CTD-like qualities given the expression of FBN1, SEMA3C, HTRA3, and C1QTNF3, (normalized effect=2.77, 3.65, 4.0, 3.58, respectively; q=6.93e41, 1.25e20, 2.53e05, 0.00012, respectively) genes that are both highly differentially expressed in this cohort and with high signal strength in our networks (Fig.4a, Supplementary Data7). FBN1 (ECM component) is strongly correlated with TGFBR3, SEMA3C, and CD248 (modulators of EMT-like processes)59,60,61. Interestingly, this group of cells co-expresses IGSF10, a marker of early osteochondroprogenitor cells62, TMEM119 (bone formation and mineralization; promotes differentiation of myeloblasts into osteoblasts)63,64, and WNT11 (bone formation)65 (Supplementary Data7).

On the other end of this continuum, we identify a subpopulation of ~70% AC cells (Fig.4a, red bar) that have elevated expression of POSTN (osteoblasts; normalized effect=2.206, q=3.60e16), CRTAC1 (chrondrocytes; normalized effect=3.22, q=3.91e26), TNFRSF11B (bone remodeling; normalized effect=0.98, q=7.31e06)66, ENG (VSMC migration; normalized effect=0.87, q=1.41e13)67, COL4A2, and COL4A1 (cell proliferation, association with CAD; normalized effect=0.98, 1.03 and q=3.17e15, 5.68e11, respectively)68,69. Collectively, the differential gene expression data and the underlying biology behind our gene co-expression networks support this group of cells as likely representing synthetic osteoblast- and chondrocyte-like VSMCs which facilitate calcification and cartilaginous matrix-secretion and reside largely in the AC.

Furthermore, gene ontology enrichment analysis provides a clear progression from muscle system processes, extracellular structure reorganization, and catabolic processes enriched in the PA to processes involved with CTD such as ossification, fat cell differentiation, and regulation of cell motility, adhesion, and cellular transdifferentiation enriched in the AC (Fig.4b, c). The shift in cell states supports a continuum of cell state changes leading to increased CTD in the atherosclerotic core.

Overall, we observe three EC subpopulations. Like VSMCs, these cells display transitory properties as they move through a continuum of cell states (Fig.4d). First, there is a group comprised near exclusively of inflammatory PA ECs that is involved in recruitment of inflammatory mediators (Fig.4d, magenta bar). This group has a greater number of cells expressing immune genes such as the cluster of HLA genes, as well as CD74 (normalized effect=1.63, q=2.07e112), a gene which forms part of the invariant chain of the MHC II complex and is a receptor for the cytokine macrophage migration inhibitory factor (MIF)70. The upregulation of MHC class II complex in this subset of PA ECs complements our previous finding of CD4+T-cell recruitment to this subpopulation of PA ECs, leading to over-activation and exhaustion via antigen-persistence.

The next group of cells is intermediate in its composition of AC (67.5%) and PA (~32.5%) ECs with a mixed gene expression profile with characteristics similar to each of the other two groups of cells (Fig.4d, green bar), likely representing dysfunctional ECs that are in transition from the inflamed subtype to the CTD subtype described below.

The final group of cells is largely comprised of ECs from the AC (96.8%) (Fig.4d, cyan bar) and is largely devoid of endothelial-marker gene EMCN71 (normalized effect=0.86, q=1.17e09). Critical EMT genes identified earlier (ITLN1, SCX, and S100A4) are predominantly expressed in this large cluster of AC ECs alongside highly correlated genes OMD, OGN, and CRTAC1, again indicating that this population of ECs likely represents the main group of transdifferentiated ECs.

Gene ontology enrichment analysis further supports this shift in EC cell state from cells primarily involved with immune response (antigen processing and presentation, adaptive immune response, etc.) to cell states predominantly involved with proliferation, migration, vascular development, and angiogenesis (Fig.4e, f).

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Decoding the transcriptome of calcified atherosclerotic plaque at single-cell resolution | Communications Biology - Nature.com

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Synthetic Stem Cells Market

Synthetic stem cells are very fragile and need careful storage, typing, and characterization before use. Synthetic stem cells operate in a very similar way to that deactivated vaccines. The membranes of the synthetic stem cells let them bypass the immune response. Nevertheless, synthetic stem cells can't amplify themselves. Therefore, we benefit from stem cell therapy without risks. The synthetic stem cells are more durable than human stem cells and can withstand severe freezing and thawing. Additionally, these cells are not derived from the patient's individual cells. Synthetic stem cells offer better therapeutic benefits as compared to natural stem cells. Furthermore, these cells have improved preservation stability and the technology is also generalized to other types of stem cells.

The increasing incidents and significant prevalence of several cardiovascular ailments around the world are accentuating the research in varied synthetic kinds of cardiac stem cells. The evolving focus on synthetic stem cell engineering has augmented the growth of the global synthetic stem cell market.

The better stability during preservation and a generalized technology for various types of stem cells are benefits that impart a large momentum to the growth of the synthetic stem cells market. However, the regulatory landscape for the development and approval of synthetic stem cells is very stringent, which poses a genuine challenge to companies hoping for rapid commercialization of the synthetic stem cells market.

The global synthetic stem cells market is divided into applications for neurological disorders, cardiovascular disease, and others (cancer, musculoskeletal disorders, gastrointestinal, and diabetes).

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By region, North America is expected to lead the global synthetic stem cells market over the forecast time period due to the presence of a leading stakeholder-North Carolina State University in the region. The Asia Pacific will experience rapid changes in the compound annual growth rate of the synthetic stem cells market and is anticipated to be one of the major shareholders globally due to the extensive research and development activities witnessed in Zhengzhou University situated in China.

With widespread research and development work being conducted in Europe, the region is expected to trail the Asia Pacific and North America. Latin America and the Middle East and Africa are expected to develop considerably in the future due to the emerging research and development works in this field.

Some key players in the global synthetic stem cells market are North Carolina State University and Zhengzhou University.

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Neurological DisordersCardiovascular DiseaseOthers (Cancer, Musculoskeletal Disorders, Gastrointestinal, and Diabetes)

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Global Synthetic Stem Cells Market Is Expected To Reach Around USD 42 Million By 2025 - openPR

Cardiac Stem Cells Comments Off on Global Synthetic Stem Cells Market Is Expected To Reach Around USD 42 Million By 2025 – openPR | October 13th, 2022

October 12, 2022 7:15 am ET

Companies on track to report data from the ongoing Phase 2 trial of mRNA-4157/V940 in combination with KEYTRUDA as adjuvant therapy in high-risk melanoma in 4Q 2022

CAMBRIDGE, M.A. and RAHWAY, N.J., October 12, 2022 Moderna, Inc. (Nasdaq: MRNA), a biotechnology company pioneering messenger RNA (mRNA) therapeutics and vaccines, and Merck (NYSE:MRK), known as MSD outside of the United States and Canada, today announced that Merck has exercised its option to jointly develop and commercialize personalized cancer vaccine (PCV) mRNA-4157/V940 pursuant to the terms of its existing Collaboration and License Agreement. mRNA-4157/V940 is currently being evaluated in combination with KEYTRUDA, Mercks anti-PD-1 therapy, as adjuvant treatment for patients with high-risk melanoma in a Phase 2 clinical trial being conducted by Moderna.

We have been collaborating with Merck on PCVs since 2016, and together we have made significant progress in advancing mRNA-4157 as an investigational personalized cancer treatment used in combination with KEYTRUDA, said Stephen Hoge, M.D., President of Moderna. With data expected this quarter on PCV, we continue to be excited about the future and the impact mRNA can have as a new treatment paradigm in the management of cancer. Continuing our strategic alliance with Merck is an important milestone as we continue to grow our mRNA platform with promising clinical programs in multiple therapeutic areas.

Under the agreement, originally established in 2016 and amended in 2018, Merck will pay Moderna $250 million to exercise its option for personalized cancer vaccines including mRNA-4157/V940 and will collaborate on development and commercialization. The payment will be expensed by Merck in the third quarter of 2022 and included in its non-GAAP results. Merck and Moderna will share costs and any profits equally under this worldwide collaboration.

This long-term collaboration combining Mercks expertise in immuno-oncology with Modernas pioneering mRNA technology has yielded a novel tailored vaccine approach, said Dr. Eliav Barr, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. We look forward to working with our colleagues at Moderna to advance mRNA-4157/V940 in combination with KEYTRUDA as it aligns with our strategy to impact early-stage disease.

About mRNA-4157/V940

Personalized cancer vaccines are designed to prime the immune system so that a patient can generate a tailored antitumor response to their tumor mutation signature to treat their cancer. mRNA-4157/V940 is designed to stimulate an immune response by generating T cell responses based on the mutational signature of a patients tumor.

About KEYNOTE-942 (NCT03897881)

KEYNOTE-942 is an ongoing randomized, open-label Phase 2 trial that enrolled 157 patients with high-risk melanoma. Following complete surgical resection, patients were randomized to mRNA-4157/V940 (9 doses every three weeks) and KEYTRUDA (200 mg every three weeks) versus KEYTRUDA alone for approximately one year until disease recurrence or unacceptable toxicity. KEYTRUDA was selected as the comparator in the trial because it is considered a standard of care for high-risk melanoma patients. The primary endpoint is recurrence-free survival, and secondary endpoints include distant metastasis-free survival and overall survival. The Phase 2 trial is fully enrolled and primary data are expected in the fourth quarter of 2022.

