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Study in mouse, human cells suggests unique anti-cancer properties of such a therapy

Pictured is a natural killer (NK) cell that researchers developed in the lab from human pluripotent stem cells. These NK cells mimic the properties of those found in the yolk sac during the earliest stages of development. Such NK cells may be more effective as immunotherapy for cancer treatment than adult NK cells that come from bone marrow, according to a new study from Washington University School of Medicine in St. Louis. White arrows point out granules that contain potent anti-cancer enzymes. Adult NK cells have very few of these granules.

Immunotherapy that involves treating cancer with the bodys own immune cells, or those of a matched donor, shows promise in clinical trials for some patients, but not all.

A new study from Washington University School of Medicine in St. Louis suggests that the age of certain immune cells used in such therapy plays a role in how effective the immunotherapy is. These cells natural killer (NK) cells appear to be more effective the earlier they are in development, opening the door to the possibility of an immunotherapy that would not utilize cells from the patient or a matched donor. Instead, they could be developed from existing supplies of what are called human pluripotent stem cells.

We are trying to improve the effectiveness of immunotherapy for more patients, said senior author Christopher M. Sturgeon, PhD, an assistant professor of medicine. This special source of natural killer cells has the potential to fill some of the gaps remaining with adult NK cell therapy. There is early evidence that they are more consistent in their effectiveness, and we would not need to process cells from a donor or the patient. They could be manufactured from existing cell supplies following the strict federal guidelines for good manufacturing practices. The characteristics of these cells let us envision a supply of them ready to pull off the shelf whenever a patient needs them.

Unlike the adult versions of NK cells used in most investigational therapies, earlier versions of such cells do not originate from bone marrow. Rather, these NK cells are a special type of short-lived immune cell that forms in the yolk sac of the early mammalian embryo. But for therapeutic purposes, such cells do not need to originate from embryos they can be developed from human pluripotent stem cells, which have the ability to give rise to many different cell types, including these specialized natural killer cells. Manufacturing such cells which many academic medical centers already have the ability to do would make them available quickly, eliminating the time needed to process the patients or donors cells, which can take weeks.

The study appears March 19 in the journal Developmental Cell.

Before a certain time point in early development, there is no such thing as bone marrow, but there is still blood being made in the embryo, Sturgeon said. Its a transient wave of blood that the yolk sac makes to keep the embryo going until bone marrow starts to form. And thats the blood cell generation thats making these unique natural killer cells. This early blood appears to be capable of things that adult blood simply cant do.

Studying mouse and human induced pluripotent stem cells that have been coaxed into forming these unique NK cells, the researchers showed that the NK cells are better at releasing specific anti-tumor chemicals a process called degranulation than their adult counterparts. Even NK cells derived from umbilical cord blood do not respond as robustly. NK cells of adult origin also release different chemicals that trigger harmful inflammation, but this response is not necessarily effective against cancer.

Past work by other groups suggested NK cells from earlier development might be more effective, but how and why this was the case remained unknown. The specific origin of these cells was also a mystery.

Now we know where these special natural killer cells come from and that we can never get them from an adult donor, only a pluripotent stem cell, Sturgeon said. Based on their unique behavior alone, there is one small clinical trial of these cells that is ongoing. Now that we know how to manufacture them and how they work, it opens the door for more trials and for improving upon their function.

According to Sturgeon, such cells could be produced from existing lines of pluripotent stem cells that would not need to come from a matched donor because, in general, NK cells do not heavily attack the bodys healthy tissues, as many T cell therapies can. T cells are another type of immune cell often used to treat blood cancer as part of a stem cell transplant, commonly called a bone marrow transplant. Even when NK cells do cause harm, they do not stay in the body for long periods of time.

From a basic science standpoint, Sturgeon also is interested in understanding why these cells are present in the early embryo in the first place and where they go in later development and after birth.

We can only speculate at this point, but its possible that during early embryonic development, when there is so much rapid cell division, these cells are there as a surveillance mechanism to protect against pediatric cancers or infection, he said.

This work was supported by the National Institutes of Health (NIH), grant numbers HL007088-41, R01DK09361, R01CA205239, P50CA171963, 5K12CA167540, and UL1TR002345; an American Society of Hematology Scholar Award; the University of Rochester; the American Cancer Society, grant number IRG-58-010-59-2; the Washington University Center of Regenerative Medicine; the technical expertise of Leah Vit; and the M. Napoleon Memorial Foundation. Electron microscopy was performed at the Washington University Center for Cellular Imaging (WUCCI). Transcriptome analyses were performed at the Genome Technology Access Center (GTAC).

Dege C, Fegan KH, Creamer JP, Berrien-Elliott MM, Luff SA, Kim D, Wagner JA, Kingsley PD, McGrath KE, Fehniger TA, Palis J, Sturgeon CM. Potently cytotoxic natural killer cells initially emerge from erythro-myeloid progenitors during mammalian development. Developmental Cell. March 19, 2020.

Washington University School of Medicines 1,500 faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Childrens hospitals. The School of Medicine is a leader in medical research, teaching and patient care, ranking among the top 10 medical schools in the nation by U.S. News & World Report. Through its affiliations with Barnes-Jewish and St. Louis Childrens hospitals, the School of Medicine is linked to BJC HealthCare.

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Immunotherapy using 'young cells' offers promising option against cancer - Washington University School of Medicine in St. Louis

Abstract

Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatographymass spectrometry (LC-MS)based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

The development of extracellular matrix (ECM)based biomaterials as surgical meshes, topical powders, and injectable hydrogels is a relatively recent pursuit compared to similar efforts with synthetic biomaterials (1). The evolutionarily conserved composition of the ECM, and its critical role in development, cell, tissue, and organ homeostasis, and the response to tissue injury provide a compelling rationale for its use as an inductive biomaterial to promote the repair of damaged tissues and organs (2). However, gaps in our understanding of the dynamic biophysical properties and composition of ECM limit our ability to design and fabricate ECM-based biomaterials that fully capitalize upon their therapeutic potential. Although advanced proteomic techniques have begun to provide an accurate profile of ECM fibrillar and soluble protein components (2), the effects of constitutive (phospho)lipids and incorporated (extracellular) RNA on matrix biology and the host tissue response following injury are largely unknown.

Extracellular vesicles (EVs) are nanometer-sized vesicles encapsulated by a membrane, which transport cell signaling molecules including microRNA (miRNA), (phospho)lipids, and proteins (3). As a potent mediator of cell signaling, EVs have been the subject of intense investigation and thought to be secreted exclusively into a liquid phase where they can be relocated freely between cells and to distant sites using biologic fluids as a mobile medium. Recently, the presence of EV firmly embedded within the ECM, termed matrix-bound nanovesicles (MBVs), was described (4). In contrast to liquid-phase EV, MBVs are secreted by tissue-resident cells and integrated into the fibrillar matrix, a feature that may define their biological purpose and mechanism of action. The nature and biologic significance of the MBV cargo and lipid membrane has not been characterized. Given that MBVs are integrated within the matrix, it is plausible that the molecular speciation of their constituent phospholipids may facilitate this interaction.

The objective of the present study is to identify similarities and differences between liquid-phase (i.e., exosomes) and matrix-bound forms (i.e., MBV) of EV. However, given that EV present in biological fluids, MBV present in native tissue ECM, and ECM-based biomaterials represent heterologous populations secreted from multiple cell sources, a direct comparative in vivo analysis between these putative EV populations is problematic. As an alternative to using body fluid or tissue-derived vesicles, ECM and conditioned medium produced in vitro by cultured cells can be isolated (5). This approach offers several advantages such as the use of a single cell type source, thereby obviating any doubts regarding vesicle origin, the ability to selectively harvest vesicles from either liquid- or solid-phase compartments, and the ability to control the cell culture environment and thus also control vesicle composition and cargo.

Here, we use a fibroblast cell culture model that allows selective harvesting of liquid-phase EV and MBV integrated into the matrix. We conduct RNA sequencing and bioinformatic analysis to characterize the differential miRNA signature and use liquid chromatographymass spectrometry (LC-MS)based lipidomics and redox lipidomics protocols to perform detailed analysis of molecular speciation of liquid-phase EV and MBV phospholipids. Results from these comparative material analyses show that MBVs represent a subpopulation of nanovesicle distinct from EV found in a liquid phase and significantly expand our understanding of EV and matrix biology. Separately, the use of ECM-based therapies can now be examined by new perspectives, and MBV can assist in the design of next-generation ECM-based materials. For example, given their nanometer size, MBV can be used in minimally invasive applications that are otherwise untenable for decellularized ECM scaffolds from which MBVs are derived, such as intravitreal injections to prevent ischemia-induced retinal ganglion cell axon degeneration (6), or incorporated into existing ECM-based devices for sustainable release of vesicles (7, 8). Furthermore, results of the present study may aid in the future development of EV-based theranostic biomaterials such as artificial EV for clinical use (9).

We used scanning electron microscopy (SEM) to provide high-resolution, high-magnification imaging of MBV embedded within an ECM bioscaffold derived from porcine urinary bladder matrix (UBM). SEM images revealed discrete spheres approximately 100 nm in diameter dispersed throughout the collagen fibers (Fig. 1A). Given their compartmentalization within ECM scaffolds, we hypothesized that MBVs deposited into a solid ECM substrate are a unique class of EV separate from EV secreted into a liquid phase. To test this hypothesis, we used an in vitro 3T3 fibroblast cell culture model that allows selective harvesting of vesicles from a liquid-phase or solid-phase extracellular compartment (Fig. 1B). Representative images from phase-contrast microscopy, and hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindolestained sections showed that no residual cells or intact nuclei were visible after decellularization of the cell culture plate (Fig. 1C). Transmission electron microscopy (TEM) imaging of liquid-phase EV harvested from the cell culture supernatant (Fig. 1D) and MBV isolated from decellularized ECM (Fig. 1E) showed that these two populations of vesicles shared a similar morphology. Moreover, nanoparticle tracking analysis (NTA) distribution plots showed similar vesicle size of both liquid-phase EV and MBV, with the majority of vesicles having a diameter of <200 nm (Fig. 1F). To determine whether MBV contained markers commonly attributed to exosomes, immunoblot analysis was performed for CD63, CD81, CD9, and Hsp70 (10). Results showed that, in contrast to liquid-phase EV, the MBV showed a marked decrease in CD63, CD81, and CD9 (Fig. 1G). Furthermore, silver staining of electrophoretically separated proteins showed that MBV contained a protein cargo that was distinctly different than the liquid-phase EV (Fig. 1H), suggesting that MBV may be a unique subpopulation of nanovesicle.

(A) SEM images of an ECM scaffold derived from urinary bladder matrix (UBM) showing discrete spherical bodies approximately 100 nm in diameter dispersed throughout the matrix. Scale bars, 1 m. (B) Illustration of the 3T3 fibroblast cell culture model used to selectively harvest vesicles from a liquid-phase or solid-phase extracellular compartment. (C) Phase-contrast microscopy, hematoxylin and eosin (H&E) staining, and 4,6-diamidino-2-phenylindole (DAPI) staining showing the absence of cells and intact cell nuclei after decellularization. (D and E) TEM of liquid-phase EV (D) and MBV (E) isolated from the 3T3 fibroblast cell culture model. Scale bars, 100 nm. (F) Size distribution plots from nanoparticle tracking analysis (NTA) of liquid-phase EV (top) and MBV (bottom) isolates from the 3T3 fibroblast cell culture. (G) Immunoblot analysis of CD9, CD63, CD81, and Hsp70 expression levels in liquid-phase EV and MBV. (H) Silverstain analysis of electrophoretically separated proteins in liquid-phase EV and MBV. M.W., molecular weight.

We used comprehensive next-generation RNA-sequencing (RNA-seq) to catalog differentially expressed miRNA in MBV and liquid-phase EV relative to the 3T3 fibroblast parent cell from which these vesicles were derived. Bioanalyzer analysis revealed the absence of 18S and 28S ribosomal RNA, and an enrichment of small RNA molecules [<200 nucleotides (nt)] in total RNA isolated from liquid-phase EV and MBV. However, the small RNA size distribution from liquid-phase EV was much broader than MBV with a marked enrichment of small RNA molecules between 100 and 200 nt in liquid-phase EV (Fig. 2A). We focused the analysis on differential miRNA signatures by conducting next-generation sequencing of miRNA libraries generated from the parental cellular RNA, the liquid-phase EV, and the MBV isolates (n = 3 per group). Principal components analysis (PCA) showed that within respective groups, the replicate miRNA profiles clustered close to one another (Fig. 2B). However, extensive differences in miRNA content were observed between the parental cell and the liquid-phase EV and MBV isolates. Overall, 28 (50.91%) miRNAs were found to be differentially expressed in MBV compared to liquid-phase EV by at least twofold (Fig. 2C). In addition, respective liquid-phase EV or MBV and the parental cellular miRNA profiles were clearly distinct (Fig. 2, B and C). To validate the results of miRNA sequencing, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to detect three up-regulated miRNAs (miR-163-5p, miR-27a-5p, and miR-92a-1-5p) and three down-regulated miRNAs (miR-451a, miR-93b-5p, miR-99b-5p) in MBV compared to liquid-phase EV isolated from 3T3 fibroblasts (Fig. 2D). As anticipated, the results showed that the level of miR-163-5p, miR-27a-5p, and miR-92a-1-5p was up-regulated, and miR-451a, miR-93b-5p, and miR-99b-5p was down-regulated in MBV compared to liquid-phase EV, thereby corroborating the results from the miRNA sequencing data. Ingenuity Pathway Analysis (IPA) of differentially enriched miRNAs in MBV compared to liquid-phase EV showed a strong association with organ and system development and function. In contrast, miRNAs differentially enriched in liquid-phase EV compared to MBV were associated with pathways involved in cellular growth, development, proliferation, and morphology (Fig. 2E).

