Stem Cells

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, as cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but currently their functional consequences in differentiating cells are scarcely described. An accurate assessment of the risk associated with these genetic variants in both research and clinical settings is therefore lacking. In this work, we established that one of these recurrent abnormalities, the loss of chromosome 18q, impairs neuroectoderm commitment and affects the cardiac progenitor differentiation of hESCs. We show that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, while transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show by gene expression analysis that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, including the WNT, NOTCH, JAK-STAT, TGF-beta and NF-kB pathways, suggesting that these cells are in an altered state of pluripotency.

Reprogramming somatic cells to pluripotency by repressing lineage-instructive transcription factors (TFs) alone has not been pursued because lineage specification is thought to be regulated by transcriptional regulatory networks (TRNs) comprising of multiple TFs rather than by single pivotal "gatekeeper" TFs. Utilizing an intra-species somatic cell hybrid model, we identified Snai2 and Prrx1 as the most critical determinants of mesenchymal commitment in rat embryonic fibroblasts (REFs) and demonstrate that siRNA-mediated knockdown of either of these master regulators is adequate to convert REFs into functional adipocytes, chondrocytes or osteocytes without requiring exogenous TFs or small molecule cocktails. Furthermore, knockdown of Snai2 alone proved sufficient to transform REFs to dedifferentiated pluripotent stem-like cells (dPSCs) that formed embryoid bodies capable of triple germ-layer differentiation. These findings suggest that inhibition of a single gatekeeper TF in a lineage committed cell is adequate for acquisition of cell plasticity and reprogramming without requiring permanent genetic modification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/999433v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1acdb83org.highwire.dtl.DTLVardef@17fb3ddorg.highwire.dtl.DTLVardef@f994c9org.highwire.dtl.DTLVardef@197541e_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic diagram depicting transdifferentiation of REFs into adipocytes, osteocytes, chondrocytes and dedifferentiation into MSCs on individual treatment with siSnai2 or siPrrx1. dPSCs were generated only in the siSnai2 group.

Mesenchymal stromal cells (MSCs) from a variety of tissue sources are widely investigated in clinical trials, and the MSCs are often administered immediately after thawing the cryopreserved product. While previous reports have examined the transcriptome of freshly-cultured MSCs from some tissues, little is known about the single-cell transcriptomic profiles of out-of-thaw MSCs from different tissue sources. Such understanding could help determine which tissue origins and delivery methods are best suited for specific indications. Here, we characterized cryopreserved MSCs, immediately post-thaw, from bone marrow (BM) and cord tissue (CT), using single-cell RNA sequencing (scRNA-seq). We show that out-of-thaw BM-vs. CT-MSCs have significant differences in gene expression. Gene-set enrichment analyses implied divergent functional potential. In addition, we show that MSC-batches can vary significantly in cell cycle status, suggesting different proliferative vs. immunomodulatory potentials. Our results provide a comprehensive single-cell transcriptomic landscape of clinically and industrially relevant MSC products. HighlightsO_LISingle cell gene expression comparison between Bone-marrow derived MSCs and Cord-tissue derived MSCs C_LIO_LIDonor effects and cell heterogeneity on tissue-specific MSC gene expression C_LIO_LISingle Cell Pooling Enhances Differential Expression Analysis for Bone marrow and Cord tissue MSC samples C_LIO_LIGene ontology reveals tissue specific unique molecular function and pathways C_LI

Interest in human mesenchymal stem cells (MSCs) as an immune therapy has been on the rise for the past two decades with cutting edge research yielding promising results, but there are currently no MSC therapies approved by the food and drug administration (FDA). Failure of MSCs to translate as a therapy has been reported by the National Cell Manufacturing Consortium (NCMC) to be due to a lack of reliable potency metrics and sufficient understanding of the mechanism of action. Here we show that cell membrane components are a good candidate to interrogate the MSC immunomodulatory mechanism of action and provide a method to increase MSC potency through the sphingolipid pathway. We found that high and low indolamine-2,3-deoxygenase (IDO) potency cells have distinct morphological signatures that is also reflected in the sphingolipid activity, with low IDO potency cell lines having low sphingomyelinase activity and high IDO potency cell lines having high sphingomyelinase activity. Perturbation of the salvage pathway with the addition of exogenous neutral sphingomyelinase not only shifted morphological signatures to a high potency profile, but also significantly increased IDO activity within both high and low IDO potency donors. These results provide a proof of concept for the engineering of MSC immunomodulation and provides further evidence for the role sphingolipids in MSC immunomodulation that can enable further investigation.