About KEYTRUDA (pembrolizumab) Injection 100 mg

KEYTRUDA is an anti-programmed death receptor-1 (PD-1) therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,600 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patients likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of adult and pediatric (12 years and older) patients with stage IIB, IIC, or III melanoma following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is:

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [Combined Positive Score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC):

Non-muscle Invasive Bladder Cancer

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options.

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC) as determined by an FDA-approved test.

Gastric Cancer

KEYTRUDA, in combination with trastuzumab, fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma.

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval of this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation either:

Cervical Cancer

KEYTRUDA, in combination with chemotherapy, with or without bevacizumab, is indicated for the treatment of patients with persistent, recurrent, or metastatic cervical cancer whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of adult patients with advanced renal cell carcinoma (RCC).

KEYTRUDA, in combination with lenvatinib, is indicated for the first-line treatment of adult patients with advanced RCC.

KEYTRUDA is indicated for the adjuvant treatment of patients with RCC at intermediate-high or high risk of recurrence following nephrectomy, or following nephrectomy and resection of metastatic lesions.

Endometrial Carcinoma

KEYTRUDA, in combination with lenvatinib, is indicated for the treatment of patients with advanced endometrial carcinoma that is not MSI-H or dMMR, who have disease progression following prior systemic therapy in any setting and are not candidates for curative surgery or radiation.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with advanced endometrial carcinoma that is MSI-H or dMMR, as determined by an FDA-approved test, who have disease progression following prior systemic therapy in any setting and are not candidates for curative surgery or radiation.

Tumor Mutational Burden-High Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) or locally advanced cSCC that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA is indicated for the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC) in combination with chemotherapy as neoadjuvant treatment, and then continued as a single agent as adjuvant treatment after surgery.

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic TNBC whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the PD-1 or the PD-L1, blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. For patients with TNBC treated with KEYTRUDA in the neoadjuvant setting, monitor blood cortisol at baseline, prior to surgery, and as clinically indicated. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA With Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis;Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy;Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss;Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis;Musculoskeletal and Connective Tissue: Myositis/polymyositis, rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica;Endocrine: Hypoparathyroidism;Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

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EAST LANSING, Mich. A chemical released during sex could become a new treatment for heart attack patients, new research reveals. Oxytocin, called the love hormone, helps heal the organ by boosting production of stem cells, researchers at Michigan State University say.

The findings are based on human tissue grown in the lab and experiments on zebrafish, which have a remarkable ability to repair themselves.

Here we show that oxytocin, a neuropeptide also known as the love hormone, is capable of activating heart repair mechanisms in injured hearts in zebrafish and human cell cultures, opening the door to potential new therapies for heart regeneration in humans, says senior author Dr. Aitor Aguirre, an assistant professor at the Department of Biomedical Engineering of Michigan State University, in a media release.

Oxytocin stimulates erections and orgasms. In women, it is believed to help sperm reach the egg. The chemical is produced by the hypothalamus in the brain. It is secreted by the pituitary gland.Abnormal amounts have a connection to sex addiction. Oxytocin is also the foundation of many pleasurable feelings, from exercise to lovemaking.

Now, the research team reports it also causes stem cells from the hearts outer layer, or epicardium, to migrate into the middle, known as the myocardium.There they develop into cardiomyocytes, muscle cells that generate heart contractions. The discovery offers hope of promoting regeneration after damaging events like a heart attack. The cells die off in great numbers after a heart attack. Highly specialized cells dont replenish themselves.

However, previous studies have shown that a subset called EpiPCs (Epicardium-derived Progenitor Cells) can undergo reprogramming, becoming cardiomyocytes or other types of heart cells.Think of the EpiPCs as the stonemasons that repaired cathedrals in Europe in the Middle Ages, Aguirre explains.

Production is inefficient for heart regeneration in humans under natural conditions, but the humble zebrafish may hold the key. They are famous for their extraordinary capacity for regenerating organs including the brain, retina, internal organs, bone, and skin.

They dont suffer heart attacks, but predators are happy to take a bite out of any organ, since zebrafish can regrow their heart when as much as a quarter of it has been lost. This is done by proliferation of cardiomyocytes and EpiPCs. The magic bullet appears to be oxytocin.

In zebrafish, within three days after the heart was exposed to cryoinjury by freezing, expression of oxytocin in the brain soared 20-fold. Scans showed the hormone travelled to the epicardium and bound to the oxytocin receptor. This triggered a molecular cascade, stimulating local cells to expand and develop into EpiPCs.

The new cells headed for the zebrafish myocardium to develop into cardiomyocytes, blood vessels, and other important heart cells, to replace those which had been lost. Crucially, the researchers found oxytocin has a similar effect on cultured human tissue. It turned human Induced Pluripotent Stem Cells (hIPSCs) into EpiPCs.

Numbers doubled due to the hormone. None of 14 other brain hormones tested worked. The effect was much stronger than other molecules tried in mice. On the other hand, genetic engineering that knocked out the oxytocin receptor prevented the regenerative activation of human EpiPCs. The link between oxytocin and the stimulation of EpiPCs was identified in a chemical pathway known to regulate the growth, differentiation and migration of cells.

These results show that it is likely that the stimulation by oxytocin of EpiPC production is evolutionary conserved in humans to a significant extent. Oxytocin is widely used in the clinic for other reasons, so repurposing for patients after heart damage is not a long stretch of the imagination. Even if heart regeneration is only partial, the benefits for patients could be enormous, Aguirre says.

Next, we need to look at oxytocin in humans after cardiac injury. Oxytocin itself is short-lived in the circulation, so its effects in humans might be hindered by that. Drugs specifically designed with a longer half-life or more potency might be useful in this setting. Overall, pre-clinical trials in animals and clinical trials in humans are necessary to move forward.

The study is published in the journal Frontiers in Cell and Developmental Biology.

South West News Service writer Mark Waghorn contributed to this report.

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'Love hormone' oxytocin could help reverse damage from heart attacks via cell regeneration - Study Finds

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The future possibilities of regenerative Medicine are endless. Know how regenerative medicine for heart diseases is better than conventional treatments.

Written by Longjam Dineshwori | Updated : October 5, 2022 9:52 AM IST

In the past few days, news of people dying due to cardiac arrest and heart attack during the festivities have been making headlines. Concerningly, increasing number of younger people, precisely adults who are in their 30s, are getting heart problems today. Health experts have been advising people to maintain a healthy lifestyle to prevent heart diseases or at least delay their onset. Also, tremendous advancements have been made in the field of cardiology making treatment of heart ailments more effective and less invasive. One of them is regenerative medicine, which is now being explored for the treatment of several diseases.

Get to more about regenerative medicine and its possibilities for treating heart diseases from Dr Pradeep Mahajan, Regenerative Medicine Researcher, Stem Rx Bioscience Solutions Pvt. Ltd, Navi Mumbai.

An alarming one out of four deaths in our country today is due to heart disease. This is largely due to our sedentary lifestyles, unhealthy eating habits, and stress. Barring the heart conditions that are present from birth (congenital) or that are passed down through the generations (inherited), heart diseases can be prevented or at least the onset can be delayed by maintaining a healthy lifestyle.

The field of cardiology (relating to the heart) has advanced tremendously, and there are several medications and surgical procedures that help patients maintain the functions of the heart. However, these call for invasive treatments and the need for life-long medications. Moreover, the side effects of medicines should also be taken into account.

Enter the field of Regenerative Cardiology! As the word suggests, this branch refers to utilising the natural healing potential of the body to repair and re-grow damaged heart tissues. Stem cells have been researched in several heart diseases to overcome the damage to the heart and facilitate healing. Not just stem cells, but cell-based products like exosomes, molecular chaperones, growth factors etc. have shown promise as well. Do not think about the technicalities, all these molecules are present in our body and researchers and clinicians are now working on how to apply these for the treatment of several diseases.

Commonly, we hear of blocks in the heart, infection, and weak muscles of the heart that do not pump blood properly leading to various diseases. With cell-based therapies, we can tackle each of these issues. Stem cells (the most basic 'unspecialized' cells of our body) can multiply and form various cells of the body, including heart cells. Similarly, cell products like exosomes are cargo packets they carry the required substances for repair and re-growth of tissues. These biological molecules have 'housekeeping functions, meaning that they ensure that any unwanted product and even bacteria/viruses are removed periodically from the body.

The possibilities of Regenerative Medicine for heart diseases are many blocks in the heart can be dissolved, blood supply can be improved, heart muscles can be strengthened, etc. because these biological molecules are capable of reducing inflammation (swelling) in the body, modify the immune system to function better, enhance the functions of other cells, etc. Since these are part of our own body, providing these molecules in the appropriate quantity at the desired site will enhance healing without side effects. In fact, there is ongoing research on growing healthy heart tissue in labs with these biological molecules to transplant them into the human body. Who knows, someday the whole heart might be grown in a lab! While the future possibilities are endless, the current cell-based therapies can be a definitive addition to enhance the outcomes of existing conventional treatments. Of course, rehabilitation and lifestyle modifications are mandatory to maintain the results.

A holistic approach is important one cannot simply rely on symptom management the core issues have to be targeted and Regenerative Medicine can do just that. The death rate due to heart disease can be reduced and patients will be able to have a better quality of life.