(A) Bioanalyzer analysis of total RNA isolated from 3T3 parental cells and their secreted liquid-phase EV and MBV. (B) Principal components analysis (PCA) comparing liquid-phase EV (green), MBV (blue), and cellular (red) RNA-seq datasets. (C) Volcano plot showing the differential expression of miRNAs in liquid-phase EV, MBV, and the parental cells. The inclusion criteria were a twofold difference of log2 (fold change) in either direction with a P value of <0.05. Each dot represents a specific miRNA transcript; green dots to the right of the vertical dashed line correspond to a relative increase in expression level, and red dots to the left correspond to a relative decrease in expression level. Blue dots indicate miRNA with no significant change in expression level. (D) RT-qPCR validation of the results of miRNA sequencing. *P < 0.05, n = 4. (E) IPA functional analysis. Significantly enriched molecular functions identified by IPA functional analysis considering differentially expressed miRNA in MBV (red) and liquid-phase EV (blue).

Results with the 3T3 fibroblast cell model showed selective packaging of miRNA within MBV deposited in the ECM compared to liquid-phase EV secreted into the cell culture supernatant. We next sought to determine whether MBV miRNA cargo is unique to the cellular origin. We characterized and compared the miRNA composition of MBV isolated from ECM produced in vitro by bone marrowderived stem cells (BMSCs), adipose stem cells (ASCs), and umbilical cord stem cells (UCSCs) isolated from different human donors using next-generation sequencing methods. A representative phase-contrast microscopy image of a decellularized BMSC cell culture plate showed the absence of cells and the presence of branched fibrillar structures (Fig. 3A). TEM imaging of isolated MBV from a decellularized BMSC cell culture plate showed the characteristic morphology attributed to EV (Fig. 3B). Furthermore, NTA showed similar distribution plots between BMSC-, ASC-, and UCSC-derived MBV, with the majority of vesicles having a diameter of <200 nm (Fig. 3, C to E). After isolation of total RNA from these samples, bioanalyzer analysis showed the absence of ribosomal RNA and an enrichment of small RNA molecules (<200 nt) (Fig. 3F). miRNA libraries were generated from the samples (BMSC, n = 3 human donors; ASC, n = 3 human donors; UCSC, n = 3 human donors) and subjected to miRNA sequencing. A PCA showed that samples clustered primarily by the cell type from which they were derived (Fig. 3G). Of note, despite the use of three separate human donors for each cell type used to generate the MBV samples, the PCA showed a high degree of homogeneity in the miRNA profile within the respective groups (Fig. 3G). In addition, volcano plots showed that fewer miRNAs were found differentially expressed between BMSC- and UCSC-derived MBV than between BMSC-ASC and UCSC-ASC.

(A) Phase-contrast microscopy image of a decellularized BMSC cell culture plate showing the absence of cells. (B) TEM of MBV isolated from the decellularized BMSC culture plate. Scale bars, 100 nm. (C to E) Size distribution plots from NTA of MBV isolated from BMSC (C), ASC (D), and UCSC (D) decellularized culture plates. (F) Bioanalyzer analysis of total RNA isolated from BMSC-, ASC-, and UCSC-derived MBV. (G) PCA comparing BMSC MBV (green), UCSC MBV (blue), and ASC MBV (red) RNA-seq datasets. (H) Volcano plot showing the differential expression of miRNAs in BMSC-, ASC-, and UCSC-derived MBV. The inclusion criterion was a twofold difference of log2 (fold change) in either direction with a P value of <0.05. Each dot represents a specific miRNA transcript; green dots to the right of the vertical dashed line correspond to a relative increase in expression level, and red dots to the left correspond to a relative decrease in expression level. Blue dots indicate miRNA with no significant change in expression level.

Several studies have characterized the lipid composition of EV (11). However, there are no data on phospholipid composition of MBV. Therefore, we performed LC-MSbased global lipidomics and redox lipidomics analyses to comparatively evaluate the phospholipid composition of MBV and liquid-phase EV compared to their 3T3 fibroblast parent cells (Fig. 4, A and D). Nine major phospholipid classes were detected in all three types of samples, with the total number of detected molecular species of 536 distributed between the following major classes: bis-monoacylglycerophosphate (BMP), 59 species; phosphatidylglycerol (PG), 37 species; cardiolipin (CL), 117 species; phosphatidylinositol (PI), 33 species; phosphatidylethanolamine (PE), 102 species; phosphatidylserine (PS), 45 species; phosphatidic acid (PA), 26 species; phosphatidylcholine (PC), 107 species; and sphingomyelin (SM), 10 species (Fig. 4D). In terms of their content of polyunsaturated fatty acid (PUFA) residues, PE, PI, PC, and PS represented the major reservoir of these polyunsaturated phospholipid species containing four to seven double bonds (Fig. 4B). These PUFA phospholipids represent the likely precursors of the signaling lipid mediators. The formation of the mediators occurs via the catalytic oxygenation of PUFA phospholipids by 5-lipoxygenase or 15-lipoxygenase to yield oxygenated phospholipids that are subsequently hydrolyzed by one of specialized phospholipases A2 to release oxygenated fatty acids, i.e., lipid mediators (12, 13). In addition, oxidized PUFA phospholipids act as signaling molecules coordinating many intracellular processes and cell responses, including apoptosis, ferroptosis, and inflammation (14). We found significant differences in molecular speciation of these phospholipids and their relative contents between liquid-phase EV and MBV (Fig. 4E). With a notable exception of SM, arachidonic acid (AA) and docosahexaenoic acid (DHA) residues were detected in all phospholipids (Fig. 4E). For many of the phospholipids, the amounts were significantly higher in MBV versus liquid-phase EV and parent cells (Fig. 4E), which identify MBV as a rich reservoir of PUFA phospholipids. PUFA phospholipids can be hydrolyzed by phospholipase A2 (PLA2), resulting in the release of free PUFA and lysophospholipids (LPLs) (15). The former can be further used by two major oxygenases, cyclooxygenase (COX) and lipoxygenase (LOX), to produce lipid mediators with pro- or anti-inflammatory capacities (13, 16, 17). This finding qualifies MBV as potential precursors for synthesis of these lipid mediators dependently on the cell/tissue context (13). Quantitatively, MBVs were enriched in PI, PS, PG, and BMP (Fig. 4C and table S1). In contrast, the content of PE, PA, and SM was higher in liquid-phase EV. PC was a predominant phospholipid in cells and liquid-phase EV. The content of a unique mitochondrial phospholipid, CL, was significantly lower in liquid-phase EV compared to MBV and parent cells (Fig. 4F). Because CL is a unique mitochondria-specific phospholipid localized predominantly in the inner mitochondrial membrane (18), this finding represents a possible link of the MBV biogenesis with the mitochondrial compartment of cells. Plasmalogen phospholipids (or ether phospholipids) are structurally different from diacyl phospholipids (or ester phospholipids) (19). In plasmalogens, vinyl ether bond is linking the sn-1 saturated or monounsaturated chain to the glycerol backbone of phospholipids (19). It has been shown that ether lipids, PE, and PC plasmalogens can facilitate membrane fusion (20) and increase membrane thickness of EV (21, 22) and therefore may play a role in nanovesicle uptake by cells. Detailed MS/MS analysis showed a high level of ether PE and PC species (plasmalogens) in both liquid-phase EV and MBV. These species were identified as PE-16:0p/20:4, PE-16:1p/20:4, PE-18:1p/20:4, PE-18:1p/22:6 and PC-16:0p/20:4, PC-18:0p/20:4, PC-20:0p/20:4, PC-18:0p/22:6, respectively (Fig. 4E).

(A) Typical total ion chromatogram of phospholipids obtained from MBV. (B) Mass spectra of the major phospholipid classes in MBV. Quantitative assessment of saturated (double bond number = 0), monounsaturated (double bond number = 1), and polyunsaturated (double bond number = 2 to 10) species of phospholipids. (C) Pie plots showing the total content of major phospholipids. Data are presented as percentage of total phospholipids. (D to F) Contents of different phospholipid molecular species. Data are presented as heat maps, autoscaled to z scores, and coded blue (low values) to red (high values). EV, exosomal vesicles; MBV, matrix-bound vesicles; PC, phosphatidylcholine; PCd, PC diacyl species; PCp, PC plasmalogens; PE, phosphatidylethanolamine; PEd, PE diacyl species; PEp, PE plasmalogens; PI, phosphatidylinositol; PS, phosphatidylserine; BMP, bis-monoacylglycerophosphate; PA, phosphatidic acid; PG, phosphatidylglycerol; SM, sphingomyelin.

LPLs, hydrolytic metabolites of phospholipids created by phospholipases A, are bioactive signaling molecules that modulate a variety of physiological responses, including macrophage activation (23), inflammation and fibrosis (24), tissue repair and remodeling (25), and wound healing (26). LC-MS analysis showed that LPLs were present in all three types of samples, albeit with their total content in MBV and liquid-phase EV being 1.7 to 1.8 times greater compared to the parent cells. More specifically, seven classes of LPL have been identified: lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphoinositol (LPI), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), and monolysocardiolipin (mCL) (Fig. 5A). MBVs were enriched in LPE, LPA, and LPG compared to parent cells (Fig. 5B). The content of LPI and mCL was significantly lower in MBV and liquid-phase EV versus cells. We found that the contents of LPA and LPG were significantly higher in MBV compared to EV. The levels of mCL and LPI in MBV were 3 and 6.3 times higher than in EV but 3.3 and 1.9 times lower compared to cells (Fig. 5, C and D). No significant changes in the contents of LPE, LPC, and LPS between MBV and EV were found. The non-oxidizable molecular species containing 16:0, 16:1, 18:0, and 18:1 were the major types found in all LPL species detected (Fig. 5C). As LPL may act as fusogenic lipids facilitating the transfer of the vesicular contents into cellular targets, this important role of LPL found in MBV should be further explored.

(A) Typical mass spectra of major LPL obtained from MBV. (B) Pie plots showing the total content of major LPL. Data are presented as percentage of total LPL. (C and D) Contents of LPL molecular species. Data are presented as heat maps, autoscaled to z scores, and coded blue (low values) to red (high values). N = 3. LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; LPA, lysophosphatidic acid; LPG, lysophosphatidylglycerol; mCL, monolysocardiolipin.

Given that exposure of murine bone marrowderived macrophages to MBV results in expression of M2-like markers, Fizz1 and Arg1, which are associated with a constructive macrophage phenotype (4), we performed LC-MS analysis of PUFA and their oxygenated products in MBV versus liquid-phase EV and parent cells. MBVs were strongly enriched in AA (20:4), DHA (22:6), and docosapentaenoic fatty acids (DPA; 22:5) (Fig. 6A). In other words, MBVs represent a reservoir of substrates for the biosynthesis of signaling lipid mediators by the respective enzymatic mechanisms, COXs and LOXs. In liquid-phase EV, the major PUFAs were linoleic (18:2) and linolenic (18:3) acids (Fig. 6A).

(A and B) Content of free PUFA (A) and their oxygenated metabolites (B) in parent cell, liquid-phase EV, and MBV. Data are means SD. *P < 0.05 versus cells or MBV. N = 3. (C) Contents of singly, doubly, and triply oxygenated phospholipid species in parent cells, liquid-phase EV, and MBV. Data are presented as heat maps, autoscaled to z scores, and coded blue (low values) to red (high values). PL, phospholipids; CL, cardiolipin.

As EVs contain enzymatic machinery for biosynthesis of AA-derived lipid mediators (27), redox lipidomics analysis of oxygenated fatty acids was performed. Higher levels of AA metabolites such as 12-HETE (12-hydroxy-eicosatetraenoic acid), 15-HETE (15-hydroxy-eicosatetraenoic acid), and lipoxin A4 (LXA4) were found in liquid-phase EV versus MBV (Fig. 6B). In the context of tissue repair, LXA4 and D-series resolvin D1 (RvD1), produced by 12/15-LOX from AA (20:4) and DHA (22:6), stimulate macrophage activation to the M2-like phenotype (28). Last, we characterized oxidized phospholipids containing oxygenated AA and DHA in MBV and liquid-phase EV. The levels of oxygenated species were higher in MBV than in liquid-phase EV, where PS, PI, and PC were represented by mono-oxygenated species. BMP, PG, and CL contained singly and doubly oxygenated AA and DHA residues; triply oxygenated PUFAs were found only in PE (Fig. 6C). Overall, lipidomics and oxidative lipidomics results show that the levels of free AA, DHA, and DPA and PUFA-containing phospholipids as well as their oxidatively modified molecular species are higher in MBV than those in liquid-phase EV.

To date, research regarding EV has been largely focused on their presence in biological fluids and their potential as biomarkers of disease, with lesser emphasis upon their therapeutic potential. These liquid-phase EVs are broadly categorized based on their biogenesis, morphology, density, or cargo (29). Although it has been shown that liquid-phase EVs contain surface proteins that can mediate binding with cells and ECM molecules (30), the integration of MBV within the fibrillar network of the ECM and trafficking of MBV across all cell membranes is largely unexplored. Traditional in vitro cell culture models neglect to differentiate the liquid-phase EV secreted into the cell culture supernatant from the MBV embedded within the ECM. Here, we fractionated vesicle populations based on their compartmentalization into either the liquid-phase cell culture medium or the solid-phase ECM substrate. In terms of composition, we found that MBV isolated from the ECM of 3T3 fibroblasts contained a differential miRNA and lipid signature compared with liquid-phase EV and with the parent cell. These data are suggestive of a scenario in which molecular sorting occurs during vesicle biogenesis to specifically distribute miRNA and lipids to vesicles destined for different extracellular locations. Moreover, the cells capacity to differentiate between a liquid interface and a solid substrate and to selectively deposit tailored subpopulations of vesicles with distinct lipid signatures into these disparate compartments provides evidence for a different and independent membrane biogenesis of MBV from the biogenesis of EV secreted into a liquid phase. Considering that MBVs were shown to be integrated within the dense fibrillar network of the ECM, it is plausible that MBVs are secreted by cells in concert with ECM components during matrix deposition, tissue development and homeostasis, and dynamic matrix remodeling following injury. Furthermore, given that the ECM is a complex mixture of proteins, proteoglycans, and glycosaminoglycans arranged in a tissue-specific three-dimensional architecture (2), it is logical that MBV cargo and lipid content are also unique to the tissue and cellular origin. We have previously shown that MBVs isolated from ECM bioscaffolds derived from anatomically distinct source tissue have differential miRNA signatures (4). Results from the present study further support this hypothesis in that MBV isolated from ECM produced in vitro by BMSC, ASC, and UCSC derived from different human donors contained a distinctive miRNA signature specific to the cell source. In addition, fewer miRNAs were found differentially expressed between BMSC- and UCSC-derived MBV than between BMSC-ASC and UCSC-ASC, a finding that may be attributed to tissue-specific differentiation potentials of ASC (31). These findings further underline the cell-specific features of MBV miRNA profiles, which were not significantly affected by the intrinsic variability of donors. However, given that the three human donors were all male, further studies to determine sex-related variations in the miRNA cargo of MBV from the stem cell samples are warranted. Gender-specific differential expression of exosomal miRNA has been observed in human subjects (32). Gender variation is just one aspect for further investigation since other variables including age (33) and disease state (34) have been shown to affect EV miRNA cargo. PCA showed a high degree of batch-to-batch consistency of the miRNA cargo from MBV deposited by specific cell types isolated from different human donors, a finding that has important implications for MBV and ECM biomaterial manufacturing for use as research tools or clinical therapeutics. For example, a major challenge in the production of EV or ECM biomaterials for preclinical or clinical use is the standardization of product characterization to meet regulatory requirements for batch consistency and for reproducibility of the manufacturing process (35, 36). Stated differently, the observed batch-to-batch consistency in miRNA cargo from MBV deposited by specific cell types may lend itself to the development of markers to verify identity and purity of MBV intended for clinical testing. Although the present study establishes that MBV integrated into the matrix are a unique subpopulation of EV, we cannot rule out the possibility of heterogeneity within the MBV subset. A similar heterogeneity has recently been described for EV secreted into cell culture medium by mesenchymal stem cells, which were shown to secrete at least three types of liquid-phase EV that could be differentially isolated based on their affinities for membrane lipidbinding ligands (37). In addition, although MBV showed a marked decrease in proteins commonly attributed to exosomes (e.g., CD63, CD81, and CD9), future proteomics studies will be required to identify the differential expression of surface and luminal proteins associated with liquid-phase EV compared to MBV.

