Stem Cell Research & Therapy

BackgroundAcute brain injury is characterized by extensive tissue damage, resulting in neuronal loss and functional deficits in patients. The capacity of nerve tissue to self-regenerate is insufficient to repair damaged tissue, thus therapies based on exogenous cells are urgently needed. Human enteric glia (EG) have interesting intrinsic properties that make them a valuable candidate for regenerative medicine. In this long-term study, we investigated whether human EG treatment induces tissue repair and improves functional recovery in a rat model of brain injury. MethodsAcute brain injury was induced by malonate injection in the motor cortex of female rats, causing extensive tissue damage and long-lasting sensorimotor deficits. Human EG were isolated from gut tissue, expanded and administered intranasally in awake immunocompetent rats. To determine the long-term safety and efficacy of human EG treatment, longitudinal evaluation of sensorimotor function, post-mortem tissue regeneration and the fate of human EG were assessed thirty-six weeks after intranasal administration. ResultsTransplanted human EG satisfied the safety criteria, non-immunogenic and non-tumorigenic, required for cell therapy; they were well tolerated in immunocompetent rats, and induced sensorimotor improvement. Importantly, thirty-six weeks post-treatment, intranasally delivered human EG were detected in the rat brain, mainly in the injured motor cortex. This indicated that transplanted human EG migrated and successfully engrafted and integrated with the host tissue. Additionally, human EG induced tissue regeneration by enhancing endogenous angiogenesis and neurogenesis. Notably, thirty-six weeks after administration, human EG generated mature neurons that were enveloped by oligodendrocytes and formed synaptic connections with the host tissue. ConclusionsTransplanted human EG induced tissue repair and showed regenerative potential after brain injury. This is the first study demonstrating the feasibility, safety and efficacy of intranasal administration of human EG for treatment of brain injury.

IntroductionOsteogenesis Imperfecta (OI) is a rare genetic disorder of connective tissue, primarily caused by mutations in the COL1A1 or COL1A2 genes. Research is hampered by the limitations of primary patient-derived cells, which are obtained through invasive methods and have finite proliferative capacity and high variability. While induced pluripotent stem cell (iPSC) models exist, direct differentiation to osteoblasts is often inefficient and may not fully replicate disease characteristics. Our study aimed to develop and characterize a robust iPSC-derived mesenchymal stem cell (iMSC) model of OI using a simple two-step differentiation protocol to serve as a platform for disease modeling and drug screening. MethodsBone marrow MSCs (BMMSCs) were isolated from two OI patients with heterozygous missense mutations, in COL1A1 (c.2299G >A) and COL1A2 (c.982G>A). The patient BMMSCs were reprogrammed into iPSCs using an integration-free Sendai virus. The resulting OI-iPSCs were characterized for pluripotency via immunofluorescence and RT-PCR, trilineage differentiation potential, and karyotype analysis. A two-step protocol was used to differentiate the OI-iPSCs into OI-iMSCs via mesodermal lineage. The OI-iMSCs were then characterized for morphology, immunophenotype by flow cytometry, and trilineage differentiation capacity. Functional osteogenic differentiation was assessed by Alizarin Red S staining and RT-PCR analysis of key osteogenic genes. Sanger sequencing confirmed the retention of patient-specific mutations in the OI-iMSCs. ResultsPatient-derived BMMSCs were successfully differentiated into adipocytes and chondrocytes but showed impaired osteogenic potential. The reprogramming process successfully generated stable OI-iPSC lines that expressed key pluripotency markers, demonstrated trilineage potential, and maintained a normal karyotype. Critically, while direct osteogenic differentiation of these iPSCs failed to recapitulate the primary cell phenotype, the two-step differentiation protocol successfully produced a homogeneous population of iMSCs. These OI-iMSCs displayed characteristics of MSC surface markers (CD73+, CD90+, CD105+) and recapitulated the disease phenotype seen in the original patient BMMSCs. Specifically, COL1A1-iMSCs exhibited cellular rolling and detachment, while COL1A2-iMSCs showed poor mineralization during osteogenic differentiation. Both OI-iMSC lines showed significantly decreased calcium deposition and downregulation of key osteogenic genes (RUNX2, ALP, COL1) compared to controls. ConclusionOur study successfully established an iMSC-based cellular model of OI that recapitulates patient-specific disease phenotypes, including impaired osteogenic differentiation. The two-stage differentiation of iPSCs to iMSCs proved more reliable than direct osteogenic differentiation for modeling the disease. iMSC model circumvents the limitations of primary cells by providing a scalable and homogeneous source of patient-specific cells. Our platform offers a valuable and robust tool for investigating OI pathophysiology and for high-throughput screening of potential therapeutic molecules, advancing efforts toward personalized therapies