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Regenerative Medicine For Heart Diseases: How It Is Better Than Conventional Treatments | TheHealthSite.co - TheHealthSite

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Inflammatory bowel disease (IBD) is increasing around the world. In 1990, around 3.7 million people had the condition; by 2017, that number had increased to 6.8 million. Nearly half of IBD patients dont respond to current treatments, and even for the lucky ones therapeutic efficacy can wane over time. As a result, there is an urgent need to develop a new generation of IBD therapies.

Unfortunately, ineffective drug development models are hampering the search for more effective treatments. Conventional two-dimensional (2D) cell models only capture bits and pieces of IBDs complexity, and many three-dimensional (3D) culture models like organoids fall short because they lack critical biological features, such as vasculature and biomechanical forces.

Animal models have their own drawbacks, as their immune systems fail to replicate many of the mechanisms associated with human immunity.

If you look at the physiology of cardiac muscle or neurons between humans and mice, theyre fairly similar, said Christopher Carman, PhD, director of Immunology at Emulate. Theres more divergence in immunology, and it can be really challenging to extract meaningful insights around immune-system-driven mechanisms. Thats why so many therapeutics fail.

To remedy this, Emulate has developed a Colon Intestine-Chip that combines primary human tissue, vasculature, mechanical forces, and (most importantly) immune cell recruitment to recapitulate the biology that drives IBD.

UNDERSTANDING HOW IBD EVOLVES

IBD begins with an unknown tissue insult, and the body responds by producing inflammatory cytokines and chemokines. In turn, these proteins recruit immune cells to the intestine, inducing further inflammation.

This process generates a cytokine cascade. Two proteins in particular, interferon gamma (IFN) and IL-22, act directly on colon epithelial cells, driving cell death, microvilli loss, and destruction of the tight junctions that guard intestinal permeability.

That is a critical hallmark of this disease, said Carman. As a result, intestinal material, including bacteria and bacterial products, leak into the interstitial space, driving even more inflammation.

MAKING THE COLON INTESTINE-CHIP

The Emulate Colon Intestine-Chip was designed to precisely recapitulate this inflammatory cascade.

This advanced, in vitro intestine model incorporates primary human biopsy tissue cultured into organoids. Critically, the cells retain their stemness, meaning they replicate the stem cell niches that are constantly regenerating in human intestines.

After the organoids are dissociated, they are seeded in the top channel of the Organ-Chip. The bottom channel contains primary human intestine-derived microvascular endothelial cells, which are in close proximity to the epithelial cells, as they would be in vivo. The channels are separated by a porous membrane coated with tissue-relevant extracellular matrix proteins.

From there, mechanical forces on the chipphysiologic flow and cyclic stretchreplicate intestinal peristalsis, which improves cell morphology and functionality while supporting more accurate gene expression.

As a result, epithelial tissues respond to microvasculature cues, and the epithelial cells differentiate into all three major epithelial types at the appropriate ratios.

With this, the Emulate Colon Intestine-Chip is able to model IBD from the initial insult to the cytokine cascade, demonstrating along the way selective immune cell recruitment, cell death, and tight junction loss. This model can be applied to study inflammation-specific immune recruitment from vasculature into epithelial tissue and subsequent downstream impacts.

We have shown that this Organ-Chip strongly reflects what we see in primary human tissue, said Carman. It develops proper tight junctions and a strong functional barrier. On the molecular level, we see transcriptional signatures that are highly reflective of primary human tissue.

This model has demonstrated the efficacy of small molecule inhibitors that target IFN and IL-22 signaling pathways, meaning researchers can use it to validate clinically relevant drug candidates designed to prevent barrier dysfunction.

SELECTIVELY GENERATING INFLAMMATION

One of the Organ-Chips most important abilities is the selective recruitment of immune cells. This selectivity comes from tissue-specific adhesion molecules on both endothelial and immune cells, which must be highly specific to bind.

Around 30% of the bodys circulating immune cells are customized for work in the intestines. They have a molecule called 47 integrin that binds to an endothelial molecule called MAdCAM-1, which is preferentially expressed in the colon endothelium and up-regulated in response to inflammatory cues.

One of the major ways the Colon Intestine-Chip replicates IBD biology is by expressing MAdCAM-1 in response to inflammatory stimuli, giving it tremendous relevance for therapeutic discovery.

The 47 integrin/MAdCAM-1 adhesion molecule axis is an important therapeutic target, said Carman. If we can interfere with that adhesion, we can potentially interrupt the inflammatory cascade. And because this mechanism is selective to the gut, any therapeutic that targets these adhesion molecules would be highly specific to the intestinal system.

One drug, AJM300, is in phase three clinical trials right now and is showing promising safety and efficacy, said Carman. We validated that efficacy in our model. We also used the model to study the corticosteroid dexamethasone, which has been a mainstay in IBD treatment for many years. We recently published the data in an application note.

The Colon Intestine-Chip provides a more complete picture of human IBD pathogenesis, delivering a human-relevant platform to test drug efficacy. However, for Emulate, its just the beginning. Inflammation plays a major role in many conditions, and creating models that effectively replicate those pathways will be essential in validating and advancing therapeutic compounds to support better care.

This IBD model is our first foray into inflammation, said Carman. Were planning on developing many variations on this theme to create better tools for a variety of inflammation-driven indications.

For more information on Emulates IBD model, please download Modeling Inflammation-Specific Immune Cell Recruitment in the Colon Intestine-Chip.

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Adult stem cells, also called somatic stem cells, are undifferentiated cells that are found in many different tissues throughout the body of nearly all organisms, including humans. Unlike embryonic stem cells, which can become any cell in the body (called pluripotent), adult stem cells, which have been found in a wide range of tissues including skin, heart, brain, liver, and bone marrow are usually restricted to become any type of cell in the tissue or organ that they reside (called multipotent). These adult stem cells, which exist in the tissue for decades, serve to replace cells that are lost in the tissue as needed, such as the growth of new skin every day in humans.

Scientists discovered adult stem cells in bone marrow more than 50 years ago. These blood-forming stem cells have been used in transplants for patients with leukemia and several other diseases for decades. By the 1990s, researchers confirmed that nerve cells in the brain can also be regenerated from endogenous stem cells. It is thought that adult stem cells in a variety of different tissues could lead to treatments for numerous conditions that range from type 1 diabetes (providing insulin-producing cells) to heart attack (repairing cardiac muscle) to neurological disease (regenerating lost neurons in the brain or spinal cord).

Efforts are underway to stimulate these adult stem cells to regenerate missing cells within damaged tissues. This approach will utilize the existing tissue organization and molecules to stimulate and guide the adult stem cells to correctly regenerate only the necessary cell types. Alternatively, the adult stem cells could be isolated from the tissue and grown outside of the body, in cultures. This would allow the cells to be easily manipulated, although they are often relatively rare and difficult to grow in culture.

Because the isolation of adult stem cells does not result in the destruction of human life, research involving adult stem cells does not raise any of the ethical issues associated with research utilizing human embryonic stem cells. Thus, research involving adult stem cells has the potential for therapies that will heal disease and ease suffering, a major focus of Notre Dames stem cell research. Combined with our efforts with induced pluripotent stem (iPS) cells, the Center for Stem Cells and Regenerative Medicine will advance the Universitys mission to ease suffering and heal disease.

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Adult Stem Cells // Center for Stem Cells and Regenerative Medicine ...

Cardiac Stem Cells Comments Off on Adult Stem Cells // Center for Stem Cells and Regenerative Medicine … | September 19th, 2022

Positive Opinion Based on Landmark ZUMA-7 Study in Which 41% of Patients Demonstrated Event-Free Survival at Two Years versus 16% for Standard of Care -

SANTA MONICA, Calif.--(BUSINESS WIRE)--Kite, a Gilead Company (Nasdaq: GILD), today announces that the European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP) has issued a positive opinion for Yescarta (axicabtagene ciloleucel) for adult patients with diffuse large B-cell lymphoma (DLBCL) and high-grade B-cell lymphoma (HGBL) that relapses within 12 months from completion of, or is refractory to, first-line chemoimmunotherapy. If approved, Yescarta will be the first Chimeric Antigen Receptor (CAR) T-cell therapy approved for patients in Europe who do not respond to first-line treatment. Although 60% of newly diagnosed LBCL patients will respond to their initial treatment, 40% will relapse or will not respond and need 2nd line treatment.

At Kite, we are committed to bringing the curative potential of cell therapy to the world, and changing the way cancer is treated, said Christi Shaw, CEO, Kite. Todays positive CHMP opinion brings us a step closer to utilizing cell therapy earlier in the treatment journey, potentially transforming the standard of care for the most common and aggressive form of non-Hodgkin lymphoma.

The European Commission will review the CHMP opinion, and a final decision on the marketing authorization is expected in the coming months.

For people with DLBCL and HGBL who do not respond to first-line treatment or have an early relapse, outcomes are often poor and there are limited curative treatment options for these patients, said Marie Jos Kersten, Professor of Hematology at Amsterdam University Medical Centers, Amsterdam. If approved, axicabtagene ciloleucel may offer a new standard of care for patients with relapsed or refractory DLBCL and HGBL. Importantly, in a randomized trial of axicabtagene ciloleucel versus the current standard of care, quality of life also showed greater improvement in the experimental arm.