As the composition and compartmentalization are different for liquid-phase EV and MBV integrated into the matrix, these subpopulations of vesicles are likely to have different biological functions. IPA network analysis of differentially enriched miRNAs in MBV compared to liquid-phase EV isolated from the 3T3 fibroblast model showed that miRNAs in MBV are associated with significant network-associated functions in organ and system development compared to miRNA enriched from liquid-phase EV. MBVs have previously been shown to recapitulate functional and phenotypical properties attributed to the ECM bioscaffolds from which they are derived, including stem cell differentiation and activation of an anti-inflammatory and pro-resolving macrophage phenotype, both of which are hallmarks of constructive tissue remodeling (2).

In contrast to EV that are secreted into body fluids and readily available for cell-cell communication, MBVs embedded within tissue ECM are stably associated with the matrix and can only be isolated following degradation of the ECM material (4). The requirement for matrix degradation to release MBV may partially define their mechanism of action, including those related to their capacity to generate pro-resolving lipid mediators. Because MBVs remain intact and attached to ECM even after decellularization, the molecular speciation of their constituent phospholipids likely plays a role in facilitating such MBV-ECM interactions. Using LC-MSbased lipidomics and redox lipidomics approaches, we performed detailed characterization of the molecular speciation of MBV phospholipids, LPL, and the oxygenated and non-oxygenated PUFA and can speculate upon the relationship of these various molecular species with their utilization as metabolic lipid signaling platforms. We report that high levels of LPL, bioactive molecules that are important for macrophage differentiation, tissue repair, remodeling, and wound healing, are a characteristic feature of MBV. In addition, as fusogenic lipids, LPL can facilitate the transfer of the vesicular contents to intracellular targets. MBVs, but not liquid-phase EVs, were enriched in PUFA non-oxygenated and oxygenated phospholipids and therefore represent a potential reservoir of oxidized and oxidizable esterified phospholipid species, the role of which has not yet been elucidated. Notably, PUFA-enriched MBV can be viewed as an important source of lipid mediators activated by different phospholipases dependent on the pro-/anti-inflammatory context of the extracellular environment.

A limitation of the present study is the use of a single cell line to evaluate differences in liquid-phase EV and MBV cargo. The 3T3 fibroblast cell line used in this study was chosen because it is a well-characterized and widely used cell line in biologic research. Furthermore, results from the RNA-seq and lipidomic analyses showed that multiple replicates of MBV derived from the 3T3 fibroblast model showed a high level of consistency in terms of miRNA and lipid cargo and that this cargo is significantly different from the cargo of the corresponding liquid-phase EV, which supports the fidelity of our results. However, further studies are required with other cell types, including primary cells, before one can derive a more definitive understanding of the biologic purpose of MBV versus liquid-phase EV. In addition, additional studies are warranted to identify and determine the biologic relevance of MBV in native (nondecellularized) tissues.

The findings of the present study may have significant clinical implications. EVs harvested from biological fluids have been used for diagnostic purposes (38), and EVs isolated from cell culture supernatant are being explored as therapeutic agents in early-phase clinical trials (39, 40). However, delineation of the MBV subpopulation may now allow new perspectives on EV-based therapeutics, especially in the design and manufacture of novel biomaterials and artificial EV for clinical use. Given the selective loading of specific miRNA and lipid cargo within MBV, further studies on MBV biogenesis may prove to be instrumental in guiding new strategies for cargo loading of EV (9) or for the incorporation of EV into ECM-based biomaterials to be used as an inductive substrate for tissue repair (7, 8).

The objective of the present study was to conduct a comparative material analysis of EV secreted into a liquid medium versus MBV integrated into the ECM using an in vitro 3T3 fibroblast cell culture model that allows selective harvesting of vesicles from liquid-phase or solid-phase extracellular compartments. 3T3 fibroblasts were seeded on polystyrene plates in the presence of ascorbic acid to induce deposition of ECM (41, 42). After 7 days, the cell culture medium containing liquid-phase EV was harvested, and the culture plates were decellularized to remove cells while maintaining the molecular composition and ultrastructure of the ECM. Following decellularization, MBVs were isolated from decellularized ECM by enzymatic digestion. We used LC-MSbased lipidomics and redox lipidomics to perform detailed analysis of liquid-phase EV and MBV phospholipids and conducted comprehensive RNA-seq and bioinformatic analysis of the intravesicular miRNA cargo.

Human BMSC, human ASC, and human UCSC ECM plates were provided by StemBioSys (San Antonio, TX) and prepared according to a published protocol (43). Briefly, human BMSCs, human ASCs, or human UCSCs were seeded onto a 75-cm2 cell culture flask coated with human fibronectin (1 hour at 37C) at a cell density of 3500 cells/cm2 and cultured in -minimum essential medium (-MEM) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 14 days. The medium was refreshed the day after initial seeding and then every 3 days. At day 7, ascorbic acid 2-phosphate (Sigma-Aldrich) was added to the medium at a final concentration of 50 M. At day 14, plates were decellularized using 0.5% Triton in 20 mM ammonium hydroxide for 5 min and rinsed two times with Hanks balanced salt solution containing both calcium and magnesium (HBSS +/+) and once with ultrapure H2O. Murine NIH 3T3 fibroblast cells were seeded onto a 75-cm2 cell culture flask at a cell density of 3500 cells/cm2 and cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with exosome-depleted FBS (44), 1% penicillin-streptomycin, and ascorbic acid 2-phosphate (Sigma-Aldrich) at a final concentration of 50 M for 7 days. At day 7, the supernatant from cultured 3T3 fibroblast cells was collected, and the culture plates were washed three times with phosphate-buffered saline (PBS), decellularized using 0.5% Triton in 20 mM ammonium hydroxide for 5 min, and then rinsed three times with ultrapure H2O.

MBVs were isolated as previously described with minor modifications (4) . Briefly, the decellularized ECM was enzymatically digested with Liberase DL (100 ng/ml; Roche) in buffer [50 mM tris (pH 7.5), 5 mM CaCl2, and 150 mM NaCl] for 1 hour at 37C. The cell culture supernatant containing the liquid-phase EV and the digested ECM containing the MBV were subjected to differential centrifugation at 500g (10 min), 2500g (20 min), and 10,000g (30 min), and the supernatant was passed through a 0.22-m filter (Millipore). The clarified supernatant containing the liberated MBV or liquid-phase EV was then centrifuged at 100,000g (Beckman Coulter Optima L-90K Ultracentrifuge) at 4C for 70 min to pellet the vesicles. The vesicle pellets were then washed and resuspended in 1 PBS and stored at 20C until further use.

UBM was prepared from market-weight pigs (Tissue Source LLC, Lafayette, IN) as previously described (4). Briefly, the tunica serosa, muscularis externa, submucosa, and muscularis mucosa were removed by mechanical delamination, and the urothelial cells of the tunica mucosa were dissociated from the basement membrane by washing with deionized water. The remaining basement membrane and the lamina propria (collectively referred to as UBM) were decellularized by agitation in 0.1% peracetic acid with 4% ethanol for 2 hours at 300 rpm followed by PBS and type 1 water washes. UBM was then lyophilized and milled using a Wiley Mill with a #60 mesh screen.

UBM was fixed in cold 2.5% glutaraldehyde for 24 hours followed by three 30-min washes in 1 PBS. Samples were then dehydrated in a graded series of alcohol (30, 50, 70, 90, and 100% ethanol) for 30 min per wash and then placed in 100% ethanol overnight at 4C. Samples were washed three additional times in 100% ethanol for 30 min each and critical pointdried using a Leica EM CPD030 critical point dryer (Leica Microsystems, Buffalo Grove, IL, USA) with carbon dioxide as the transitional medium. Samples were then sputter-coated with a 4.5-nm-thick gold/palladium alloy coating using a 108 Auto sputter coater (Cressington Scientific Instruments, UK) and imaged with a JEOL JSM6330F scanning electron microscope (JEOL, Peabody, MA, USA).

TEM imaging was conducted on MBV or liquid-phase EV loaded on carbon-coated grids and fixed in 4% paraformaldehyde as previously described (4). Grids were imaged at 80 kV with a JEOL 1210 TEM with a high-resolution Advanced Microscopy Techniques digital camera. The size of MBV was determined from representative images using JEOL TEM software.

Particle size and concentration of the liquid-phase EV and the MBV were calculated using a NanoSight (NS300) instrument equipped with fast video capture and particle-tracking software. Samples were diluted 1:500 to a final volume of 1000 l using particle-free water. A syringe pump was used to dispense the sample into the system. Measurements were performed from three captures of 45 s each sample. For the video processing and particle calculation, the detection threshold was adjusted to 4. Data are presented as concentration versus particle size for each of the evaluated samples.

Total RNA was isolated from 3T3 cells, liquid-phase EV, and MBV using the RNeasy Mini Kit (Qiagen) according to the manufacturers instructions. Before RNA isolation, liquid-phase EV and MBV samples were treated with ribonuclease A (10 g/ml) at 37C for 30 min to degrade any contaminating RNA. RNA quantity was determined using a NanoDrop spectrophotometer, and its quality was determined by Agilent Bioanalyzer 2100 (Agilent Technologies).

The miRNA library preparation was initiated with 100 ng of each sample and the QIAseq miRNA Library Kit (Qiagen) following the manufacturers instructions. Briefly, mature miRNAs were ligated to adapters on their 3 and 5 ends. The ligated miRNAs were then reverse-transcribed to complementary DNA (cDNA) using a RT primer with unique molecular indices. The cDNA was then cleaned up to remove adapter primers, followed by amplification of the library with a universal forward primer and one of 48 reverse primers that assign a sample index. A presequencing quality control was performed using the Agilent RNA ScreenTape System. Next-generation sequencing was performed on a NextSeq 500 instrument with a loading concentration of 2.5 pM. Bioinformatic analysis was conducted by Genevia Technologies (Tampere, Finland). The quality of the sequencing reads was inspected using FastQC software. TrimGalore! (version 0.4.5) was used to remove the adapter sequences, with default settings, on all the samples. All reads were shortened to 21 bases, the typical size of miRNAs, using the fastx_trimmer software (FASTX-Toolkit by Hannon Lab, version 0.0.14). The reads of each sample were then aligned against the corresponding reference genome (hg38, GRCm38). Tables of miRNA counts across samples were created using the softwares bowtie (version 1.2.2) and miRDeep2 (version 0.0.8). In this process, precursor miRNA and mature miRNA sequences for each species involved in the study were taken from miRbase. Counts of mature miRNAs were obtained by taking the median of all precursor miRNAs associated with them. The counts of mature miRNAs of all samples were normalized using DESeq2. To ensure data quality before further analyses, PCA was performed and the results were visualized using ggplot2, separately for murine and human samples. Normalization of mature miRNA data and statistical testing between sample groups were performed with DESeq2. P values were corrected for multiple testing using the Benjamini-Hochberg method. miRNAs with adjusted P value of <0.05 and absolute log2 fold change >1 were considered as significantly differentially expressed. Tables of differentially expressed miRNAs were annotated with their targets and their confidences using the mirTARbase database of experimentally tested miRNA target interactions. Differentially expressed miRNAs were also annotated with predicted targets using the R package miRNAtap. miRNAtap aggregates the miRNA target predictions from five different databases (PicTar, DIANA, TargetScan, miRanda, and miRDB) and calculates an overall miRNA target score. The minimum amount of database sources required for a potential miRNAtarget interaction to be included into the annotations was 3.

IPA software (version 01-14) was used for functional analysis of differentially expressed miRNAs. miRNA targets were identified using the IPA Core Analysis. The filter was set to Experimentally Observed findings to obtain information about significantly enriched molecular and cellular functions and physiological system development functions that were affected by the miRNAs.

RT and qPCR were performed using the TaqMan Advanced miRNA Assays Protocol (Applied Biosystems). Briefly, 10 ng of total RNA was used with the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, catalog no. A28007) to synthesize and adapt a 3-poly(A) tail to the miRNAs. Universal RT primers recognizing the poly(A) tail were used to synthesize the cDNA in the RT reaction, followed by a miR-AMP step, using miR-AMP forward and reverse universal primers, to increase the number of cDNA molecules. The qPCR was made on a QuantStudio system machine using the TaqMan Fast Advanced Master Mix (Applied Biosystems, catalog no. 4444556) and specific TaqMan Advanced miRNA Assays (Applied Biosystems, catalog no. A25576) recognizing mmu-miR-163-5p, mmu-miR-27a-5p, mmu-miR-92a-1-5p, mmu-miR-451a, mmu-miR-93-5p, and mmu-miR-99b-5p. Fold change expression on the MBV sample was calculated for each of the specific targets using liquid-phase EV as a reference.