Mesenchymal stem cells (MSCs) cultured in hypoxic conditions have been suggested to have more therapeutic efficacy than those cultured under normoxic conditions, and there is growing interest in using hypoxic MSCs for clinical treatment, particularly human umbilical cord (hUC)-MSCs. We investigated how hUC-MSCs and human bone marrow (hBM)-MSCs change from normoxia to hypoxia (1% O2) for 2 weeks of culture. In the growth speed and population doubling time, hUC-MSCs cultured under hypoxia exhibited a significantly higher proliferation rate beyond cancerous cells, such as human glioblastoma and breast cancer cells, while hBM-MSCs did not show a significant difference between normoxia and hypoxia, and were statistically slower than these cancerous cells. Notably, hypoxic hUC-MSCs showed upregulation of genes related to metabolic reprogramming (cholesterol biosynthesis and fatty acid metabolism pathways) and cancer stem cell-like phenotype (factors related to Wnt and Hedgehog signaling pathways, cell proliferation drivers, and apoptosis-resistance), and lesser migration and homing to the traumatic brain injury than normoxic hUC-MSCs after intravenous injection. Thus, whether hUC-MSCs cultured under hypoxia offer clinical benefits and use are safe, given their extremely accelerated proliferation rate and partial cancer stem cell-like traits, requires comprehensive and careful investigation.

BackgroundMesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A-to assess the impact on MSC characteristics when different laboratories performed analysis on the same MSC material using harmonised culture conditions and B-to understand source-specific differences. MethodsThree independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). ResultsBy implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. ConclusionThese results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of standardised culture procedures can reduce but not eliminate inter-lab and operator differences. HighlightsIn this study, we have: - Provided a harmonised manufacturing workflow that has demonstrated reproducible results across three independent laboratories when expanding MSCs. - Defined a multi-assay matrix capable of identifying functional differences in terms of angiogenesis, wound healing abilities and immunosuppressive properties. - Demonstrated similar in vivo biodistribution properties regardless of cell origin.

Summary/AbstractHuman pluripotent stem cells are a tremendous tool to model early human development and disease including their use in the in vitro generation of blood cell fates. Hematopoietic progenitors and stem cells are the primary source of blood and the immune system from early development to adulthood and arise through successive waves of hemogenic mesoderm either in the yolk sac or embryo proper. Researchers have long sought a tractable human model for observing and distinguishing these waves of hematopoiesis in the dish for human developmental and disease modeling. Here we report a high-efficiency method for differentiating human pluripotent stem cells into an aorta-gonad-mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells. The hematopoietic progenitor and stem cells exhibit robust multilineage in vitro colony forming potential. Gene expression analysis and single cell sequencing strongly support the developmental timing and notion that the pluripotent stem cell derived hematopoietic stem and progenitors are strikingly like bone fide hematopoietic stem cells. The hematopoietic progenitors can be subsequently differentiated into polarized macrophage and T-cells in vitro. Minimal silencing was observed upon differentiation of the pluripotent stem cells to hematopoietic lineages when conducting gene editing. Finally, upon engraftment into immunodeficient animals the hematopoietic progenitors and stem cells differentiate into multiple lineages including B-cells, T-cells, NK-cells, and monocytes.

IntroductionCyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM and UC upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility. MethodsMSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of three healthy female donors (BM-MSC-2-4), and from umbilical cords of two healthy male term infants (UC-MSC-1-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC and UC-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures. Given robust upregulation of CCL2 mRNA expression in HESCs exposed to the BM- and UC-MSC secretomes, transwell migration and invasion assays were performed to determine the effect of recombinant CCL2 on HESC motility. Statistical significance was defined as p<0.05. ResultsIndirect co-culture of HESCs with BM- or UC-MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. However, effects on cellular proliferation varied among the MSC donors. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,139 genes with FDR < 0.05. There was overlap among 4350 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2. Within four biological pathways enriched in HESCs, 4 genes (CCL2, HGF, PLAU, and BDKRB2) were differentially expressed in HESCs that had been cocultured with BM-MSC-1 and BM-MSC-2. qRT-PCR showed significantly increased mRNA expression of CCL2 in HESCs exposed to BM-MSC-1 (5-fold) and BM-MSC-2 (7.7-fold). In contrast, the increase in HGF expression was significant after exposure to BM-MSC-2 (1.8-fold) but not BM-MSC-1. Exposure to the UC-MSC secretome had similar effects on HESC-derived CCL2 and HGF levels. CCL2 expression was significantly increased (6.5-fold) by UC-MSC-2 but not by UC-MSC-1; HGF expression was significantly increased (1.6-fold) by UC-MSC-2 but not by UC-MSC-1. Validation studies indicated that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration (1.2-fold) and invasion (1.4-fold). These data suggest that CCL2 is a key factor in mediating MSC-induced HESC motility. ConclusionIncreased HESC motility by the secretome of BM- and UC-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by upregulated CCL2 expression in HESC. Together, our data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.

Ionizing radiation (IR) and chemotherapies severely impair hematopoietic stem and progenitor cell (HSPC) function, causing bone marrow failure and secondary malignancies. Mesenchymal stromal cells (MSCs) within the hematopoietic niche support HSPC survival and regeneration, but the underlying pro-survival mechanisms remain incompletely understood. Here, we show that MSCs suppress IR-induced apoptosis in human HSPCs and preserve their regenerative capacity. Transcriptomic analyses identified a robust induction of CREB target genes in HSPCs upon MSC contact, driven by MSC-secreted prostaglandin E2 (PGE2) via cAMP signaling. While MSC-derived PGE2 predominantly protected quiescent HSPCs from IR-induced apoptosis, direct pharmacological elevation of cAMP with Forskolin/IBMX (FSKN/IBMX) effectively shielded both quiescent and cycling HSPCs, significantly enhancing their engraftment and self-renewal. Mechanistically, cAMP pathway activation reduced pro-apoptotic ASPP1 and PUMA expression, elevated p21, and stabilized anti-apoptotic MCL1 and BCL-XL proteins. Collectively, our study uncovers an MSC-driven PGE2/CREB signaling pathway critical for human HSPC regeneration, highlighting pharmacological modulation of this axis as a promising strategy to mitigate DNA damage-induced myelosuppression and improve transplantation outcomes.

Generation of pure pancreatic progenitor cells (PPs) is critical for clinical translation of stem cell derived islets. Herein, we performed PP differentiation with and without AKT/P70 inhibitor AT7867 and characterized the resulting cells at protein and transcript level in vitro and in vivo upon transplantation into diabetic mice. AT7867 treatment increased the percentage of PDX1+NKX6.1+ (-AT7867: 50.9% [IQR 48.9%-53.8%]; +AT7867: 90.8% [IQR 88.9%-93.7%]; p=0.0021) and PDX1+GP2+ PP cells (-AT7867: 39.22% [IQR 36.7%-44.1%; +AT7867: 90.0% [IQR 88.2%-93.6%]; p=0.0021). Transcriptionally, AT7867 treatment significantly upregulated PDX1 (p=0.0001), NKX6.1 (p=0.0005) and GP2 (p=0.002) expression compared to controls, while off-target markers PODXL (p<0.0001) and TBX2 (p <0.0001) were significantly downregulated. Transplantation of AT7867 treated PPs resulted in faster hyperglycemia reversal in diabetic mice compared to controls (time and group: p<0.0001). Overall, our data shows that AT7867 enhances PP cell differentiation leading to accelerated diabetes reversal.