The positive opinion for Yescarta is based on the primary results of the landmark Phase 3 ZUMA-7 study, the largest and longest trial of a CAR T-cell therapy versus standard of care (SOC) in second-line LBCL. Results demonstrated that at a median follow-up of two years, Yescarta-treated patients had a four-fold greater improvement in the primary endpoint of event-free survival (EFS; hazard ratio 0.40; 95% CI: 0.31-0.51, P<0.001) over the current SOC (8.3 months v 2.0 months). Additionally, Yescarta demonstrated a 2.5 fold increase in patients who were alive at two years without disease progression or need for additional cancer treatment vs SOC (41% v 16%). Improvements in EFS with Yescarta were consistent across key patient subgroups, including elderly patients (HR: 0.28 [95% CI: 0.16-0.46]), primary refractory patients (HR: 0.43 [95% CI: 0.32- 0.57]), high-grade B cell lymphoma including double-hit and triple-hit lymphoma patients (HGBL; HR: 0.28 [95% CI: 0.14-0.59]), and double expressor lymphoma patients (HR: 0.42 [95% CI: 0.27-0.67]).

In a separate, secondary analysis of Patient-Reported Outcomes (PROs) published in Blood patients receiving Yescarta and eligible for the PROs portion of the study (n=165) showed statistically significant improvements in Quality of Life (QoL) at Day 100 compared with those who received SOC (n=131), using a pre-specified analysis for three PRO-domains (EORTC QLQ-C30 Physical Functioning, EORTC QLQ-C30 Global Health Status/QOL, and EQ-5D-5L visual analog scale [VAS]). There was also a trend toward faster recovery to baseline QoL in the Yescarta arm versus SOC.

In the ZUMA-7 trial, Yescarta had a manageable safety profile that was consistent with previous studies. Among the 170 Yescarta-treated patients evaluable for safety, Grade 3 cytokine release syndrome (CRS) and neurologic events were observed in 6% and 21% of patients, respectively. No Grade 5 CRS or neurologic events occurred. In the SOC arm, 83% of patients had high-grade events, mostly cytopenias (low blood counts).

About ZUMA-7

ZUMA-7 is an ongoing, randomized, open-label, global, multicenter (US, Australia, Canada, Europe, Israel) Phase 3 study of 359 patients at 77 centers, evaluating the safety and efficacy of a single-infusion of Yescarta versus current SOC for second-line therapy (platinum-based salvage combination chemotherapy regimen followed by high-dose chemotherapy and autologous stem cell transplant in those who respond to salvage chemotherapy) in adult patients with relapsed or refractory LBCL within 12 months of first-line therapy. The primary endpoint is event free survival (EFS) as determined by blinded central review, and defined as the time from randomization to the earliest date of disease progression per Lugano Classification, commencement of new lymphoma therapy, or death from any cause. Key secondary endpoints include objective response rate (ORR) and overall survival (OS). Additional secondary endpoints include patient reported outcomes (PROs) and safety.

About Yescarta

Yescarta was first approved in Europe in 2018 and is currently indicated for three types of blood cancer: Diffuse Large B-Cell Lymphoma (DLBCL); Primary Mediastinal Large B-Cell Lymphoma (PMBCL); and Follicular Lymphoma (FL). For the full European Prescribing Information, please visit: https://www.ema.europa.eu/en/medicines/human/EPAR/yescarta

Please see full US Prescribing Information, including BOXED WARNING and Medication Guide.

YESCARTA is a CD19-directed genetically modified autologous T cell immunotherapy indicated for the treatment of:

U.S. IMPORTANT SAFETY INFORMATION

BOXED WARNING: CYTOKINE RELEASE SYNDROME AND NEUROLOGIC TOXICITIES

CYTOKINE RELEASE SYNDROME (CRS)

CRS, including fatal or life-threatening reactions, occurred. CRS occurred in 90% (379/422) of patients with non-Hodgkin lymphoma (NHL), including Grade 3 in 9%. CRS occurred in 93% (256/276) of patients with large B-cell lymphoma (LBCL), including Grade 3 in 9%. Among patients with LBCL who died after receiving YESCARTA, 4 had ongoing CRS events at the time of death. For patients with LBCL in ZUMA-1, the median time to onset of CRS was 2 days following infusion (range: 1-12 days) and the median duration was 7 days (range: 2-58 days). For patients with LBCL in ZUMA-7, the median time to onset of CRS was 3 days following infusion (range: 1-10 days) and the median duration was 7 days (range: 2-43 days). CRS occurred in 84% (123/146) of patients with indolent non-Hodgkin lymphoma (iNHL) in ZUMA-5, including Grade 3 in 8%. Among patients with iNHL who died after receiving YESCARTA, 1 patient had an ongoing CRS event at the time of death. The median time to onset of CRS was 4 days (range: 1-20 days) and the median duration was 6 days (range: 1-27 days) for patients with iNHL.

Key manifestations of CRS ( 10%) in all patients combined included fever (85%), hypotension (40%), tachycardia (32%), chills (22%), hypoxia (20%), headache (15%), and fatigue (12%). Serious events that may be associated with CRS include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), renal insufficiency, cardiac failure, respiratory failure, cardiac arrest, capillary leak syndrome, multi-organ failure, and hemophagocytic lymphohistiocytosis/macrophage activation syndrome.

The impact of tocilizumab and/or corticosteroids on the incidence and severity of CRS was assessed in 2 subsequent cohorts of LBCL patients in ZUMA-1. Among patients who received tocilizumab and/or corticosteroids for ongoing Grade 1 events, CRS occurred in 93% (38/41), including 2% (1/41) with Grade 3 CRS; no patients experienced a Grade 4 or 5 event. The median time to onset of CRS was 2 days (range: 1-8 days) and the median duration of CRS was 7 days (range: 2-16 days). Prophylactic treatment with corticosteroids was administered to a cohort of 39 patients for 3 days beginning on the day of infusion of YESCARTA. Thirty-one of the 39 patients (79%) developed CRS and were managed with tocilizumab and/or therapeutic doses of corticosteroids with no patients developing Grade 3 CRS. The median time to onset of CRS was 5 days (range: 1-15 days) and the median duration of CRS was 4 days (range: 1-10 days). Although there is no known mechanistic explanation, consider the risk and benefits of prophylactic corticosteroids in the context of pre-existing comorbidities for the individual patient and the potential for the risk of Grade 4 and prolonged neurologic toxicities.

Ensure that 2 doses of tocilizumab are available prior to YESCARTA infusion. Monitor patients for signs and symptoms of CRS at least daily for 7 days at the certified healthcare facility, and for 4 weeks thereafter. Counsel patients to seek immediate medical attention should signs or symptoms of CRS occur at any time. At the first sign of CRS, institute treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as indicated.

NEUROLOGIC TOXICITIES

Neurologic toxicities (including immune effector cell-associated neurotoxicity syndrome) that were fatal or life-threatening occurred. Neurologic toxicities occurred in 78% (330/422) of all patients with NHL receiving YESCARTA, including Grade 3 in 25%. Neurologic toxicities occurred in 87% (94/108) of patients with LBCL in ZUMA-1, including Grade 3 in 31% and in 74% (124/168) of patients in ZUMA-7 including Grade 3 in 25%. The median time to onset was 4 days (range: 1-43 days) and the median duration was 17 days for patients with LBCL in ZUMA-1. The median time to onset for neurologic toxicity was 5 days (range:1- 133 days) and the median duration was 15 days in patients with LBCL in ZUMA-7. Neurologic toxicities occurred in 77% (112/146) of patients with iNHL, including Grade 3 in 21%. The median time to onset was 6 days (range: 1-79 days) and the median duration was 16 days. Ninety-eight percent of all neurologic toxicities in patients with LBCL and 99% of all neurologic toxicities in patients with iNHL occurred within the first 8 weeks of YESCARTA infusion. Neurologic toxicities occurred within the first 7 days of infusion for 87% of affected patients with LBCL and 74% of affected patients with iNHL.

The most common neurologic toxicities ( 10%) in all patients combined included encephalopathy (50%), headache (43%), tremor (29%), dizziness (21%), aphasia (17%), delirium (15%), and insomnia (10%). Prolonged encephalopathy lasting up to 173 days was noted. Serious events, including aphasia, leukoencephalopathy, dysarthria, lethargy, and seizures occurred. Fatal and serious cases of cerebral edema and encephalopathy, including late-onset encephalopathy, have occurred.

The impact of tocilizumab and/or corticosteroids on the incidence and severity of neurologic toxicities was assessed in 2 subsequent cohorts of LBCL patients in ZUMA-1. Among patients who received corticosteroids at the onset of Grade 1 toxicities, neurologic toxicities occurred in 78% (32/41), and 20% (8/41) had Grade 3 neurologic toxicities; no patients experienced a Grade 4 or 5 event. The median time to onset of neurologic toxicities was 6 days (range: 1-93 days) with a median duration of 8 days (range: 1-144 days). Prophylactic treatment with corticosteroids was administered to a cohort of 39 patients for 3 days beginning on the day of infusion of YESCARTA. Of those patients, 85% (33/39) developed neurologic toxicities, 8% (3/39) developed Grade 3, and 5% (2/39) developed Grade 4 neurologic toxicities. The median time to onset of neurologic toxicities was 6 days (range: 1-274 days) with a median duration of 12 days (range: 1-107 days). Prophylactic corticosteroids for management of CRS and neurologic toxicities may result in a higher grade of neurologic toxicities or prolongation of neurologic toxicities, delay the onset of and decrease the duration of CRS.