Liquid-phase EV and MBV, derived from three separate cultures of 3T3 fibroblasts, were respectively pooled and quantified by nanotracking particle analysis. For both immunoblot and silverstain analysis, an equal number of vesicles for both the liquid-phase EV and MBV samples were loaded onto the gel. MBV or liquid-phase EV (21 1011) was mixed with 2 Laemmli buffer (R&D Systems) containing 5% mercaptoethanol (Sigma-Aldrich), resolved on a 4 to 20% gradient SDSpolyacrylamide gel electrophoresis (Bio-Rad), and then transferred onto a polyvinylidene difluoride membrane. Membranes were incubated overnight with the following primary antibodies: rabbit anti-CD63, rabbit anti-CD81, rabbit anti-CD9, and rabbit anti-Hsp70, at 1:1000 dilution (System Biosciences). Membranes were washed three times for 15 min each before and after they were incubated with goat anti-rabbit secondary antibody, at 1:5000 dilution (System Biosciences). The washed membranes were exposed to chemiluminescent substrate (Bio-Rad) and then visualized using a ChemiDoc Touch instrument (Bio-Rad). Silver staining of gels was performed using the Silver Stain Plus Kit (Bio-Rad) according to the manufacturers instruction and visualized using a ChemiDoc Touch instrument (Bio-Rad).

Lipids were extracted from 3T3 cells, exosomes, and MBV by Folch procedure (45). MS analysis of phospholipids and their oxygenated products was performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific), as previously described (46). Briefly, phospholipids were separated on a normal-phase column [Luna 3 m Silica (2) 100 , 150 2.0 mm (Phenomenex)] at a flow rate of 0.2 ml/min on a Dionex Ultimate 3000 HPLC system. The column was maintained at 35C. The analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water (285:215:5, v/v/v), and solvent B contained propanol:hexane:water (285:215:40, v/v/v). All solvents were LC-MS grade. The column was eluted for 0 to 23 min with a linear gradient from 10 to 32% B, 23 to 32 min using a linear gradient of 32 to 65% B, 32 to 35 min with a linear gradient of 65 to 100% B, 35 to 62 min held at 100% B, and 62 to 64 min with a linear gradient from 100 to 10% B followed by an equilibration from 64 to 80 min at 10% B. Spectra were acquired in negative ion mode. Deuterated phospholipids were used as internal standards (Avanti Polar Lipids). Three technical replicates for each sample were run to evaluate reproducibility. Analysis of LC-MS data was performed using the software package Compound Discoverer (Thermo Fisher Scientific) with an in-house generated analysis workflow and nonoxidized/oxidized phospholipid database. Lipids were further filtered by retention time and confirmed by fragmentation mass spectrum.

Free fatty acids were analyzed by LC-MS using a Dionex Ultimate 3000 HPLC system coupled online to a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA), as previously described (47). Briefly, fatty acids and their oxidative derivatives were separated by a C18 column (Acclaim PepMap RSLC, 300 m 15 cm, Thermo Fisher Scientific) using gradient solvents A [methanol (20%)/water (80%) (v/v)] and B [methanol (90%)/water (10%) (v/v)], both containing 5 mM ammonium acetate. The column was eluted at a flow rate of 12 l/min using a linear gradient from 30% solvent B to 95% solvent B over 70 min, held at 95% B from 70 to 80 min, followed by a return to initial conditions by 83 min and re-equilibration for an additional 7 min. Spectra were acquired in negative ion mode. Analytical data were acquired and analyzed using Xcalibur software. A minimum of three technical replicates for each sample was run to increase the reproducibility.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We gratefully acknowledge T. Block and S. Griffey from StemBioSys for providing the ECM plates and K.-L. Aho from Genevia for the bioinformatic analysis. This project used the University of Pittsburgh HSCRF Genomics Research Core. We thank H. Deborah for conducting the small RNA-seq assay. Funding: This work was supported by NIH (HL114453-06, U19AI068021) and by Russian academic excellence program 5-100. S.F.B. and G.S.H. were supported, in part, by NIH R01AR073527, Mechanisms of functional skeletal muscle repair: Critical role of matrix associated IL-33. Author contributions: G.S.H., V.E.K., and S.F.B. conceptualized and designed the research. G.S.H., C.P.M., M.C.C., Y.Y.T., V.A.T., Y.C.L., S.O.E.-M., M.H.M., and P.S.T. performed the experiments. G.S.H., C.P.M., Y.Y.T., V.A.T., Y.C.L., P.S.T., V.E.K., and S.F.B. analyzed the data and interpreted the results of experiments. G.S.H., C.P.M., Y.Y.T., and V.E.K. prepared the figures. G.S.H., V.E.K., and S.F.B. drafted the manuscript. Competing interests: S.F.B. is the chief scientific officer and equity holder in ECM Therapeutics Inc., which has license rights to MBV technology from the University of Pittsburgh. S.F.B. and G.S.H. are inventors on several patents related to this work filed by the University of Pittsburgh. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data are available from the authors upon request.

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Lipidomics and RNA sequencing reveal a novel subpopulation of nanovesicle within extracellular matrix biomaterials - Science Advances

With coronavirus dominating the headlines, its easy to forget what else is going on in the world.

And it can be even harder to see any good news as most of the globe tackles the pandemic.

But in recent weeks there has been plenty of positive news to help with the coronavirus worries.

These are eight good news stories from across the world since the coronavirus outbreak began:

A million seagrass seeds were planted off the Welsh coast to help tackle climate change.

The seagrass seeds are being planted in a new 20,000sqm meadow off Pembrokeshire.

Seagrass can absorb carbon dioxide faster than trees, and scientists hope it can boost fish numbers and life in the sea.

In the summer of 2019, one million seeds were collected from areas like Porthdinllaen in the Lln Peninsula by a group of volunteers.

The seagrass, which is found in shallow, sheltered areas along the coast, was reached by snorkelling, diving and wading.

After being taken to Swansea University, the seeds were placed in hessian bags ready to be planted again at Dale Bay.

In more good news for the planet, carbon emissions from the power sector across the world have seen their biggest drop since 1990.

Research from environmental think tank Ember, emissions dropped 2% in 2019.

They said the historic decline was largely caused by the US and Europe moving away from coal power which saw a global drop of 3% - the largest decrease in three decades.

The budget doesnt normally bring much good news. But this year it was announced VAT on all sanitary products will be abolished from 2021.

Although small, it could mean women could save around 40 in a lifetime.

Scientists said a former chef living in the UK has become the second person in the world to be 'cured' of HIV.

Adam Castillejo has remained free of HIV for two-and-a-half years since he was given ground-breaking therapy at Hammersmith Hospital in west London.

The 40-year-old, who was born in Venezuela but lives in England, received a transplant of bone marrow stem cells that rid him of the AIDS-causing virus, the Mirror reported.

Amid the sombre news being reported on the TV was the annual Crufts show.

Viewers saw Maisie the wire-haired Dachshund beat tough competition and be crowned best in show.

The lovable pup even then did her business on the floor during her victory lap in front of a giggling crowd.

Around 28,000 pooches were put through their paces at the biggest dog show in the world at Birmingham's NEC.

In more animal related good news, a seal pup that spent two months in RSPCA care was released back into the wild.

Underweight Graham was looked after for around eight weeks after being rescued in Pembrokeshire.

RSPCA animal collection officer and wildlife officer Ellie West, who released Graham back into the sea at Port Eynon, said: This seal pup who when found was very underweight initially spent a few days coughing up sea shells!

He came into the centre weighing just 16.6kg and needed fluids and antibiotics for a chest infection and was treated for lungworm. He put on weight pretty steadily after starting to eat by himself on day six and then didnt look back.

Ellie released Graham back into the sea on March 7.

The fast-food chain has announced the bargain offer will last for five weeks, meaning customers have until April 19 to grab the discount.

Those wanting a meal for less will get a mini burger, regular fries and two hot wings for just 1.99 instead of the usual 3.99.

The offer was spotted by eagle-eyed deal hunters and posted on the money-saving website LatestDeals.co.uk.

A cleaver Australian teen has come up with a clever way to get rid of plastic waste - by using prawn shells.

17-year-old Angelina Arora has used prawn shells to create a plastic that will decompose in landfill in an average of 33 days.

The teen said she is now in talks with supermarkets to use her products, News.Au reported.

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Eight feel good stories you may have missed during the coronavirus outbreak - Wales Online

The latest Stem Cell Banking market study offers an all-inclusive analysis of the major strategies, corporate models, and market shares of the most noticeable players in this market. The study offers a thorough analysis of the key persuading factors, market figures in terms of revenues, segmental data, regional data, and country-wise data. Thhttps://jewishlifenews.com/uncategorized/stem-cell-bankinindustry-players/is study can be described as most wide-ranging documentation that comprises all the aspects of the evolving Stem Cell Banking market.

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Stem cell banking or preservation is a combined process of extraction, processing and storage of stem cells, so that they may be used for treatment of various medical conditions in the future, when required. Stem cells have the amazing power to get transformed into any tissue or organ in the body. In recent days, stem cells are used to treat variety of life-threatening diseases such as blood and bone marrow diseases, blood cancers, and immune disorders among others.

The market of stem cell banking is anticipated to grow with a significant rate in the coming years, owing to factors such as, development of novel technologies for stem cell preservation and processing, and storage; growing awareness on the potential of stem cells for various therapeutic conditions. Moreover, increasing investments in stem cell research is also expected to propel the growth of the stem cell banking market across the globe. On other hand rising burden of major diseases and emerging economies are expected to offer significant growth opportunities for the players operating in stem cell banking market.

Key Players

The stem cell banking market report also includes the profiles of key companies engaged with stem cell banking along with their SWOT analysis and market strategies. In addition, the report focuses on leading industry players with information such as company profiles, products and services offered, financial information of last 3 years, key development in past five years. Some of the key players influencing the market are Cordlife, ViaCord (A Subsidiary of PerkinElmer), Cryo-Save AG, StemCyte India Therapeutics Pvt. Ltd., Cryo-Cell International, Inc., SMART CELLS PLUS, Vita 34, LifeCell, Global Cord Blood Corporation, CBR Systems, Inc.

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The study conducts SWOT analysis to evaluate strengths and weaknesses of the key players in the Stem Cell Banking market. Further, the report conducts an intricate examination of drivers and restraints operating in the market. The report also evaluates the trends observed in the parent market, along with the macro-economic indicators, prevailing factors, and market appeal according to different segments. The report also predicts the influence of different industry aspects on the Stem Cell Banking market segments and regions.

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Stem Cell Banking Market Segmented by Region/Country: North America, Europe, Asia Pacific, Middle East & Africa, and Central & South America

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The Insight Partnersis a one stop industry research provider of actionable intelligence. We help our clients in getting solutions to their research requirements through our syndicated and consulting research services. We are committed to provide highest quality research and consulting services to our customers. We help our clients understand the key market trends, identify opportunities, and make informed decisions with our market research offerings at an affordable cost.

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Stem Cell Banking Market: Industry Analysis And Detailed Profiles Of Top Industry Players - Jewish Life News

A research team led by Professor Michael Sieweke, from the Center for Regenerative Therapies TU Dresden (CRTD) and the Center of Immunology of Marseille Luminy (CNRS, INSERM, Aix-Marseille University), has uncovered a surprising property of blood stem cells that contributes to boosting our immunity.

Not only do the stem cells ensure the continuous renewal of blood cells and contribute to the immune response triggered by an infection, but they can also remember previous infectious encounters to drive a more rapid and more efficient immune response in the future.

These cells are found within the soft tissue, or bone marrow, in the centre of large bones such as the hip and thigh bones.

The new findings should have a significant impact on future vaccination strategies and pave the way for new treatments of an underperforming or over-reacting immune system.

Stem cells in our bodies act as reservoirs of cells that divide to produce new stem cells, as well as a myriad of different types of specialised cells that are required to secure tissue renewal and function.

Commonly called blood stem cells, the hematopoietic stem cells (HSC) are found in the bone marrow, the soft tissue that is in the centre of large bones such as the hips or thighs. The role of the cells is to renew the repertoire of blood cells, including cells of the immune system, which are crucial to fight infections and other diseases.

Work from Professor Michael Siewekes laboratory and others over the past years has proven the dogma that HSCs were unspecialised cells, blind to external signals such as infections, was wrong, and has shown that HSCs can actually sense external factors to specifically produce subtypes of immune cells on demand to fight an infection.

Beyond their role in an emergency immune response, the question remained as to the function of HSCs in responding to repeated infectious episodes. The immune system is known to have a memory that allows it to better respond to returning infectious agents. The present study now establishes a central role for blood stem cells in this memory.

Professor Michael Sieweke, Humboldt Professor at TU Dresden, CNRS Research Director and last author of the publication, explained how they found the memory was stored within the cells: The first exposure to LPS causes marks to be deposited on the DNA of the stem cells, right around genes that are important for an immune response. Much like bookmarks, the marks on the DNA ensure that these genes are easily found, accessible and activated for a rapid response if a second infection by a similar agent was to come.

The authors further explored how the memory was inscribed on the DNA, and found C/EBPb to be the major actor, describing a new function for this factor, which is also important for emergency immune responses. Together, these findings should lead to improvements in tuning the immune system or better vaccination strategies.

Sieweke concluded: The ability of the immune system to keep track of previous infections and respond more efficiently the second time they are encountered is the founding principle of vaccines.

Now that we understand how blood stem cells bookmark immune response circuits, we should be able to optimise immunisation strategies to broaden the protection to infectious agents. It could also more generally lead to new ways to boost the immune response when it underperforms or turn it off when it overreacts.

The results of this research are published in Cell Stem Cellon March 12, 2020.

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Continued here:
Stem cells: what do bones have to do with boosting immunity? - Health Europa

A BUCKLEY man is one step closer to a clean bill of health after receiving life-saving treatment.

The Leader previously reported that Matt Davies was given 12-months to live without a stem cell transplant, which sparked a massive support network, with thousands signing up to become a donor.

Wife Sarah Davies urged people to sign up to become a donor, which could save the lives of many people and over 7,000 had signed up from her link alone.