Induced pluripotent stem cells (iPSCs) can provide a biological resource for functional and conservation research in various species. This expectation has led to generation of iPSCs from various species, including those identified as endangered species. However, the understanding of species variation in mammalian iPSCs is largely unknown. Here, to gain insight into the species variation in iPSCs, we the first generated iPSCs from the endangered species Grevys zebra (Equus grevyi; gz-iPSCs) for the first time in the world. We isolated primary fibroblasts cell from an individual that had died of natural causes at a zoo and reprogrammed the fibroblasts into iPSCs. We confirmed their pluripotency and differentiation potential and performed RNA sequencing analysis. The gz-iPSC transcriptome showed that the generated gz-iPSCs robustly expressed genes associated with pluripotency and reprogramming processes, including epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions. Comparative transcriptomics with other species revealed patterns of gene expression among mammalian PSCs and detected evolutionary conservation of pluripotency-associated genes and the plausible importance of the translation process. This study provides new insights into the evolution of mammalian PSCs, and the species conservation and variation of PSCs will advance our understanding of the early development of mammals.

Endometrial mesenchymal-like stem cells (eMSCs) are adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b+CD146+) have been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity and molecular program controlling their expansion and differentiation in vitro remains largely unclear. In this study, we applied 10X genomics single-cell RNA sequencing to eMSCs cultured in vitro after microbeading from 7 donors to investigate cellular heterogeneity in an unbiased manner. Corresponding clonogenic progenies of eMSCs after culture for 14 days were also sequenced to construct the in vitro differentiation trajectory of eMSCs. Transcriptomic expression based clustering revealed several subpopulations in eMSCs. Each subpopulation manifested distinct functional characteristics associated with immunomodulation, proliferation, extracellular matrix organization and cell differentiation. Pseudotime trajectory analysis on eMSCs and their differentiated progenies identified in vitro differentiation hierarchy of eMSCs. Further ligand-receptor pair analysis found that WNT signaling, NOTCH signaling, TGF-beta signaling and FGF signaling were important regulatory pathways for eMSC self-renewal and differentiation. By comparing eMSCs to Whartons Jelly MSCs and adipose-derived MSCs, we found these 3 kinds of MSCs expressed largely overlapping differentiation (CD) genes and highly variable genes. In summary, we reveal for the first time high molecular and cellular heterogeneity in cultured eMSCs, and identify the key signaling pathways that may be important for eMSC differentiation.

1In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery and increased activity of the senescence-associated {beta}-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

Hematopoietic stem cell (HSC) transplantation is extensively studied in mouse models, but their limited scale presents challenges for effective engraftment and comprehensive evaluations. Rats, due to their larger size and anatomical similarity to humans, offer a promising alternative. In this study, we establish a rat model with the KitV834M mutation, mirroring KitW41 mice often used in KIT signaling and HSC research. KitV834M rats are viable and fertile, displaying anemia and mast cell depletion similar to KitW41 mice. The mutation affects myeloid cell proliferation and differentiation, as seen in the colony-forming unit granulocyte-macrophage assay. Importantly, KitV834M rats support donor rat-HSC engraftment without irradiation. Competitive transplantation assays reveal reduced reconstruction capacity in KitV834M HSCs. Leveraging the larger scale of this rat model enhances our understanding of HSC biology and transplantation dynamics, potentially advancing our knowledge in this field.

Pluripotent state can be established via reprogramming of somatic nuclei by factors within an oocyte or by ectopic expression of a few transgenes. Considered as being extensive and intensive, the full complement of genes to be reprogrammed, however, has never been defined, nor has the degree of reprogramming been determined quantitatively. Here, we propose a new concept of reprogramome, which is defined as the full complement of genes that need to be reprogrammed to the expression levels found in pluripotent stem cells (PSCs). This concept in combination with RNA-seq enables us to precisely profile reprogramome and sub-reprogramomes, and study the reprogramming process with the help of other available tools such as GO analyses. With reprogramming of human fibroblasts into PSCs as an example, we have defined the full complement of the human fibroblast-to-PSC reprogramome. Furthermore, our analyses of the reprogramome revealed that WNT pathways and genes with roles in cellular morphogenesis have to be extensively and intensely reprogrammed for the establishment of pluripotency. We further developed the first mathematical model to quantitate the overall reprogramming, as well as reprogramming in a specific cellular feature such as WNT signaling pathways and genes regulating cellular morphogenesis. We anticipate that our concept and mathematical model may be applied to study and quantitate other reprogramming (pluripotency reprogramming from other somatic cells, and lineage reprogramming), as well as transcriptional and epigenetic differences between any two types of cells including cancer cells and their normal counterparts.