Monitor patients for signs and symptoms of neurologic toxicities at least daily for 7 days at the certified healthcare facility, and for 4 weeks thereafter, and treat promptly.

REMS

Because of the risk of CRS and neurologic toxicities, YESCARTA is available only through a restricted program called the YESCARTA and TECARTUS REMS Program which requires that: Healthcare facilities that dispense and administer YESCARTA must be enrolled and comply with the REMS requirements and must have on-site, immediate access to a minimum of 2 doses of tocilizumab for each patient for infusion within 2 hours after YESCARTA infusion, if needed for treatment of CRS. Certified healthcare facilities must ensure that healthcare providers who prescribe, dispense, or administer YESCARTA are trained in the management of CRS and neurologic toxicities. Further information is available at http://www.YescartaTecartusREMS.com or 1-844-454-KITE (5483).

HYPERSENSITIVITY REACTIONS

Allergic reactions, including serious hypersensitivity reactions or anaphylaxis, may occur with the infusion of YESCARTA.

SERIOUS INFECTIONS

Severe or life-threatening infections occurred. Infections (all grades) occurred in 45% of patients with NHL; Grade 3 infections occurred in 17% of patients, including Grade 3 infections with an unspecified pathogen in 12%, bacterial infections in 5%, viral infections in 3%, and fungal infections in 1%. YESCARTA should not be administered to patients with clinically significant active systemic infections. Monitor patients for signs and symptoms of infection before and after infusion and treat appropriately. Administer prophylactic antimicrobials according to local guidelines.

Febrile neutropenia was observed in 36% of all patients with NHL and may be concurrent with CRS. In the event of febrile neutropenia, evaluate for infection and manage with broad-spectrum antibiotics, fluids, and other supportive care as medically indicated.

In immunosuppressed patients, including those who have received YESCARTA, life-threatening and fatal opportunistic infections including disseminated fungal infections (e.g., candida sepsis and aspergillus infections) and viral reactivation (e.g., human herpes virus-6 [HHV-6] encephalitis and JC virus progressive multifocal leukoencephalopathy [PML]) have been reported. The possibility of HHV-6 encephalitis and PML should be considered in immunosuppressed patients with neurologic events and appropriate diagnostic evaluations should be performed.

Hepatitis B virus (HBV) reactivation, in some cases resulting in fulminant hepatitis, hepatic failure, and death, can occur in patients treated with drugs directed against B cells, including YESCARTA. Perform screening for HBV, HCV, and HIV in accordance with clinical guidelines before collection of cells for manufacturing.

PROLONGED CYTOPENIAS

Patients may exhibit cytopenias for several weeks following lymphodepleting chemotherapy and YESCARTA infusion. Grade 3 cytopenias not resolved by Day 30 following YESCARTA infusion occurred in 39% of all patients with NHL and included neutropenia (33%), thrombocytopenia (13%), and anemia (8%). Monitor blood counts after infusion.

HYPOGAMMAGLOBULINEMIA

B-cell aplasia and hypogammaglobulinemia can occur. Hypogammaglobulinemia was reported as an adverse reaction in 14% of all patients with NHL. Monitor immunoglobulin levels after treatment and manage using infection precautions, antibiotic prophylaxis, and immunoglobulin replacement. The safety of immunization with live viral vaccines during or following YESCARTA treatment has not been studied. Vaccination with live virus vaccines is not recommended for at least 6 weeks prior to the start of lymphodepleting chemotherapy, during YESCARTA treatment, and until immune recovery following treatment.

SECONDARY MALIGNANCIES

Secondary malignancies may develop. Monitor life-long for secondary malignancies. In the event that one occurs, contact Kite at 1-844-454-KITE (5483) to obtain instructions on patient samples to collect for testing.

EFFECTS ON ABILITY TO DRIVE AND USE MACHINES

Due to the potential for neurologic events, including altered mental status or seizures, patients are at risk for altered or decreased consciousness or coordination in the 8 weeks following YESCARTA infusion. Advise patients to refrain from driving and engaging in hazardous occupations or activities, such as operating heavy or potentially dangerous machinery, during this initial period.

ADVERSE REACTIONS

The most common non-laboratory adverse reactions (incidence 20%) in patients with LBCL in ZUMA-7 included fever, CRS, fatigue, hypotension, encephalopathy, tachycardia, diarrhea, headache, musculoskeletal pain, nausea, febrile neutropenia, chills, cough, infection with an unspecified pathogen, dizziness, tremor, decreased appetite, edema, hypoxia, abdominal pain, aphasia, constipation, and vomiting.

The most common adverse reactions (incidence 20%) in patients with LBCL in ZUMA-1 included CRS, fever, hypotension, encephalopathy, tachycardia, fatigue, headache, decreased appetite, chills, diarrhea, febrile neutropenia, infections with an unspecified, nausea, hypoxia, tremor, cough, vomiting, dizziness, constipation, and cardiac arrhythmias.

The most common non-laboratory adverse reactions (incidence 20%) in patients with iNHL in ZUMA-5 included fever, CRS, hypotension, encephalopathy, fatigue, headache, infections with an unspecified, tachycardia, febrile neutropenia, musculoskeletal pain, nausea, tremor, chills, diarrhea, constipation, decreased appetite, cough, vomiting, hypoxia, arrhythmia, and dizziness.

About Kite

Kite, a Gilead Company, is a global biopharmaceutical company based in Santa Monica, California, with manufacturing operations in North America and Europe. Kites singular focus is cell therapy to treat and potentially cure cancer. As the cell therapy leader, Kite has more approved CAR T indications to help more patients than any other company. For more information on Kite, please visit http://www.kitepharma.com. Follow Kite on social media on Twitter (@KitePharma) and LinkedIn.

About Gilead Sciences

Gilead Sciences, Inc. is a biopharmaceutical company that has pursued and achieved breakthroughs in medicine for more than three decades, with the goal of creating a healthier world for all people. The company is committed to advancing innovative medicines to prevent and treat life-threatening diseases, including HIV, viral hepatitis and cancer. Gilead operates in more than 35 countries worldwide, with headquarters in Foster City, California.

Forward-Looking Statements

This press release includes forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 that are subject to risks, uncertainties and other factors, including the ability of Gilead and Kite to initiate, progress or complete clinical trials within currently anticipated timelines or at all, and the possibility of unfavorable results from ongoing and additional clinical trials, including those involving Yescarta; uncertainties relating to regulatory applications and related filing and approval timelines, including the risk that the European Commission may not grant marketing authorization for Yescarta for use in second-line DLBCL and HGBL in a timely manner or at all; the risk that any regulatory approvals, if granted, may be subject to significant limitations on use; the risk that physicians may not see the benefits of prescribing Yescarta for the treatment of LBCL; and any assumptions underlying any of the foregoing. These and other risks, uncertainties and other factors are described in detail in Gileads Quarterly Report on Form 10-Q for the quarter ended June 30, 2022 as filed with the U.S. Securities and Exchange Commission. These risks, uncertainties and other factors could cause actual results to differ materially from those referred to in the forward-looking statements. All statements other than statements of historical fact are statements that could be deemed forward-looking statements. The reader is cautioned that any such forward-looking statements are not guarantees of future performance and involve risks and uncertainties and is cautioned not to place undue reliance on these forward-looking statements. All forward-looking statements are based on information currently available to Gilead and Kite, and Gilead and Kite assume no obligation and disclaim any intent to update any such forward-looking statements.

U.S. Prescribing Information for Yescarta including BOXED WARNING, is available at http://www.kitepharma.com and http://www.gilead.com .

Kite, the Kite logo, Yescarta and GILEAD are trademarks of Gilead Sciences, Inc. or its related companies .

View source version on businesswire.com: https://www.businesswire.com/news/home/20220916005209/en/

Jacquie Ross, Investorsinvestor_relations@gilead.com

Anna Padula, Mediaapadula@kitepharma.com

Source: Gilead Sciences, Inc.

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CCL7 as a novel inflammatory mediator in cardiovascular disease, diabetes mellitus, and kidney disease - Cardiovascular Diabetology - Cardiovascular...

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Pluripotent embyronic cell group giving rise to diverse cell lineages

Neural crest cells are a temporary group of cells unique to vertebrates that arise from the embryonic ectoderm germ layer, and in turn give rise to a diverse cell lineageincluding melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia.[1][2]

After gastrulation, neural crest cells are specified at the border of the neural plate and the non-neural ectoderm. During neurulation, the borders of the neural plate, also known as the neural folds, converge at the dorsal midline to form the neural tube.[3] Subsequently, neural crest cells from the roof plate of the neural tube undergo an epithelial to mesenchymal transition, delaminating from the neuroepithelium and migrating through the periphery where they differentiate into varied cell types.[1] The emergence of neural crest was important in vertebrate evolution because many of its structural derivatives are defining features of the vertebrate clade.[4]

Underlying the development of neural crest is a gene regulatory network, described as a set of interacting signals, transcription factors, and downstream effector genes that confer cell characteristics such as multipotency and migratory capabilities.[5] Understanding the molecular mechanisms of neural crest formation is important for our knowledge of human disease because of its contributions to multiple cell lineages. Abnormalities in neural crest development cause neurocristopathies, which include conditions such as frontonasal dysplasia, WaardenburgShah syndrome, and DiGeorge syndrome.[1]

Therefore, defining the mechanisms of neural crest development may reveal key insights into vertebrate evolution and neurocristopathies.