Before Christmas, Matt was given the news that there was a match for him and he could start his treatment in January.

She told the Leader: It was a success, at the moment the cells in his body are 99.5 per cent donor and 0.5 per cent his. In time they will be 100 per cent donor so its definitely working which is fantastic.

We are on day 67 and on day 100 we can have a bone marrow scan to find out what stage we are.

GvHD is our biggest worry at the moment. Small amounts after a transplant can be good because it means his body is fighting but in huge amounts it can be damaging. It is starting to affect his gut now.

Because we live in Wales and have done for several years, we couldnt get the funding for the therapy which is what the Christie does, but we are now in the process of getting the drug for him, we are in constant talks so its a frustrating game at the moment.

We need to start this medication to get rid of this GvHD before it becomes chronic, so we are still in the process of getting that drug but hes doing really well.

Graft versus host disease (GvHD) is a condition that might occur after a transplant. In GvHD, the donated bone marrow or peripheral blood stem cells view the recipient's body as foreign, and the donated cells then attack the body.

Matt was diagnosed with cancer last year and beat it, however less than eight months later after having his three-monthly routine bone marrow results he was told the leukaemia was back and his only option was a stem cell transplant.

He has since made significant progress however the pair say they are worried about the latest coronavirus outbreak due to Matt essentially having no immune system.

Sarah said: At the moment with coronavirus its very scary because he has a low immune system, he is basically starting from scratch with his immune system so cant get immunisations until he is one year old. We have decided to take the kids out of school because we dont want him catching anything.

Hes done absolutely fantastic and is now back to eating.

Matt has been really lucky. They are pleased with his progress, but they would like his GvHD levels to be lower.

Although Matt faced no real complications during the treatment however has lost a significant amount of weight.

A JustGiving Page has been set up to raise funds for the Christie in Manchester where Matt has been receiving his treatment.

Sarah added: Even still now I will be walking somewhere and random people who Ive never met before will ask me how he is doing. Its actually been so positive. I dont think people realise how much it has helped, just them asking it has really helped us get through this and knowing that a lot of people are supporting us.

On social media we have spoken to so many people in similar situations as ours, its about helping one another, and we have made friends for life.

Thank you so much for your support, it means a lot to us and its lovely for us to read all the comments, even if we cannot reply to them all.

Matts progress can be found on social media via the Team Davies Facebook and Instagram page.

See the article here:
Buckley couple thank community for their support as dad-of-two receives life-saving treatment | The Leader - LeaderLive

Today, we will see why Fate Therapeutics (FATE) is an attractive pick in March 2020.

Fate Therapeutics is a clinical-stage biopharmaceutical company focused on the development of next-generation cellular immunotherapies for cancer and immune disorders. The company has pioneered proprietary iPSC (induced pluripotent stem cell) platform technology to develop off-the-shelf cell-based cancer immunotherapy products. Current patient-derived autologous and allogeneic cell therapies suffer from drawbacks such as high costs, manufacturing complexity, product heterogeneity, and high turnaround time. These methods, including patient and donor-derived approaches to cell therapy, also require batch-to-batch sourcing and engineering of millions of primary cells.

Fate Therapeutics aims to be the game-changer in cell-based cancer immunotherapy space by enabling the development of off-the-shelf cell products derived from master cell lines. The company aims to develop less costly, homogenous, and multi-dose or multi-cycle cell therapies with small turnaround time. The resultant cell therapy products are expected to be well-defined and uniform in the composition and can be mass-produced at a significant scale in a cost-effective manner and can be delivered off-the-shelf for broad patient accessibility.

The company's cell therapy pipeline comprises immune-oncology programs including off-the-shelf NK- and T-cell product candidates derived from master iPSC lines, and immuno-regulatory programs, including product candidates to prevent life-threatening complications in patients undergoing hematopoietic cell transplantation and to promote immune tolerance in patients with autoimmune disease.

Human-induced Pluripotent Stem cells are generated by reprogramming adult somatic cells to a pluripotent state. Fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells. They are reprogrammed using retroviruses. Pluripotent cells are capable of differentiating in all cell types that make up the body.

A single human iPSC can potentially differentiate into more than 200 cell types and provides a renewable source for making cells.

NK (natural killer) cells are the body's first line of defense against tumors and various pathogens. Fate Therapeutics is leveraging its iPSC platform to produce off-the-shelf NK cell therapy products.

FT500 is Fate Therapeutics' first off-the-shelf iPSC-derived NK-cell product candidate. The FT500 study is an open-label, multi-dose Phase 1 clinical trial designed to evaluate FT500 for the treatment of advanced solid tumors.

The dose-escalation stage of the study was originally designed to assess the safety and tolerability of three once-weekly doses of FT500, without IL-2 cytokine support, as a monotherapy and in combination with one of three FDA-approved ICI (immune checkpoint inhibitor) therapies in patients that have failed prior ICI therapy.

Data for the first 12 patients in the Phase 1 study has demonstrated clean safety for the iPSC platform. The cutoff date considered was November 28, 2019. It was seen that there were no reported dose-limiting toxicities, no FT500 related Grade 3 or greater adverse events or serious adverse events, and no incidents of cytokine release syndrome, neurotoxicity, or graft-versus-host disease.

Further, the trial also involved the evaluation of a multi-dose treatment course consisting of outpatient lympho-conditioning followed by three once-weekly doses of FT500 over up to two 30-day treatment cycles. Here, based on patients' T-cell and antibody repertoire, no anti-product immune responses against FT500 were evident over the multi-dose treatment course.

A total of 62 doses of FT500 were administered to these 12 patients in a safe and well-tolerated manner. Initial clinical data thus provides strong evidence that multiple doses of iPSC-derived NK-cells can be delivered off-the-shelf without patient matching.

In December 2019, the company disclosed plans to amend the trial protocol by including IL-2 cytokine support with each dose of FT500 after completion of 300 million cells per dose cohort in the ICI combination arm. The company has commenced dose-expansion part of Phase 1 trial with 300 million cells per dose and is focusing on enrolling NSCLC patients who are refractory to or have relapsed following CBT. This tumor type is highly susceptible to NK-cell recognition and killing. The study is enrolling at three clinical sites in the U.S. Fate Therapeutics expects expansion data readout from the trial in the second half of 2020.

Fate Therapeutics is studying the second product candidate from iPSC product platform and off-the-shelf NK-cell cancer immunotherapy, FT516, in an open-label, multi-dose Phase 1 trial. This product has been engineered to augment antibody-dependent cellular cytotoxicity.

In December 2019, the company announced results for two patients dosed with FT516. FT516 was administered as a monotherapy to the first patient who was suffering from relapsed/refractory AML (acute myeloid leukemia). The company dosed FT516 in combination with rituximab to the second patient who was suffering from high-risk DLBCL (diffuse large B-cell lymphoma) and had relapsed after multiple rituximab combination regimens, autologous hematopoietic stem cell transplant, and CAR (chimeric antigen receptor) T-cell therapy. The patients had received a first treatment cycle consisting of outpatient lympho-conditioning, three once-weekly doses of FT516 and IL-2 to better promote NK-cell activity.

Initial clinical data based on bone marrow biopsy at day 42 demonstrated no morphologic evidence of leukemia. There was even evidence of hematopoietic recovery following the completion of the first FT516 treatment cycle in the AML patient. There was also no circulating leukemia cells in the patient's peripheral blood. The patient even reported the recovery of neutrophils without growth factor support. The data did not demonstrate dose-limiting toxicities, although serious adverse events were seen. Initial dose escalation data may be read out in the second half of 2020.

This initial clinical evidence highlights the high probability of engineered iPSC-derived NK-cells demonstrating anti-tumor activity in AML indication. Besides, there is a body of data that has demonstrated clinical proof-of-concept for donor-derived NK-cell therapy in relapsed refractory AML and relapsed refractory DLBCL.

In December 2019, FDA accepted FT516's second IND application for studying the product in combination with PDL1, PD1, EGFR and HER2-targeting monoclonal antibody therapies in solid tumor indications. Initially, the company plans to prioritize the combination of FT516 and avelumab in patients with advanced solid tumors who are refractory to or have relapsed following, at least one line of anti-PDL1 monoclonal antibody therapy. The company plans to initiate enrollment in a clinical trial for FT516 and avelumab in mid-2020.

Fate Therapeutics is studying off-the-shelf multi-antigen targeted CAR NK-cell product candidate, FT596, in solid tumor indications.

In December 2019, Fate Therapeutics reported favorable in vivo preclinical data for FT596.

Here, in humanized mouse models of lymphoma and leukemia, FT596's efficacy was comparable to that of primary CAR T-cells in promoting tumor clearance and extending survival. FT596 combined with rituximab also showed the enhanced killing of lymphoma cells in vivo as compared to rituximab alone. FT596 can thus emerge to be best-in-class off-the-shelf treatment in B-cell malignancies. Fate Therapeutics has started enrolling patients in the open-label Phase I study. Initial dose escalation data readout on FT596 is expected in the second half of 2020.

Fate Therapeutics has high hopes for FT596, considering that initial clinical data from a donor-derived CAR19 NK-cell program at MD Anderson, demonstrated a 73% overall response rate in patients with relapsed refractory non-Hodgkin's lymphoma and chronic lymphocytic leukemia with no major toxicities. Hence, while the efficacy seemed similar to CAR T therapy, the safety profile was differentiated in favor of CAR NK-cell therapies.

Although early, this data has highlighted CAR NK-cells' capacity to confer a high level of efficacy without the CAR-T cell therapy-related toxicities. Fate Therapeutics expects FT596 to effectively replace patient-specific and allogeneic CAR19 T-cell immunotherapies. The latter single-antigen specific and hence pose a risk of disease relapse due to antigen escape as well as cause significant toxicities due to off-target activity. FT596, on the other hand, has been engineered with three active anti-tumoral functional components.

Fate Therapeutics aims to be the first company to introduce off-the-shelf iPSC-derived CAR T-cell therapy to patients, FT819, by submitting IND in the second quarter of 2020. The company expects to file an IND application for off-the-shelf CRISPR-edited, iPSC-derived NK-cell product candidate, FT538, by early May 2020. The company has also planned IND submission for FT576 in the second half of 2020.

Although Fate Therapeutics is pioneering a revolutionary approach for mass production of off-shelf cell therapy products, its pipeline is very early stage. There has not been sufficient data from its clinical programs to make an informed estimate about the success probability of these programs. In this backdrop, the company is exposed to significant R&D failure risks. In case data readouts from FT500 and FT596 clinical programs do not match expectations, the company may witness increased share price volatility.

At the end of 2019, the company had cash worth $261 million on its balance sheet. The company spent cash worth $83.2 million on operating activities in 2019. This is a proxy for the 2019 cash burn rate. We assume that the annual cash burn rate in 2020 will be around $120 million, considering that three assets have entered in-human trials. Hence, the company seems to have cash that can sustain operations until the end of 2021. However, if cash is needed at a faster pace, the company may land up requiring more funds. This can lead to equity dilution.

According to finviz, the 12-month consensus target price of Fate Therapeutics is $37.94. On March 4, Citi analyst Yigal Nochomovitz reiterated the "Buy" rating and increased target price from $26 to $41. On March 4, Barclays analyst Peter Lawson also initiated coverage of Fate Therapeutics with an Overweight rating and $40 price target.

On March 3, BMO Capital analyst Do Kim raised the firm's price target on Fate Therapeutics to $28 from $22 and reiterated the "Market Perform" rating. On March 3, Guggenheim analyst Michael Schmidt reiterated the "Buy" rating and increased target price from $25 to $41. On March 3, Roth Capital analyst Tony Butler reiterated the "Neutral" rating but increased the target price from $20 to $30. On March 3, BTIG analyst Amanda Murphy reiterated the "Buy" rating and increased target price from $27 to $42. The analyst has also raised the estimated value of the company's iPSC platform from $740 million to $2.0 billion.

On March 3, Oppenheimer analyst Matthew Biegler reiterated the "Outperform" rating and increased the target price from $27 to $36. Piper Sandler analyst, Edward Tenthoff also reiterated the "Overweight" rating and raised the target price from $28 to $57.

In September 2019, Fate Therapeutics launched in-house GMP (Good Manufacturing Practices) manufacturing facility at headquarters in San Diego, California. This is custom designed to use clonal master iPSC lines as a renewable cell source for the consistent and scaled manufacture of off-the-shelf NK-cell and CAR T-cell products. The company has already produced hundreds of cryopreserved, infusion-ready doses of FT500, FT516, and FT596 at a low cost per dose. Currently stored in inventory, these doses are immediately available for use in the clinical settings.

The full control of cGMP production and the technical expertise to genetically engineer iPSCs and create qualified clonal master lines for clinical use implies that the company has operational expertise and redundancies required for the consistent cost-effective manufacturing and clinical supply of off-the-shelf cell products.

I believe that the 12-month target price of $30 fairly reflects the growth potential as well as risks associated with early-stage Fate Therapeutics. I consider this company to be a good pick for aggressive biotech investors with an investment horizon of at least one year.

Disclosure: I/we have no positions in any stocks mentioned, and no plans to initiate any positions within the next 72 hours. I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.

See the rest here:
Fate Therapeutics: Potential Catalysts Ahead - Seeking Alpha

For their study, Terzic and colleagues analyzed the hearts of mice that received cardiopoietic stem cell therapy as well as those that did not. They used an algorithmic approach to map the proteins in the heart muscle, identifying 4,000 proteins. Ten percent of these were damaged during a heart attack.

The investigators found that the therapy either fully or partially reversed two-thirds of the changes caused by the event. And about 85% of cellular functional categories impacted by infarction responded positively to treatment, the authors wrote. They also noted that new blood vessels and heart tissue began to grow as a result of the intervention.

In the United States, someone has a heart attack every 40 seconds, according to the study, which kills this precious cardiac tissue and leads to a significantly weaker heart. Although cardiopoietic stem cells are still being investigated in advanced clinical trials in human patients, this most recent study is a big step in the right direction.

The current findings will enrich the base of knowledge pertinent to stem cell therapies and may have the potential to guide therapeutic regimens in the future," Terzic concluded.

Read the original:
Stem cell therapy revives cardiac muscle damaged during heart attacks - Cardiovascular Business

The northern white rhino population is in jeopardy

The northern white rhino is one of the animal kingdoms many majestic giants, but years of poaching has taken a toll on their population. From 1970 to 1980, their numbers plummeted from 500 to 15 as illegal hunters pursued white rhinos for the ivory of their horns.