Thymic epithelial cells (TECs) are a critical functional component of the thymuss ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpression of the transcription factor FOXN1 initiates reprogramming of fibroblasts into TEC-like cells (iTECs) that support T cell differentiation in culture or after transplant. In this study, we characterized iTEC reprogramming at the cellular and molecular level to determine how reprogramming proceeds and identify mechanisms that can be targeted for improving this process. These data show that iTEC reprogramming consists of discrete gene expression changes that differ in early and late reprogramming, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages, although mTEC differentiation is blocked at a progenitor stage. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows induction of mTEC differentiation. Finally, we show that a major difference between iTEC and fetal TEC is the expression of MHCII. This study supports future efforts to improve iTEC reprogramming for both research and translational uses.

During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

The crisis of biodiversity loss in the anthropogenic era requires new tools for studying non-model organisms. Elephants, for example, are both an endangered species and excellent models studying complex phenotypes like size, social behavior, and longevity, but they remain severely understudied. Here we report the first derivation of elephant (Elephas maximus) induced pluripotent stem cells (emiPSCs) achieved via a two-step process of chemical-media induction and colony selection, followed by overexpression of elephant transcription factors OCT4, SOX2, KLF4, MYC {+/-} NANOG and LIN28A, and modulation of the TP53 pathway. Since the seminal discovery of reprogramming by Shinya Yamanaka, iPSCs from many species including the functionally extinct northern white rhinocerous have been reported, but emiPSCs have remained elusive. While for multiple species the reprogramming protocol was adopted with little changes compared to model organisms like mouse and human, our emiPSC protocol requires a longer timeline and inhibition of TP53 expansion genes that are hypothesized to confer unique cancer resistance in elephants. iPSCs unlock tremendous potential to explore cell fate determination, cell and tissue development, cell therapies, drug screening, disease modeling, cancer development, gametogenesis and beyond to further our understanding of this iconic megafauna. This study opens new frontiers in advanced non-model organism cellular models for genetic rescue and conservation.

Aging leads to a decline in function of hematopoietic stem cells (HSCs) and increases susceptibility to hematological disease. We found CD61 to be highly expressed in aged HSCs. Here we investigate the role of CD61 in identifying distinct subpopulations of aged HSCs and assess how expression of CD61 affects stem cell function. We show that HSCs with high expression of CD61 are functionality superior and retain self-renewal capacity in serial transplantations. A population of aged HSCs with highest CD61 expression is functionally comparable to young HSCs. These CD61High HSCs display a notably higher quiescence compared to their CD61Low counterparts. We also show that CD61High and CD61Low HSCs are transcriptomically distinct populations within aged HSCs. Collectively, our research identifies CD61 as a key player in maintaining stem cell quiescence during aging, ensuring the preservation of their functional integrity and potential. Moreover, CD61 emerges as a marker to prospectively isolate a superior, highly dormant population of young and aged HSCs, making it a valuable tool both in fundamental and clinical research. HighlightsO_LICD61 expression marks a functionally superior population of long-term hematopoietic stem cells and plays a pivotal role in the maintenance of aged LT-HSCs by regulating their dormancy C_LIO_LIAged LT-HSCs with high expression of CD61 are functionally comparable to their young counterparts C_LI

This study investigated the molecular mechanisms underpinning the transition from primed to naive pluripotency in human pluripotent stem cells (hPSCs) by utilizing a dual genetic switch combining hormone-dependent STAT3-ERT2 and a chimeric GCSFR:gp130 receptor. Upon activation with tamoxifen and G-CSF, hPSCs demonstrated upregulation of naive markers, and sustained self-renewal independent of LIF. Transcriptomic analyses revealed distinct clusters of early response genes, with a subset of 26 genes significantly enriched in the human epiblast. Knockdown experiments further delineated essential regulators that modulate differentiation and proliferation during naive reprogramming including interferon gamma inducible protein (IFI)16 and interferon-induced transmembrane (IFITM) proteins. Moreover, functional assays using mutant chimeric GCSFR:gp130 receptor highlighted the critical role of JAK kinase recruitment in orchestrating epigenomic remodeling and early gene activation. This approach establishes a robust platform to dissect the signaling dynamics governing the accession of hPSCs to naive pluripotency.