Neural crest was first described in the chick embryo by Wilhelm His Sr. in 1868 as "the cord in between" (Zwischenstrang) because of its origin between the neural plate and non-neural ectoderm.[1] He named the tissue ganglionic crest since its final destination was each lateral side of the neural tube where it differentiated into spinal ganglia.[6] During the first half of the 20th century the majority of research on neural crest was done using amphibian embryos which was reviewed by Hrstadius (1950) in a well known monograph.[7]

Cell labeling techniques advanced the field of neural crest because they allowed researchers to visualize the migration of the tissue throughout the developing embryos. In the 1960s Weston and Chibon utilized radioisotopic labeling of the nucleus with tritiated thymidine in chick and amphibian embryo respectively. However, this method suffers from drawbacks of stability, since every time the labeled cell divides the signal is diluted. Modern cell labeling techniques such as rhodamine-lysinated dextran and the vital dye diI have also been developed to transiently mark neural crest lineages.[6]

The quail-chick marking system, devised by Nicole Le Douarin in 1969, was another instrumental technique used to track neural crest cells.[8][9] Chimeras, generated through transplantation, enabled researchers to distinguish neural crest cells of one species from the surrounding tissue of another species. With this technique, generations of scientists were able to reliably mark and study the ontogeny of neural crest cells.

A molecular cascade of events is involved in establishing the migratory and multipotent characteristics of neural crest cells. This gene regulatory network can be subdivided into the following four sub-networks described below.

First, extracellular signaling molecules, secreted from the adjacent epidermis and underlying mesoderm such as Wnts, BMPs and Fgfs separate the non-neural ectoderm (epidermis) from the neural plate during neural induction.[1][4]

Wnt signaling has been demonstrated in neural crest induction in several species through gain-of-function and loss-of-function experiments. In coherence with this observation, the promoter region of slug (a neural crest specific gene) contains a binding site for transcription factors involved in the activation of Wnt-dependent target genes, suggestive of a direct role of Wnt signaling in neural crest specification.[10]

The current role of BMP in neural crest formation is associated with the induction of the neural plate. BMP antagonists diffusing from the ectoderm generates a gradient of BMP activity. In this manner, the neural crest lineage forms from intermediate levels of BMP signaling required for the development of the neural plate (low BMP) and epidermis (high BMP).[1]

Fgf from the paraxial mesoderm has been suggested as a source of neural crest inductive signal. Researchers have demonstrated that the expression of dominate-negative Fgf receptor in ectoderm explants blocks neural crest induction when recombined with paraxial mesoderm.[11] The understanding of the role of BMP, Wnt, and Fgf pathways on neural crest specifier expression remains incomplete.

Signaling events that establish the neural plate border lead to the expression of a set of transcription factors delineated here as neural plate border specifiers. These molecules include Zic factors, Pax3/7, Dlx5, Msx1/2 which may mediate the influence of Wnts, BMPs, and Fgfs. These genes are expressed broadly at the neural plate border region and precede the expression of bona fide neural crest markers.[4]

Experimental evidence places these transcription factors upstream of neural crest specifiers. For example, in Xenopus Msx1 is necessary and sufficient for the expression of Slug, Snail, and FoxD3.[12] Furthermore, Pax3 is essential for FoxD3 expression in mouse embryos.[13]

Following the expression of neural plate border specifiers is a collection of genes including Slug/Snail, FoxD3, Sox10, Sox9, AP-2 and c-Myc. This suite of genes, designated here as neural crest specifiers, are activated in emergent neural crest cells. At least in Xenopus, every neural crest specifier is necessary and/or sufficient for the expression of all other specifiers, demonstrating the existence of extensive cross-regulation.[4] Moreover, this model organism was instrumental in the elucidation of the role of the Hedgehog signaling pathway in the specification of the neural crest, with the transcription factor Gli2 playing a key role.[14]

Outside of the tightly regulated network of neural crest specifiers are two other transcription factors Twist and Id. Twist, a bHLH transcription factor, is required for mesenchyme differentiation of the pharyngeal arch structures.[15] Id is a direct target of c-Myc and is known to be important for the maintenance of neural crest stem cells.[16]

Finally, neural crest specifiers turn on the expression of effector genes, which confer certain properties such as migration and multipotency. Two neural crest effectors, Rho GTPases and cadherins, function in delamination by regulating cell morphology and adhesive properties. Sox9 and Sox10 regulate neural crest differentiation by activating many cell-type-specific effectors including Mitf, P0, Cx32, Trp and cKit.[4]

The migration of neural crest cells involves a highly coordinated cascade of events that begins with closure of the dorsal neural tube.

After fusion of the neural fold to create the neural tube, cells originally located in the neural plate border become neural crest cells.[17] For migration to begin, neural crest cells must undergo a process called delamination that involves a full or partial epithelial-mesenchymal transition (EMT).[18] Delamination is defined as the separation of tissue into different populations, in this case neural crest cells separating from the surrounding tissue.[19] Conversely, EMT is a series of events coordinating a change from an epithelial to mesenchymal phenotype.[18] For example, delamination in chick embryos is triggered by a BMP/Wnt cascade that induces the expression of EMT promoting transcription factors such as SNAI2 and FoxD3.[19] Although all neural crest cells undergo EMT, the timing of delamination occurs at different stages in different organisms: in Xenopus laevis embryos there is a massive delamination that occurs when the neural plate is not entirely fused, whereas delamination in the chick embryo occurs during fusion of the neural fold.[19]

Prior to delamination, presumptive neural crest cells are initially anchored to neighboring cells by tight junction proteins such as occludin and cell adhesion molecules such as NCAM and N-Cadherin.[20] Dorsally expressed BMPs initiate delamination by inducing the expression of the zinc finger protein transcription factors snail, slug, and twist.[17] These factors play a direct role in inducing the epithelial-mesenchymal transition by reducing expression of occludin and N-Cadherin in addition to promoting modification of NCAMs with polysialic acid residues to decrease adhesiveness.[17][21] Neural crest cells also begin expressing proteases capable of degrading cadherins such as ADAM10[22] and secreting matrix metalloproteinases (MMPs) that degrade the overlying basal lamina of the neural tube to allow neural crest cells to escape.[20] Additionally, neural crest cells begin expressing integrins that associate with extracellular matrix proteins, including collagen, fibronectin, and laminin, during migration.[23] Once the basal lamina becomes permeable the neural crest cells can begin migrating throughout the embryo.

Neural crest cell migration occurs in a rostral to caudal direction without the need of a neuronal scaffold such as along a radial glial cell. For this reason the crest cell migration process is termed free migration. Instead of scaffolding on progenitor cells, neural crest migration is the result of repulsive guidance via EphB/EphrinB and semaphorin/neuropilin signaling, interactions with the extracellular matrix, and contact inhibition with one another.[17] While Ephrin and Eph proteins have the capacity to undergo bi-directional signaling, neural crest cell repulsion employs predominantly forward signaling to initiate a response within the receptor bearing neural crest cell.[23] Burgeoning neural crest cells express EphB, a receptor tyrosine kinase, which binds the EphrinB transmembrane ligand expressed in the caudal half of each somite. When these two domains interact it causes receptor tyrosine phosphorylation, activation of rhoGTPases, and eventual cytoskeletal rearrangements within the crest cells inducing them to repel. This phenomenon allows neural crest cells to funnel through the rostral portion of each somite.[17]

Semaphorin-neuropilin repulsive signaling works synergistically with EphB signaling to guide neural crest cells down the rostral half of somites in mice. In chick embryos, semaphorin acts in the cephalic region to guide neural crest cells through the pharyngeal arches. On top of repulsive repulsive signaling, neural crest cells express 1and 4 integrins which allows for binding and guided interaction with collagen, laminin, and fibronectin of the extracellular matrix as they travel. Additionally, crest cells have intrinsic contact inhibition with one another while freely invading tissues of different origin such as mesoderm.[17] Neural crest cells that migrate through the rostral half of somites differentiate into sensory and sympathetic neurons of the peripheral nervous system. The other main route neural crest cells take is dorsolaterally between the epidermis and the dermamyotome. Cells migrating through this path differentiate into pigment cells of the dermis. Further neural crest cell differentiation and specification into their final cell type is biased by their spatiotemporal subjection to morphogenic cues such as BMP, Wnt, FGF, Hox, and Notch.[20]

Neurocristopathies result from the abnormal specification, migration, differentiation or death of neural crest cells throughout embryonic development.[24][25] This group of diseases comprises a wide spectrum of congenital malformations affecting many newborns. Additionally, they arise because of genetic defects affecting the formation of neural crest and because of the action of Teratogens [26]

Waardenburg's syndrome is a neurocristopathy that results from defective neural crest cell migration. The condition's main characteristics include piebaldism and congenital deafness. In the case of piebaldism, the colorless skin areas are caused by a total absence of neural crest-derived pigment-producing melanocytes.[27] There are four different types of Waardenburg's syndrome, each with distinct genetic and physiological features. Types I and II are distinguished based on whether or not family members of the affected individual have dystopia canthorum.[28] Type III gives rise to upper limb abnormalities. Lastly, type IV is also known as Waardenburg-Shah syndrome, and afflicted individuals display both Waardenburg's syndrome and Hirschsprung's disease.[29] Types I and III are inherited in an autosomal dominant fashion,[27] while II and IV exhibit an autosomal recessive pattern of inheritance. Overall, Waardenburg's syndrome is rare, with an incidence of ~ 2/100,000 people in the United States. All races and sexes are equally affected.[27] There is no current cure or treatment for Waardenburg's syndrome.