Things started to turn around during the 1990s and 2000s, groups and individuals began to crack down on poachers within the white rhinos range. As a result, the population of white rhinos in the wild recovered slightly, peaking at around 32 individuals.

Since 2003, the rate of white rhino poaching has been on the rise and has affected the animals numbers. As of 2008, northern white rhinos have been declared extinct in the wild, and in 2018, the last male northern white rhino died. Now, there are only two of these magnificent beasts left on Earth. Both of them are females.

Najin and Fatu are the last two northern white rhinos in existence. They live at the Ol Pejeta Conservancy in Kenya, and they could be the species last hope for the future. In 2014, keepers in the Czech Republic collected sperm samples from a male northern white rhino living in their care.

Those samples were frozen and stored, and later, they were used in an attempt to breed Najin and Fatu. Both attempts at inducing pregnancies in the two female rhinos were unsuccessful, forcing scientists to consider new methods of approach for saving the white rhinos from extinction.

Typically, when a species is placed on the endangered list, a recovery plan is established by whatever local conservancy group oversees the population. From there, breeding programs of captive individuals are used to begin bolstering the number of individuals on the planet.

When healthy breeding populations have been established, in most cases, reintroduction begins. Small populations of the species are released into the wild to begin repopulation. However, in the case of the northern white rhinos, scientists and conservationists alike have been stuck at step two for decades.

Unwillingness and inability to breed arent uncommon among captive species and individuals, and in most cases, zoos can jockey animals around until a pair matches and produces offspring. In the case of Najin and Fatu, the options for procreation are far more limited. Even the fallback of artificial insemination isnt working for them, so what are scientists to do?

Weve revived entire species from the dead before, but it has never been an easy task. Fortunately, the world of reproductive sciences has been evolving quickly, and conservationists and animal experts now have myriad options to choose from when it comes to creating new life.

Neither surviving female is healthy enough to birth live young. Aside from that, there is the added challenge of finding an option that preserves the northern white rhino genome while maintaining high enough levels of viability.

One possible route to repopulation involves approaching conventional methods from a new and enlightened angle. Although neither Najin nor Fatu can bear young, they both still produce viable egg cells, which can be harvested, frozen, and kept in a lab.

Much like humans undergoing fertility therapy or other conception aids, the grandmother-granddaughter pair or northern white rhinos can hope for success through in-vitro fertilization. This method of conception combines sperm and multiple egg cells in an external environment before implanting them in a host mother.

By using multiple eggs during the in-vitro process, the chances for success, even in females with fertility issues, is significantly increased. In some fortunate cases, the method is so effective, and it results in multiple pregnancies. Once the sperm has fertilized the eggs, the cells are transferred to a living host.

While Najin and Fatu may not be the physical mothers of any of their calves, modern reproductive science has made it possible for their genes to be passed on to another generation.

How? with modern science, a surrogate mother from the thriving population of southern white rhinos could become the mother to their children.The two types of animals have similar enough reproductive organs and their eggs could be used in place of Najin or Fatus.

While the animals are compatible, gathering eggs from them is a far more complicated procedure.

Researchers working on bringing back the northern white rhinos have managed to gather a few eggs so far, but not nearly enough to repopulate an entire species.

Its no secret that rhinoceroses are large animals. Just as cattle and horses have significantly larger hearts than we humans do, rhinos have much larger reproductive organs. Locating and withdrawing eggs from a rhinos ovaries is a far greater ordeal than it is for humans.

To complicate matters further, the ovaries of a southern white rhino are located three to four feet from her rump, and the veterinarian seeking to collect the eggs must guide a probe that distance up her rectum and into an ovary before using a catheter to remove the eggs.

The procedure is anything but easy. In addition to the difficulty involved in the process of extracting eggs, the success rate of current methods is hardly ideal. Researchers working on bringing back the northern white rhinos have managed to gather a few eggs so far, but not nearly enough to repopulate an entire species.

The odds of reestablishing a sustainable population of northern white rhinos through in-vitro fertilization and surrogacy currently seem pretty slim. Fortunately for the rhinos, science has a few other methods up its sleeve.

In the last decade, stem cell research has gone from a thing of whimsy to an advanced field of study that continues to improve by leaps and bounds with every passing year. Its applications are seemingly endless, and they just might be the answer that the northern white rhino conservationists have been looking for.

Stem cells are sort of like biological canvases. They come in different varieties: Totipotent, pluripotent, multipotent, oligopotent, and unipotent. Each of these types has unique limitations and can be found in various sources from embryonic tissue to adult bone marrow.

To make baby rhinos, scientists have been focused on induced pluripotent stem cells, which are gathered and grown from the skin of adult white rhinos

A cell from your bicep and a cell from your gametes (sperm or egg) both hold the same blueprints; they just come in different packaging.

Pluripotent cells behave similarly to embryonic stem cells, which can be coaxed into becoming just about any other type of cell. In this case, even though the original cells were taken from the skin of adult rhinos, they can be trained to become something different, such as egg cells.

Using what knowledge we currently have of stem cells and their manipulation, scientists can tell a northern white rhinos skin cell to become a viable egg or sperm cell. From there, they can attempt in-vitro fertilization and implantation into a surrogate, even without fertile parents.

The method is still in its infancy, but it has been successfully carried out more than once.

With stem cells as a backup and surrogates abound, Najin and Fatu have plenty of options. In late 2019, conservationists and rhinos alike received promising news. Eggs gathered from the two northern white rhinos had been fertilized and resulted in successful embryos. Those embryos were frozen in liquid nitrogen and prepared for a long journey.

Waiting down in southern Africa are the lucky mamas who will become the surrogates for the next generation of northern white rhinos. The embryos have quite a ways to travel before they can be implanted. After that, they can grow within their new mother for the 16 to 18-month gestation period typical of white rhinos.

Although the methods of creating viable embryos are currently long, challenging, and not terribly efficient, these babies-to-be are incredibly promising first steps. In addition to the two successful in-vitro attempts in September, December of 2019 saw the creation of a third viable embryo.

2020 will undoubtedly see further attempts at creating more embryos. With luck, we can soon hope to hear news of successful implantations in surrogate moms. In 2021, we can throw a worldwide baby shower for some bouncing baby northern white rhinos, whose births will serve as a beacon of hope for a dying species.

Read the original:
Can hybrid embryos save the white rhinos from extinction? - Science 101

UPDATE:

SPOKANE, Wash. -- Life-saving cells for alocal father of six are on their way to him from Poland. His family has been panicked after a travel ban was put in place by the Polish Government. They say they were told the status of the transport was stalled, and with time slipping away, they needed immediate action.

Jared Weeks was diagnosed withAcute Myeloid Leukemia back in October. His wife Janet contacted 'Help Me Hayley' on Saturday. On Sunday morning, Janet got word that the cells were on their way. She reached out to many government officials and is still trying to sort how and who helped make this happen for her husband.

"I heard that relief in (my husband's) voice and that's all I needed," she said. "I'm so thankful to everyone who shared the story, sent us prayers. I felt it. I really did. People are so overwhelmingly beautiful."

Janet says her husband will have the stem-cell transplant on Tuesday.

"I will be traveling over to Seattle on Monday evening to be there for his 're-birthday,'" she said of the procedure. "I'm so grateful."

PREVIOUS COVERAGE:

SPOKANE, Wash. -- A local father of six desperately needs help receiving life-saving cells provided by an overseas donor. His family says his life depends on it.

His wife Janet sent our Hayley Guenthner this 'Help Me Hayley' request:

"Dear Help Me Hayley,

My children and I are desperate to save my husband. He was diagnosed with Acute Myeloid Leukemia on 10/15/2019 (on his 42nd birthday of all days) since then he has been in the hospital. At the beginning of February we started our journey to the west side of the state to be under the care of Seattle Cancer Care Alliance and to make a long story short, we are now in the transplant stage of his disease.

My husband, Jared Weeks, went inpatient to the University of Washington Medical Center (UWMC) on behalf of the Seattle Cancer Care Alliance. He started his myeloablative chemo regimen on March 10th with the expectation of receiving an Unrelated Allogeneic Peripheral Blood Stem Cell transplant. He had the highest dose of chemotherapy to eliminate his disease and replace his immune system with a 38-year-old female peripheral blood stem cell donation from Poland. Because of the travel ban put in place by the Polish Government in response to the outbreak of the Novel COVID-19 virus, it is becoming impossible to transport these LIFE-SAVING cells that have been extracted from my husband's donor and brought back to the United States. I have left messages for Senator Cathy McMorris-Rodgers, Governor Jay Inslee, Mayor Woodward and Senator Maria Cantwell. I was able to speak personally with State Senator Shelly Short who is passing on this to some of her contacts in the cabinet. I reached out to the Polish Government agency handling the travel ban restrictions and have spoken with an Overseas Citizen Services Safety Officer out of Krakow Poland at the US Embassy-State Department. The travel ban has been put in place but I have been told that roads are still open as well as trains and planes, but as of midnight tonight (not sure if our time or their time) the borders will be closed until March 25th, and maybe extended depending on the COVID-19 outbreak. The cells have been collected from the donor and we are desperate to get them here. Please help us!! God help us.

My husband, Jared Weeks, was diagnosed with Acute Myeloid Leukemia on October 15, 2019 and is in DIRE need of these stem cells to survive.

We need some assistance from the "powers that be" to get these life-saving stem cells to my husband in Washington State ASAP.

His life depnds on it."

There have many people offering to test to see if they are a local match for Jared. Unfortunately, the family doesn't have the kind of time required to find a new donor.

"They would need to go to bethematch.org , however, it is too late in the game to be a donor for Jared but there are hundreds of others that need this life-saving donation as well," Janet said. "The HLA TYPING that is done can take weeks to complete and for Jared, we don't have that kind of time."

Janet is currently in Spokane with their children. She said she is doing everything she can to stay strong for her husband.

"(Jared) is one heck of a dad," Janet said. "He is hardworking, loves the outdoors, fishing, boating and taking his kids on adventures. He is amazing to us and is the center of gravity for our rather large family. He has been through hell and back with this cancer, and is still trusting God completely."

Seattle Cancer Cancer Care Alliance sent KHQ a statement on Jared and other cancer patients relying on life-saving bone marrow transplants during the COVID-19 outbreak.

"The COVID-19 outbreak is an evolving and fluid situation, and the global medical community is collaborating to address the needs of people who are relying on bone marrow transplants for their treatment and survival.

"Seattle Cancer Care Alliance is evaluating every patient who is currently connected with an international or USA-based donor to ensure we have an alternative solution for their treatment should the need arise.

"We are committed to continuing to coordinate with the National Marrow Donor Program and the World Marrow Donor Association, along with donor representatives in various countries, to prevent potential disruptions of critical medical transport so that every cancer patient has access to the life-saving treatment they need.

"SCCA is dedicated to providing the highest-quality cancer care, and we take that responsibility very seriously. We continue to work very closely with our alliance partners -Fred Hutch, UW Medicine and Seattle Childrens- and sharing our approach and best practices with other transplant centers around the country who may face similar unprecedented challenges."

See the article here:
HELP ME HAYLEY: Spokane father of six receiving life-saving cells from Poland donor - KHQ Right Now

An HIV patient recovered with a stem cell transplant. He became the second patient in the world to recover from this disease.

an HIV patient who received a stem cell transplant is now cured. He has become the second patient in the world to recover from the disease, his doctors announced Tuesday, March 10.

Almost ten years after the first confirmed case of an HIV patient who managed to get rid of it, this second case, known as the London patient, has shown no signs of the virus for 30 months, according to the results published in the journal The Lancet HIV.

In March 2019, Professor Ravindra Gupta, of the University of Cambridge, announced that this man diagnosed with HIV in 2003 was in remission, having shown no sign of the virus for 18 months. The doctor had however called for caution, insisting on the term of remission and not healing, asking for more time.

We suggest that our results represent a cure for HIV

A year later, his team took this step. We suggest that our results represent a cure for HIV, they write, after testing samples of blood, tissue, sperm. We tested a fairly considerable number of places where the virus likes to hide and practically everything was negative, apart from a few fossil remains of non-active virus, Pr Gupta told AFP. It is hard to imagine that all traces of a virus that infects billions of cells have been eliminated, he said.

Like the Berlin patient, the American Timothy Ray Brown considered cured in 2011, this London patient underwent a bone marrow transplant to treat blood cancer, and thus received stem cells from donors carrying a rare genetic mutation that prevents HIV from taking hold, CCR5.

Scientists point out that the procedure used for the two recovered patients is very cumbersome and risky, asking ethical questions, as Professor Gupta points out.

Is the London patient really healed? Asked Sharon Lewin of the University of Melbourne. The data () is of course exciting and encouraging, but in the end, only time will tell, she noted, saying it would take more than a handful of HIV-cured patients to assess the likelihood of a late and unexpected resumption of virus replication .

Almost 38 million people are living with HIV worldwide, but only 62% are receiving triple therapy. Nearly 800,000 people died in 2018 from HIV-related conditions. The emergence of drug-resistant forms of HIV is also a growing concern.

The rest is here:
For the second time in the world, an HIV patient is cured - Medical Progress - Medical Progress

By: Cobey Bartels

Date: 16.03.2020

Friend of Owner//Driver and owner of the iconic Filthy White 4000 we covered last year, Mick Moo Lake, was diagnosed with cancer on Christmas Eve.

Mick and Mel Lake, with 'Filthy'

Since the devastating news, Mick and his wife Mel have been struggling to balance treatment and operating their business Truckin Stainless setting up a GoFundMe page to help cover the costs of hospital care.

Mick has Double Myeloma, a form of cancer that develops from plasma cells in the bone marrow, and has been receiving ongoing treatment since New Years Eve.

In 10 weeks Mick is having a Stem Cell Blood Transfusion, which will put him out of work for at least a month and will put significant strain on his business and familys livelihood.

At the time of writing, the GoFundMe page has raised $3,195 and Mick and Mel say the money will go towards covering the cost of treatment and help keep their business afloat.

Mick has been working through the treatment, where possible, to keep Truckin Stainless kicking along with the help of his good mate Steve who travelled down from Mackay to help lighten the load.

"He heard about what was going on when he was down here at the time, and he decided to stay and help while I was getting treatment," Mick says.

"Ive been working through it, going to hospital for treatments then back to the workshop - i havent stopped.

"In respect of all the wonderful people that have had to endure this terrible disease, we understand that asking for donations seems a bit steep, but if you would like to donate please do so, or think to donate to any cancer foundation," he says.

You can donate or find out more HERE.

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Moo trucks on through cancer battle - Owner//Driver

Immune cells by fluorescence microscopy: Blood stem cells remember a previous attack and produce more immune cells like these macrophages to fight a new infection. Credit: Sieweke lab/CIML

These findings should have a significant impact on future vaccination strategies and pave the way for new treatments of an underperforming or over-reacting immune system. The results of this research are published in Cell Stem Cell on March 12, 2020.

Stem cells in our bodies act as reservoirs of cells that divide to produce new stem cells, as well as a myriad of different types of specialized cells, required to secure tissue renewal and function. Commonly called blood stem cells, the hematopoietic stem cells (HSC) are nestled in the bone marrow, the soft tissue that is in the center of large bones such as the hips or thighs. Their role is to renew the repertoire of blood cells, including cells of the immune system which are crucial to fight infections and other diseases.

Until a decade ago, the dogma was that HSCs were unspecialized cells, blind to external signals such as infections. Only their specialized daughter cells would sense these signals and activate an immune response. But work from Prof. Michael Siewekes laboratory and others over the past years has proven this dogma wrong and shown that HSCs can actually sense external factors to specifically produce subtypes of immune cells on demand to fight an infection. Beyond their role in an emergency immune response, the question remained as to the function of HSCs in responding to repeated infectious episodes. The immune system is known to have a memory that allows it to better respond to returning infectious agents. The present study now establishes a central role for blood stem cells in this memory.

We discovered that HSCs could drive a more rapid and efficient immune response if they had previously been exposed to LPS, a bacterial molecule that mimics infection, said Dr. Sandrine Sarrazin, Inserm researcher and senior-author of the publication. Prof. Michael Sieweke, Humboldt Professor at TU Dresden, CNRS Research Director and last author of the publication, explained how they found the memory was stored within the cells: The first exposure to LPS causes marks to be deposited on the DNA of the stem cells, right around genes that are important for an immune response. Much like bookmarks, the marks on the DNA ensure that these genes are easily found, accessible and activated for a rapid response if a second infection by a similar agent was to come.

The authors further explored how the memory was inscribed on the DNA, and found C/EBP? to be the major actor, describing a new function for this factor, which is also important for emergency immune responses. Together, these findings should lead to improvements in tuning the immune system or better vaccination strategies.

The ability of the immune system to keep track of previous infections and respond more efficiently the second time they are encountered is the founding principle of vaccines. Now that we understand how blood stem cells bookmark immune response circuits, we should be able to optimize immunization strategies to broaden the protection to infectious agents. It could also more generally lead to new ways to boost the immune response when it underperforms or turn it off when it overreacts, concluded Prof. Michael Sieweke.

The research group of Prof. Michael Sieweke works at the interface of immunology and stem cell research. The scientists focus on the study of hematopoietic stem cells and macrophages, long-lived mature cells of the immune system that fulfil an important role in tissue regeneration. In 2018, Prof. Michael Sieweke received the most valuable research award in Germany: the Alexander von Humboldt Professorship, which brings top international researchers to German universities. In addition to his position as Research Director at the Centre for Immunology at the University of Marseille Luminy, he now acts as Deputy Director at the Center for Regenerative Therapies at TU Dresden (CRTD). CRTD is academic home for scientists from more than 30 nations. Their mission is to discover the principles of cell and tissue regeneration and leveraging this for recognition, treatment and reversal of diseases. The CRTD links the bench to the clinic, scientists to clinicians to pool expertise in stem cells, developmental biology, gene-editing and regeneration towards innovative therapies for neurodegenerative diseases such as Alzheimers and Parkinsons disease, hematological diseases such as leukaemia, metabolic diseases such as diabetes, retina and bone diseases.

Reference: C/EBP-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells by Brengre de Laval, Julien Maurizio, Prashanth K. Kandalla, Gabriel Brisou, Louise Simonnet, Caroline Huber, Gregory Gimenez, Orit Matcovitch-Natan, Susanne Reinhardt, Eyal David, Alexander Mildner, Achim Leutz, Bertrand Nadel, Christophe Bordi, Ido Amit, Sandrine Sarrazin and Michael H.Sieweke, 12 March 2020, Cell Stem Cell.DOI: 10.1016/j.stem.2020.01.017

This study was funded by TU Dresden / CRTD through the German Excellence Initiative, the German Research Foundation as well as through an ERC Advanced Grant from the European Research Council and the Alexander von Humboldt Foundation. The study was further supported by funding from the Institut National de la Sante et de la Recherche Medicale, the Centre National de la Recherche Scientifique, Aix-Marseille University, the Agence Nationale de la Recherche, the Foundation ARC pour la Recherche sur le Cancer, an INSERM-Helmholtz cooperation programme and the Einstein Foundation.

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Newly Discovered Memory in Our Bones: Keeping a Record of Previous Infections to Boost Immunity - SciTechDaily

Revolutionary Gene-Editing Tool

Cardiovascular or heart disease (CVDs) is the leading cause of death across the world. Heart attacks resulting due to CVDs can cause death, and severe damage to cardiac muscle a muscle that forms the wall of the heart in survivors. However, researchers claim that they have discovered stem-cell activated mechanisms that promote healing after a heart attack.

According to the study by researchers from Mayo Clinic, stem cells were found to reverse the damage and restore cardiac muscle back to its condition before a heart attack. Human cardiopoietic cells obtained from stem cells within the bone marrow were found to hone in on damaged proteins and reverse intricate changes that a heart attack caused.

"The response of the diseased heart to cardiopoietic stem cell treatment revealed development and growth of new blood vessels, along with new heart tissue," said Dr. Kent Arrell, first author of the study, in a statement.

For the study, the researchers examined the diseased hearts of mice. The hearts of mice that received human cardiopoietic stem cell therapy were compared with those of that did not. Nearly 4,000 cardiac proteins were identified using a data science technique to map proteins found in the cardiac muscle. Over 10 per cent of the discovered proteins were found to suffer damage as a result of a heart attack.

"While we anticipated that the stem cell treatment would produce a beneficial outcome, we were surprised how far it shifted the state of diseased hearts away from disease and back toward a healthy, pre-disease state," said Dr. Arrell.

While the organs in the human body have the ability to repair their damaged cells, they may be unable to restore the loss entirely, and this holds good for cardiac cells as well. Dr. Andre Terzic, senior author of the study, said: "The extent of change caused by a heart attack is too great for the heart to repair itself or to prevent further damage from occurring."

He explained that upon the administration of cardiopoietic stem cell therapy to mice, a partial or complete reversal of nearly two-thirds of the damage caused by a heart attack was noted. Around 85 per cent of all cellular functional categories struck by the disease responded favorably to the treatment.

According to the World Health Organisation (WHO), CVDs claim nearly 18 million lives every year, which translates to 31 per cent of all deaths. The findings of the study provide an improved understanding of the restoration of heart health using stem cells and provide a framework for wider utilization of stem cell therapy for the treatment of various conditions.

Stressing that the actual mechanism behind the repair of diseased organs by stem cells is poorly understood, Dr. Terzic added: "This study sheds light on the most intimate, yet comprehensive, regenerative mechanisms paving a road map for responsible and increasingly informed stem cell application."

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Stem cells can reverse damage caused by heart attack; repair mechanism discovered: Study - International Business Times, Singapore Edition

A 40-year-old HIV patient has been declared cured after a promising treatment has left him with no active virus. The man, Adam Castillejo, was the subject of extensive research in early 2019 after doctors failed to find HIV in his body over an 18-month period after previously being diagnosed in 2003.

Castillejo, known by the nickname London Patient lived with the disease for many years, taking medicine to manage it since 2012. That same year he was diagnosed with Hodgkins Lymphoma and later endured a bone marrow transplant. That operation may have ultimately cured him of HIV and appears to have made him only the second person to ever be cured of the disease that causes AIDS.

As ScienceAlert reports, the bone marrow transplant that doctors performed on Castillejo used cells from a donor with a very special genetic quirk. The cells are thought to work against HIV in the body, but there was no guarantee that the transplant would provide any concrete benefits beyond treating the cancer.

However, it appears as though the decision to treat Castillejo with the unique stem cells worked in more ways than one and last year doctors announced they couldnt find the virus in his body after 18 months. At the time, they were hesitant to declare the London Patient cured, but after a new round of testing returned the same results, they are more confident that the active form of the virus has indeed been defeated.

This is a unique position to be in, a unique and very humbling position, Castillejo told the New York Times. I want to be an ambassador of hope.

While this sounds like incredible news and for Castillejo, it certainly is the treatment is not an option for everyone. With cancer limiting their options, doctors used the stem cell transplant as a last resort to keep him alive. Its a serious operation and one that was only performed because Castillejos condition was so dire.

Castillejo and the other HIV patient who had similar results, known as the Berlin Patient, may be uniquely fortunate. The doctors note that there are others who have had the same transplant performed but did not improve as rapidly as the others. There are obviously many factors at work here and as exciting as it is to see a second person cured of this terrible disease, theres a lot more work to be done before we can say HIV has been truly beaten.

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HIV patient appears to be cured after stem cell treatment - New York Post

Although most of the news from the world of health and medicine has been quite bleak lately, there are still major strides being made in the sector in an effort to combat the worst illnesses that plague humankind. One such stride was just announced, and its certainly worth celebrating: A second person has been cured of HIV.

In a study published in the medical journal, The Lancet, which comes via Medical News Daily, researchers in London say theyve been able to cure a patient of HIV; meaning the patient tested negative for HIV for an extended period of time (30 months as of March, 2020) despite the lack of antiretroviral therapy.

The person whos been cured, Adam Castillejo, was formerly known only as the London patient in order to protect his identity. But Castillejo, who lives in London, came forward recently, and said that he aims to be an ambassador of hope.

The first person to be cured of HIV, Timothy Ray Brown, an American known originally as the Berlin patient, revealed his identity in 2010, saying that I wanted to do what I could to make [a cure] possible. My first step was releasing my name and image to the public. Brown lived and was treated in Berlin. Incidentally, he is technically the second Berlin patient because the results from treatment of the first one are debatable.

AIDS Policy Project with Timothy Ray Brown (third from left with sunglasses). Griffin Boyce.

Castillejo, as well as Brown, were cured of HIV not by antiretroviral medications, which are often able to drastically mitigate the effects, and transmission rate of, HIV, but rather by stem cell transplants from donor bone marrow. Both Castillejo and Brown hadand may still have, that is unclearcancer along with HIV, and were treated with the stem cell transplants primarily to tackle the former disease. (It seems in Castillejos case doctors and researchers were hoping to cure both simultaneously.)

Both Brown and Castillejo underwent a procedure known as a Hematopoietic stem cell transplantation (or HSCT), which involves injecting bone marrow stem cells from a donor, whos often times a parent or sibling, into the recipients bloodstream. Castillejos HSCT treatment was different from Browns, as well as many others, because it was performed with cells that expressed the CCR5 gene.

A video from the MD Anderson Cancer Center that gives a brief outline of how bone marrow stem cell transplants work.

In Castillejos case, stem cells with genomes that express the CCR5 gene were selected because of the fact that it allows for the production of the CCR5 protein: a protein that makes people far more resistant to HIV-1, which accounts for the vast majority of global HIV infections.

While Castillejo received stem cells that did express the CCR5 gene, Brown did notat least according to the study in The Lancet. In fact, according to a 2017 article in New Scientist (which says that Brown received cells with a mutated CCR5 gene, rather than an unexpressed CCR5 gene), some experts believe the curing of Browns HIV was actually due to a potential side effect of his procedure, known as graft-versus-host disease. According to New Scientist, these experts believe that the donor cells attacked Browns native, HIV-infected immune cells, subsequently killing off the virus.

In Castillejos case, on the other hand, it seems there was no graft-versus-host issue that could account for his diminishment of HIV infection levels beyond whats expected to be detectable. Instead, the authors of the study say that one of the implications here is that the Long-term remission of HIV-1 can be achieved utilizing these kinds of cells. The authors also say this method does not require total body irradiation, which would usually be required in cases like these to weaken a recipients immune system in order to allow them to accept donor cells.

An HIV-infected T cell. NIAID

Unfortunately, it seems the treatment that cured Castillejo of HIV is a nonstarter when it comes to mass deployment. There are fatal side effects associated with HSCT, with host-versus-graft chief among them, and doctors say that it should only be performed when there are no other options left.

Prof. Ravindra Kumar Gupta from the University of Cambridge in the U.K., the lead author of the study, told Medical News Daily that [Its] important to note that this curative treatment is high risk and only used as a last resort for patients with HIV who also have life threatening hematological [blood] malignancies.

But Gupta and the other authors of the study still appear to be optimistic that this stands as a proof-of-concept for the idea of using CCR5 gene editing to cure HIV on a larger scale. They warn in their study, however, that several barriers, including the need for increased gene editing efficiency and a lack of robust safety data, still stand in the way of something that could be used as a scalable strategy for tackling HIV.

What do you think about this method of treating HIV? Do you think gene editing will play a big role in curing HIV, or do you think there are other, more promising treatments worth pursuing instead? Let us know your thoughts in the comments.

Feature image: C. Goldsmith / Eliot Lash

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A Second Person Has Been Cured of HIV - Nerdist

Newswise The number of COVID-19 cases are expected to continue to grow across the globe in the upcoming months and that means more people will have to take extra measures to help protect themselves and reduce the transmission of the disease. This is particularly important for people with cancer, whose immune systems have often been weakened by their cancer treatments.

But does that mean people with cancer should stockpile hand sanitizer and face masks?

Oncologists Gary Schiller, MD, and Joshua Sasine, MD, PhD, help explain what cancer patients need to know about COVID-19.

Dr. Schiller is a professor of hematology/oncology at the David Geffen School of Medicine at UCLA and director of the hematological malignancies/stem cell transplantation unit, and Dr. Sasine is an assistant professor of medicine and director of the CAR T cell program at the UCLA Jonsson Comprehensive Cancer Center.