Also implicated in defects related to neural crest cell development and migration is Hirschsprung's disease (HD or HSCR), characterized by a lack of innervation in regions of the intestine. This lack of innervation can lead to further physiological abnormalities like an enlarged colon (megacolon), obstruction of the bowels, or even slowed growth. In healthy development, neural crest cells migrate into the gut and form the enteric ganglia. Genes playing a role in the healthy migration of these neural crest cells to the gut include RET, GDNF, GFR, EDN3, and EDNRB. RET, a receptor tyrosine kinase (RTK), forms a complex with GDNF and GFR. EDN3 and EDNRB are then implicated in the same signaling network. When this signaling is disrupted in mice, aganglionosis, or the lack of these enteric ganglia occurs.[30]

Prenatal alcohol exposure (PAE) is among the most common causes of developmental defects.[31] Depending on the extent of the exposure and the severity of the resulting abnormalities, patients are diagnosed within a continuum of disorders broadly labeled Fetal Alcohol Spectrum Disorder (FASD). Severe FASD can impair neural crest migration, as evidenced by characteristic craniofacial abnormalities including short palpebral fissures, an elongated upper lip, and a smoothened philtrum. However, due to the promiscuous nature of ethanol binding, the mechanisms by which these abnormalities arise is still unclear. Cell culture explants of neural crest cells as well as in vivo developing zebrafish embryos exposed to ethanol show a decreased number of migratory cells and decreased distances travelled by migrating neural crest cells. The mechanisms behind these changes are not well understood, but evidence suggests PAE can increase apoptosis due to increased cytosolic calcium levels caused by IP3-mediated release of calcium from intracellular stores. It has also been proposed that the decreased viability of ethanol-exposed neural crest cells is caused by increased oxidative stress. Despite these, and other advances much remains to be discovered about how ethanol affects neural crest development. For example, it appears that ethanol differentially affects certain neural crest cells over others; that is, while craniofacial abnormalities are common in PAE, neural crest-derived pigment cells appear to be minimally affected.[32]

DiGeorge syndrome is associated with deletions or translocations of a small segment in the human chromosome 22. This deletion may disrupt rostral neural crest cell migration or development. Some defects observed are linked to the pharyngeal pouch system, which receives contribution from rostral migratory crest cells. The symptoms of DiGeorge syndrome include congenital heart defects, facial defects, and some neurological and learning disabilities. Patients with 22q11 deletions have also been reported to have higher incidence of schizophrenia and bipolar disorder.[33]

Treacher Collins Syndrome (TCS) results from the compromised development of the first and second pharyngeal arches during the early embryonic stage, which ultimately leads to mid and lower face abnormalities. TCS is caused by the missense mutation of the TCOF1 gene, which causes neural crest cells to undergo apoptosis during embryogenesis. Although mutations of the TCOF1 gene are among the best characterized in their role in TCS, mutations in POLR1C and POLR1D genes have also been linked to the pathogenesis of TCS.[34]

Neural crest cells originating from different positions along the anterior-posterior axis develop into various tissues. These regions of neural crest can be divided into four main functional domains, which include the cranial neural crest, trunk neural crest, vagal and sacral neural crest, and cardiac neural crest.

Cranial neural crest migrates dorsolaterally to form the craniofacial mesenchyme that differentiates into various cranial ganglia and craniofacial cartilages and bones.[21] These cells enter the pharyngeal pouches and arches where they contribute to the thymus, bones of the middle ear and jaw and the odontoblasts of the tooth primordia.[35]

Trunk neural crest gives rise two populations of cells.[36] One group of cells fated to become melanocytes migrates dorsolaterally into the ectoderm towards the ventral midline. A second group of cells migrates ventrolaterally through the anterior portion of each sclerotome. The cells that stay in the sclerotome form the dorsal root ganglia, whereas those that continue more ventrally form the sympathetic ganglia, adrenal medulla, and the nerves surrounding the aorta.[35]

The vagal and sacral neural crest cells develop into the ganglia of the enteric nervous system and the parasympathetic ganglia.[35]

Cardiac neural crest develops into melanocytes, cartilage, connective tissue and neurons of some pharyngeal arches. Also, this domain gives rise to regions of the heart such as the musculo-connective tissue of the large arteries, and part of the septum, which divides the pulmonary circulation from the aorta.[35]The semilunar valves of the heart are associated with neural crest cells according to new research.[37]

Several structures that distinguish the vertebrates from other chordates are formed from the derivatives of neural crest cells. In their "New head" theory, Gans and Northcut argue that the presence of neural crest was the basis for vertebrate specific features, such as sensory ganglia and cranial skeleton. Furthermore, the appearance of these features was pivotal in vertebrate evolution because it enabled a predatory lifestyle.[38][39]

However, considering the neural crest a vertebrate innovation does not mean that it arose de novo. Instead, new structures often arise through modification of existing developmental regulatory programs. For example, regulatory programs may be changed by the co-option of new upstream regulators or by the employment of new downstream gene targets, thus placing existing networks in a novel context.[40][41] This idea is supported by in situ hybridization data that shows the conservation of the neural plate border specifiers in protochordates, which suggest that part of the neural crest precursor network was present in a common ancestor to the chordates.[5] In some non-vertebrate chordates such as tunicates a lineage of cells (melanocytes) has been identified, which are similar to neural crest cells in vertebrates. This implies that a rudimentary neural crest existed in a common ancestor of vertebrates and tunicates.[42]

Ectomesenchyme (also known as mesectoderm):[43] odontoblasts, dental papillae, the chondrocranium (nasal capsule, Meckel's cartilage, scleral ossicles, quadrate, articular, hyoid and columella), tracheal and laryngeal cartilage, the dermatocranium (membranous bones), dorsal fins and the turtle plastron (lower vertebrates), pericytes and smooth muscle of branchial arteries and veins, tendons of ocular and masticatory muscles, connective tissue of head and neck glands (pituitary, salivary, lachrymal, thymus, thyroid) dermis and adipose tissue of calvaria, ventral neck and face

Endocrine cells:chromaffin cells of the adrenal medulla, glomus cells type I/II.

Peripheral nervous system:Sensory neurons and glia of the dorsal root ganglia, cephalic ganglia (VII and in part, V, IX, and X), Rohon-Beard cells, some Merkel cells in the whisker,[44][45] Satellite glial cells of all autonomic and sensory ganglia, Schwann cells of all peripheral nerves.

Enteric cells:Enterochromaffin cells.[46]

Melanocytes and iris muscle and pigment cells, and even associated with some tumors (such as melanotic neuroectodermal tumor of infancy).

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Rise In Research And Development Projects In Various Regions Such As East Asia, South Asia Are Expected To Offer An Opportunity Of US $ 0.5 Bn In 2022-2026 Period.

Fact.MR A Market Research and Competitive Intelligence Provider: The global induced pluripotent stem cell (iPSC) market was valued at US $ 1.8 Bn in 2022, and is expected to witness a value of US $ 2.3 Bn by the end of 2026.

Moreover, historically, demand for induced pluripotent stem cells had witnessed a CAGR of 6.6%.

Rise in spending on research and development activities in various sectors such as healthcare industry is expected to drive the adoption of human Ips cell lines in various applications such as personalized medicine and precision.

Moreover, increasing scope of application of human iPSC cell lines in precision medicine and emphasis on therapeutic applications of stem cells are expected to be driving factors of iPSC market during the forecast period.

Surge in government spending and high awareness about stem cell research across various organizations are predicted to impact demand for induced pluripotent stem cells. Rising prevalence of chronic diseases and high adoption of stem cells in their treatment is expected to boost the market growth potential.

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Besides this, various cells such as neural stem cells, embryonic stem cells umbilical cord stem cells, etc. are anticipated to witness high demand in the U.S. due to surge in popularity of stem cell therapies.

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Many key players in the market are increasing their investments in R&D to provide offerings in stem cell therapies, which are gaining traction for the treatment of various chronic diseases.

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Cardiac Mapping System Market - The advent of cardiac mapping systems enabled electrophysiologists to target and treat complex arrhythmias more effectively than ever. According to the Centers for Disease Control and Prevention, more than 600,000 people die of heart disease in the United States, every year, making it essential for the medical professionals to take strong awareness initiatives to tell people about the availability of cardiac mapping technology, in a move to control the ever-growing cardiac deaths.

Cardiac Ablation Technologies Market - A surge in atrial fibrillation cases is proving to be a growth generator as cardiac ablation technology is prominently used for its treatment. Revenue across the radiofrequency segment is expected to gather considerable momentum, anticipated to account for over50%of global revenue.

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Cardiac Resynchronization Therapy Market - Newly-released Cardiac Resynchronization Therapy industry analysis report by Fact.MR shows that global sales of Cardiac Resynchronization Therapy in 2021 were held atUS$ 5.7 Bn. With7.9%, the projected market growth during 2022-2032 is expected to be slightly lower than the historical growth. CRT-Defibrillator is expected to be the higher revenue-generating product, accounting for an absolute dollar opportunity of nearlyUS$ 4 Bnduring 2022 2032.