Which cancer patients should be concerned about coronavirus?

Sasine: The patients most at risk are those with bone marrow cancers or who have had a bone marrow transplant within the last 12 months. If patients have cancer and are on active chemotherapy, they are also at a higher risk than the general population. This is especially true if they are over the age of 60.

Schiller: Bone marrow transplant recipients who received bone marrow from other people are the most immunocompromised patients we take care of and the group at greatest risk for sustaining a life-threatening complication from an infection.

What does it mean to have a compromised immune system?

Sasine: The body's white blood cells normally clear out infections, like bacteria, viruses, and fungi. When the cells have either decreased in number, function, or both, the immune system is compromised. This can be due to having cancer, HIV, getting chemotherapy, and many other situations. This means that a person is more likely than others to contract an infection and the infection is likely to do more harm than average. It might also last longer.

Are there precautions cancer patients should be taking?

Schiller: Patients who are immunocompromised need to be wary of going into crowds, should maintain good hand washing techniques and should stay away from individuals who have a cough.

Sasine: For most events, canceling plans is ideal. However, sometimes one must weigh the risks and benefits. If there is a very important event (son or daughter is getting married, etc.) this might be a risk worth taking.

Should cancer patients delay travel plans?

Schiller: For patients with malignancies of the blood and bone marrow, and patients who had bone marrow transplants, I absolutely tell them to delay travel. Dont travel right now.

Is it safe for patients to come to the hospital and clinics for treatment?

Schiller: Yes. Weve been working to develop better isolation procedures and policies to isolate the potentially sick patients from our immunocompromised patients. For example, bringing the potentially sick patients in through a different entrance to isolate them in the waiting room and put them in an isolation room for evaluation.

Should patients be wearing a mask or stockpile hand sanitizer?

Schiller: A mask is not sufficient protection and were concerned that if you wear a mask, especially one that is insufficiently protective, then you have a false sense of security and you may put yourself in a position that might compromise your safety. In regards to hand sanitizer, I would like my patients to stockpile on soap and water. That would be more effective than using hand sanitizer repeatedly.

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Cancer and COVID-19: What you should know - Newswise

NEW YORK, March 10, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited(Nasdaq: MESO; ASX:MSB) today announced that it plans to evaluate its allogeneic mesenchymal stem cell (MSC) product candidate remestemcel-L in patients with acute respiratory distress syndrome (ARDS) caused by coronavirus (COVID-19) in the United States, Australia, China and Europe. The Company is in active discussions with various government and regulatory authorities, medical institutions and pharmaceutical companies to implement these activities.

Mortality in COVID-19 infected patients with the inflammatory lung condition acute respiratory distress syndrome (ARDS) is reported to approach 50%, and is associated with older age, co-morbidities such as diabetes, higher disease severity, and elevated markers of inflammation.1 Current therapeutic interventions do not appear to be improving in-hospital survival.1

Remestemcel-L has potential for use in the treatment of ARDS, which is the principal cause of death in COVID-19 infection.1 This is supported by recently published results from an investigator-initiated clinical study conducted in China which reported that allogeneic MSCs cured or significantly improved functional outcomes in all seven treated patients with severe COVID-19 pneumonia.2

Additionally, in post-hoc analyses of a 60-patient randomized controlled study in chronic obstructive pulmonary disease (COPD), remestemcel-L infusions were well tolerated, significantly reduced inflammatory biomarkers, and significantly improved pulmonary function in those patients with elevated inflammatory biomarkers. Since the same inflammatory biomarkers are also elevated in COVID-19, these data suggest that remestemcel-L could be useful in the treatment of patients with ARDS due to COVID-19.The COPD study results have been submitted for presentation at an international conference, with full results to be submitted for publication shortly.

Remestemcel-L is being studied in numerous clinical trials across several inflammatory conditions, including in elderly patients with lung disease and adults and children with steroid-refractory acute graft versus host disease (aGVHD).3-5 This product candidate is currently being reviewed by the United States Food and Drug Administration (FDA) for potential approval in the treatment of children with steroid-refractory aGVHD.

Remestemcel-L Remestemcel-L is being developed for rare pediatric and adult inflammatory conditions. It is an investigational therapy comprising culture-expanded MSCs derived from the bone marrow of an unrelated donor and is administered in a series of intravenous infusions. Remestemcel-L is believed to have immunomodulatory properties to counteract the inflammatory processes that are implicated in several diseases by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

Intellectual PropertyMesoblasts intellectual property (IP) portfolio encompasses over 1,000 patents or patent applications in all major markets and includes the use of MSCs obtained from any source for patients with acute respiratory distress syndrome (ARDS),and for inflammatory lung disease due to coronavirus (COVID-19), influenza and other viruses. Additionally, these patents cover Mesoblasts manufacturing processes that yield industrial-scale cellular medicines.This IP position is expected to provide Mesoblast with substantial commercial advantages as it develops its product candidates for these conditions.

References1. Liu Y et al. Clinical features and progression of acute respiratory distress syndrome in coronavirus disease 2019. Medrxiv 2020; https://doi.org/10.1101/2020.02.17.200241662. Leng Z, et al. Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia[J]. Aging and Disease, 10.14336/AD.2020.02283. Kurtzberg J et al. Annual Meeting of the American Society for Transplantation Cell Therapy, 2020.4. Chaudhury S et al. A Phase 3 Single-Arm, Prospective Study of Remestemcel-L, Ex-Vivo Cultured Adult Human Mesenchymal Stromal Cells, for the Treatment of Steroid Refractory Acute GVHD in Pediatric Patients. Biol Blood Marrow Transplant 2018; 24:S119S290.5. Kurtzberg J et al. Allogeneic human mesenchymal stem cell therapy (remestemcel-L, Prochymal) as a rescue agent for severe refractory acute graft-versus-host disease in pediatric patients. Biol Blood Marrow Transplant. 2014 Feb;20(2):229-35.

About MesoblastMesoblast Limited (Nasdaq: MESO; ASX: MSB) is a world leader in developing allogeneic (off-the-shelf) cellular medicines. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of commercial products and late-stage product candidates. Mesoblasts proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Mesoblast has filed a Biologics License Application to the United States Food and Drug Administration (FDA) to seek approval of its product candidate RYONCIL (remestemcel-L) for steroid-refractory acute graft versus host disease (acute GvHD). Remestemcel-L is also being developed for other rare diseases. Mesoblast is completing Phase 3 trials for its product candidates for advanced heart failure and chronic low back pain. If approved, RYONCIL is expected to be launched in the United States in 2020 for pediatric steroid-refractory acute GVHD. Two products have been commercialized in Japan and Europe by Mesoblasts licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

Forward-Looking StatementsThis announcement includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. Forward- looking statements include, but are not limited to, statements about: the initiation, timing, progress and results of Mesoblasts preclinical and clinical studies, and Mesoblasts research and development programs; Mesoblasts ability to advance product candidates into, enroll and successfully complete, clinical studies, including multi-national clinical trials; Mesoblasts ability to advance its manufacturing capabilities; the timing or likelihood of regulatory filings and approvals, manufacturing activities and product marketing activities, if any; the commercialization of Mesoblasts product candidates, if approved; regulatory or public perceptions and market acceptance surrounding the use of stem-cell based therapies; the potential for Mesoblasts product candidates, if any are approved, to be withdrawn from the market due to patient adverse events or deaths; the potential benefits of strategic collaboration agreements and Mesoblasts ability to enter into and maintain established strategic collaborations; Mesoblasts ability to establish and maintain intellectual property on its product candidates and Mesoblasts ability to successfully defend these in cases of alleged infringement; the scope of protection Mesoblast is able to establish and maintain for intellectual property rights covering its product candidates and technology; estimates of Mesoblasts expenses, future revenues, capital requirements and its needs for additional financing; Mesoblasts financial performance; developments relating to Mesoblasts competitors and industry; and the pricing and reimbursement of Mesoblasts product candidates, if approved. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblasts actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. We do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

Release authorized by the Chief Executive.

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Mesoblast To Evaluate Anti-Inflammatory Cell Therapy Remestemcel-L For Treatment Of COVID-19 Lung Disease - BioSpace

Genes may determine what characteristics are passed down from parent to offspring, but each cell expresses these genes differently based on external epigenetic modifications. Epigenetics dont alter the gene sequence (genotype), but they do influence cell behavior and function (phenotype). The study of epigenetics helps us understand how phenotypic changes lead to disease, stem cell differentiation, and essentially, what drives the fate of every cell in the human body.The epigenome is not consistent between cells, or even between cells of the same type. Individual modifications come and go throughout a cells lifetime. Therefore, scientists are faced with the steep challenge as they try to decipher the role of epigenetics in disease and development.[i] Understanding intercellular heterogeneity is key here. The epigenome must be examined at single-cell resolution.

Now, with the advancement of single-cell sequencing methods like the single-cell assay for transposase accessible chromatin (scATAC-seq), researchers have access to sophisticated techniques to map large cell populations, one cell at a time. The resulting epigenomic information provides unprecedented insight into the different cell types that come together to form organs and organ systems, as well as pathogenic modifications associated with disease.

Every single cell has unique epigenomic instructions that guide how it expresses its genes and these instructions are subject to change. A map locating epigenetic modifications in the genome would help scientists understand how epigenetics drives cellular differentiation. But until recently, epigenetic assays mainly focused on select regions of DNA or gave bulk results across an entire sample of cells.[ii] These assays were not designed to detect epigenetic patterns in individual cells.

Single-cell tools like scATAC-seq help us get a grasp on intracellular heterogeneity, differentiate between cell populations and map the role of epigenetics in the larger context of an organism. By building a collection of scATAC-seq data, scientists have begun generating a cell atlas to provide insight into the role of epigenetics during the intricate biological processes that occur throughout the human lifetime.

During ATAC-seq, a hyperactive transposase mutant, Tn5, binds to open chromatin (euchromatin) regions. Wherever Tn5 binds, it cleaves the DNA and attaches sequencing adapters. Then, after PCR amplification, ATAC fragments are sequenced to identify open chromatin regions. ATAC results indicate where nucleosomes are typically positioned in the cell sample and which regions of the genome are open for transcription factors to bind. As such, scientists use ATAC-Seq as a first-pass screening approach to identify changes in chromatin accessibility between samples.

ATAC-seq has many practical applications, but it cant account for the cell-to-cell variability thats often an important aspect of developmental processes and disease. So, researchers developed a new assay in which microfluidic technology is used to isolate individual cells before ATAC-seq.[iv] This assay provides epigenomic information at single cell resolution, earning it the name scATAC-seq.

The key to the scATAC-seq method is that it isolates genomes of individual cells early on to perform a separate ATAC-seq reaction on each individual cell. Then, open regions of the genome are cleaved by the Tn5 transposase, tagged with sequencing adapters and amplified with barcoded cell-identifying primers. Subsequently, the barcoded libraries of ATAC fragments, (each representing an individual cell) are pooled together and sequenced to reveal open chromatin regions of thousands of individual cells.

The first droplet-based iteration of the scATAC-seq method (dscATAC-seq) uses a single cell isolator to encapsulate thousands of individual nuclei in nanoliter-sized droplets for ATAC sequencing. It uses a custom Tn5 transposase to enhance library complexity and signal resolution. Compared to the original microfluidic method, the new workflow is faster and yields greater biological insight with less time and effort spent on sequencing. To demonstrate its power and potential, this technique has been used to conduct an unbiased analysis of the many different cell types and regulatory elements in a mouse brain. [v]

Figure 1:In scATAC-seq, droplet-based technology partitions thousands of whole cells or nuclei into individual nanoliter-sized droplets, enabling researchers to prepare a library of ATAC fragments for sequencing to reveal open chromatin regions. Credit:Bio-Rad Laboratories.

To capture single cell data on a truly massive scale, combinatorial indexing was next introduced into the dscATAC-seq workflow. This new method, called dsciATAC-seq, enables researchers to assess up to 50,000 cells in a single assay. Assaying a large volume of cells is possible because, in dsciATAC-seq, the hyperactive mutant transposase integrates a first set of barcodes as it cleaves open regions of chromatin in each nucleus. Because every cells DNA already carries a barcode, multiple cells can be loaded into a single droplet. Then, as usual, ATAC fragments are amplified with a second set of barcoded primers. After sequencing these fragments, the two sets of barcodes are used to derive epigenomic profiles for tens of thousands of cells.

Putting the dsciATAC-seq method to the test, researchers have studied immune cell clusters from human bone marrow derived cells to illustrate how the chromatin accessibility landscape in these cells changes according to different stimulants at the single cell level.5Although the number of cells that a single scATAC-seq experiment can evaluate has grown dramatically, it will take a continued concerted effort from scientists across many disciplines to create a comprehensive map of the human epigenome, encompassing data from trillions of cells.[vi] Furthermore, to help decode the patterns we find in the human epigenome, it may be valuable to gather information about the epigenomes of animals commonly used as research models. As each of these maps become increasingly detailed, scientists will gain a more thorough understanding of how biological process work and may apply this knowledge towards developing better treatments for complex diseases.

Reference:

[i] Egger, G., et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004, 429, 457463. doi:10.1038/nature02625[ii] DeAngelis, J. T., Farrington, W. J., & Tollefsbol, T. O. An overview of epigenetic assays. Molecular biotechnology, 2008, 38(2), 179183. doi:10.1007/s12033-007-9010-y[iii] Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods, 2013, 10(12):1213-8. doi: 10.1038/nmeth.2688.[iv] Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature, 2015, 523(7561):486-90. doi: 10.1038/nature14590.[v] Lareau, C.A., Duarte, F.M., Chew, J.G. et al. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature Biotechnology 37, 916924 (2019) doi:10.1038/s41587-019-0147-6.[vi] Bianconi, E., Piovesan, A., Facchin F., Beraudi, A., Casadei. R., Frabetti, F., Vitale, L., Pelleri, M., Tassani. S., Piva, F., Perez-Amodio, S, Strippoli, P. & Canaider, S. An estimation of the number of cells in the human body. Annals of Human Biology, 2013, 40:6, 463-471. doi: 10.3109/03014460.2013.807878.

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Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

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Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

The regional analysis covers:

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Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

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Stem Cell Therapy Market Report on Recent Adoption 2025 - 3rd Watch News