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Rise In Number Of CROS In Various Regions Such As Europe Is Expected To Fuel The Growth Of Induced Pluripotent Stem Cell Market At An Impressive CAGR...

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Healthy fasting is therapeutic if appropriately done, and evidence supports this. Our body can cure itself if given the correct nourishment, movement, sleep, emotional wellness, and surroundings; fasting boosts its curing capabilities. Its vital for holistic health.

It has beneficial effects on physical, emotional, brain, and spiritual health. In fact, it exists as a practice in most religions (religious fasting). For example, Muslims reduce caloric intake for a period of time during Ramadan to cleanse the mind, body, and soul. Other religious fasts include Christians, Greek Orthodox Christians, Jews, Hindus, and Buddhists, reducing caloric intake on certain days of the week or year.

Fasting has been performed for millennia with favorable effects, but only lately have studies shown its significance in adaptive cellular responses that minimize oxidative damage and inflammation, optimize energy metabolism and heart health, and bolster cellular defense. Furthermore, it helps with weight loss because it depletes liver glycogen, causing lipolysis and ketone body production, which reduces body fat (fat percentage) and hip circumference.

Fasting is such a popular scientific research topic today that the number of these studies demonstrating how good it is for holistic health keeps growing. The outcomes of these studies show that it can make you smarter, increase longevity by slowing down the aging process, and heal diseases, digestive issues, neurodegenerative disorders, and neurological disorders (mood disorders). Other health effects include the prevention of cardiovascular disease and chronic diseases.

Fasting activates our inner intelligence via calorie restriction. Its straightforward science. Fasting lets the digestive system rest by halting calorie intake. This break saves energy that would have gone toward digesting food. This conserved energy is used for repair, recovery, development, rejuvenation, and healing, which are needed for curing every human disease.

What happens first when were sick? Reduced appetite. So, what does this tell us? Our body reduces appetite to save energy that would have gone to digestion for mending and repair instead. Fasting does the same thing. It activates good genes with protective mechanisms, such as the SIRT1 gene, which regulates longevity, inflammation, fat and glucose metabolism, and other health effects.

A PLOS One study found that fasting reduces hunger hormones, improves metabolism, and helps people lose weight. Chicago researchers tested intermittent fasting on 20 obese adults for eight weeks. It enhanced the participants insulin resistance and glucose regulation, reduced cravings, and increased the feeling of fullness. Furthermore, they felt better overall and experienced no side effects.

Most people today overeat by incessantly munching and nibbling. Constant and excessive eating and out-of-balance dietary intake can overload the digestive system, leading to illness and a majority of health-related problems. Fasting helps mend this damage.

Chronic fasting (long-term fasting) enhances the lower eukaryote lifetime by altering metabolic and stress resistance pathways. Intermittent fasting (short-term fasting) protects against diabetes, malignancies, heart disease, neurodegeneration, obesity, hypertension, asthma, and rheumatoid arthritis.

Most people fast by only drinking water, dubbed water fasting. Other versions include juice fasting (apple cider vinegar, lemonade, carrot juice, celery juice, etc.) and eating light, where participants primarily eat vegetables, fruits, and lean meats like fish and chicken. However, real fasting involves going without food, solid, and liquid (aside from water) for at least 12 hours.

Several variations exist. Sometimes spiritual disciplines like prayer and meditation are included, turning it into a ritual. These disciplines make the process easier by calming the psyche.

As mentioned, various methods (diets) exist; all deliver positive effects. Here are a few examples:

This is the most common style of fasting and the most accurate form. Except for water, no solids or liquids are consumed. For those doing an extended water fast (over three days), sometimes herbal teas, tonics, and broths are consumedbut absolutely no caffeine or alcohol.

People following this diet will only drink vegetable and fruit juices for the duration of the fast.

This variation allows anything liquid, like broth or pureed soups, smoothies, and juices.

Its odd to call this one a fast because you can eat. Nevertheless, this diet is for people looking to purify their bodies. They must eliminate all non-plant-based foods (only things like fruits, vegetables, nuts, seeds, and legumes are allowed).

Skipping meals regularly, known as intermittent fasting or partial fasting, is becoming increasingly popular worldwide. People realize its physical and mental health benefits. It enhances energy, moods, sleep, and sex life. However, it involves a set daily fasting time.

Intermittent fasting also has the following benefits:

There are over twenty variations of intermittent fasting. The most popular include:

This strategy entails daily periods of fastingof 18 hours and then eating a light meal every other day. On alternate days you can eat healthy things like vegetables, berries, nuts, lean protein, etc.

Every day, you consume within specific periods of time. For example, your daily fast may be limited to eating from midday to 8:00 p.m..

You follow a schedule of regular eating for five days, then two days of fasting (preferably water fasting).

This fast allows one meal a day, but not breakfast. It is also commonly referred to as the One Meal a Day diet (OMAD).

You designate a six-hour window per day in which you can eat.

Most people fast to shed weight, regulate blood sugar, cleanse themselves of toxins, or regain mental clarity and emotional stability. However, it is a difficult thing to do alone. For those that need a little motivation, inspiration, and guidance, there are many fasting or detox retreats worldwide.

In addition, a growing number of medical clinics are offering guided fasting treatments. During these rehabilitation sessions, physicians supervise patients while undertaking water-only or very low-calorie (less than 200 kcal/day) fasting periods of one week or more. People participate for help in weight management or disease treatment and prevention.

Mexico has fasting pods, aka Fast incubators. These locations surround individuals with nature and block out food odors and noise. One can fast for 10 to 30 days. As a result, various disorders have reportedly healed faster. Many even experience improved eyesight and hearing.

While fasting is a simple concept, it can perplex many people due to the abundance of claims, methods, and precautions floating around the internet. However, it does not have to be challenging. On the contrary, it should be second nature to us.

Circadian rhythm fasting is the most natural and realistic technique to fast. In laymans terms, sunset to sunrise fasting involves eating ones last meal of the day early (near to or with sundown) and breaking it after sunrise. This provides for a minimum of 12-hour fasting and is one of the most efficient strategies to incorporate the practice into your lifestyle.

If you are still not hungry after 12 hours, gently extend your fast until you experience actual physical hunger, and then break youre fast correctly. You are not required to have breakfast if you arent hungry. Not feeling hungry in the morning indicates that your body is still detoxifying and processing your evening meal. Respect your body by fasting accordingly.

Fasting while sleeping is ideal since all critical detoxification, repair, and recovery processes occur during deep sleep. Our bodies detoxify at night, and the physical health benefits are more noticeable when fasting.

When you want to break the fast, however, it is entirely up to you and the signs your body is sending. Some people wake up hungry, while others do not till the afternoon. Pay attention to your body. There is a distinct distinction between fasting and starvation. If you are not hungry, respect your hunger and continue your fast for a few more hours.

Breaking a fast gently awakens your digestive system. So, gorging after a fast is terrible. It could overwhelm your stomach. Water breaks a dry fast best. Take a few sips, then eat fruit or 1-2 fresh dates. After 30-40 minutes, cook a wholesome meal. This is particularly important for long fasts.

Some fasters drink tea, coffee, or juice. Acidic drinks can damage stomach linings. Therefore, one should fast appropriately or not at all. If opting for juice fast, stick with vegetable juice like celery, green juice, or non-acidic fruits. Likewise, teas should be caffeine-free and herbal only (lavender, jasmine, etc.).

Theres no one-size-fits-all answer. Some find fasted workouts beneficial, while others find them hazardous. Fasted workouts depend on objectives, energy and hunger levels, training, and health conditions. However, do it if you can because fasted workouts are fantastic for insulin resistance, weight loss, and abdominal fat.

Note: Your body needs time to acclimate to a fast before you experience mental changes. You may get headaches or discomfort early on. Your brain is granted a cleaner bloodstream after your body eliminates toxins. This improves your thoughts, emotions, memory, and other senses.

Fasting causes ketogenesis, promotes potent changes in metabolic pathways and cellular processes such as stress resistance, lipolysis, and autophagy, and can have medical applications that are as effective as approved drugs, such as dampening seizures and seizure-associated brain damage, alleviating rheumatoid arthritis, and maximizing holistic health, as explained in the rest of this page.

Fasting uses up excess carbohydrates. The body burns fat. The metabolic rate rises, unlike with caloric restrictionweight loss results.

Half of our energy goes into digestion. This energy can be used to heal and regenerate, which happens during a fast. The human body recognizes what needs mending.

Sick and weaker cells are killed after 24-36 hours via apoptosis and autophagy, then recycled into new cells. Its natural. Apoptosis kills 50 to 70 billion human cells daily. Fasting boosts this rate.

Stem cell production and activation rise after fasting. The number of new stem cells and HGH peak during days 3-5 of a fast, then fall. Additional research shows that new white blood cells are created with increased stem cell growth, boosting the immune system.

Besides fat burning and strengthening the immune system, it reduces inflammation, rebalances the gut microbiome and hormones, protects the brain from neurological diseases, reduces cancer risk, slows aging, and promotes cell maintenance and repair.

Fasting is the best medicine, and its free!

Fasting has many powerful benefits, but its not for everyone. It should be avoided or done only under medical supervision in the following situations. People who are:

If you think you can do it, go for it! Fasting is the bodys natural stem cell therapy, renewing and regenerating the body. It is the ultimate biohack. Theres no better method to restore cells, improve healing, and increase energy and focus.

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Discover the Mental and Physical Health Benefits of Fasting - Intelligent Living

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