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

BackgroundHaematopoietic stem cell transplant (HCT) is a curative therapy for various paediatric conditions but is associated with significant morbidity and mortality, particularly in children requiring intensive care, facing delayed immune reconstitution or prolonged viral reactivation. Due to the rarity and heterogeneity of paediatric HCT, traditional randomised controlled trials are challenging. Adaptive platform trials (APTs), which evaluate multiple interventions across multiple subgroups, offer a solution, but typically rely on a shared short-term primary outcome that is relevant to clinicians, patients and families and can be used for all interventions/subgroups. No such outcome currently exists in paediatric HCT. In this article we propose and validate four novel ordinal outcomes to assess HCT-related morbidity and mortality within the first 100 days post-transplant for use in An adaptive platform trial Designed to Improve the COmplications, cost-effectiveness and health Outcomes for children receiving a stem cell Transplant (BANDICOOT) APT, designed. MethodsThe proposed outcomes were validated using real-world data from n=202 paediatric patients who underwent allogeneic HCT. The validation process included examining the distribution of patients across outcome categories, assessing the association with key long-term outcomes post HCT, and evaluating whether exposures with known efficacy had the expected associations with the proposed endpoints and whether the proportional odds assumption used in the analysis is likely to be reasonable. We also sought feedback on the outcomes from clinicians and family representatives. ResultsThe results showed strong associations between each ordinal endpoint and long-term HCT complications, including relapse, chronic-graft-versus-host disease, and death. Associations with key exposures (e.g. donor type and positive minimal residual disease pre HCT) were mostly in the expected direction. Moreover, expert feedback from clinicians and family representatives indicates that one of the proposed endpoints, which incorporates viral-related patient states and single/multi-organ support days, was both feasible and relevant for use in BANDICOOT. ConclusionsThe selected ordinal endpoint provides a robust and clinically applicable framework for evaluating interventions in paediatric HCT that offers broad applicability across various HCT outcomes.

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

Abnormalities in skeletal muscle repair lead to poor function and complications such as scarring or heterotopic ossification (HO). Here, we use fibrodysplasia ossificans progressiva (FOP), a disease of progressive HO caused by ACVR1R206H (Activin receptor type-1 receptor) mutation, to elucidate how ACVR1 affects skeletal muscle repair. Rare and unique primary FOP human muscle stem cells (Hu-MuSCs) isolated from cadaveric skeletal muscle demonstrated increased ECM marker expression, and showed skeletal muscle-specific impaired engraftment and regeneration ability. Human induced pluripotent stem cell (iPSC)-derived muscle stem/progenitor cells (iMPCs) Single cell transcriptome analyses from FOP also revealed unusually increased ECM and osteogenic marker expression compared to control iMPCs. These results show that iMPCs can recapitulate many aspects of Hu-MuSCs for detailed in vitro study, that ACVR1 is a key regulator of Hu-MuSC function and skeletal muscle repair; and that ACVR1 activation in iMPCs or Hu-MuSCs contributes to HO by changing the local tissue environment.

Boosting stem cell resilience against an extrinsically harsh recipient environment is critical to therapeutic efficiency of stem cell-based transplantation innovations in liver disease contexts. We aimed to establish the efficacy of a transient plasmid-based preconditioning strategy to boost mesenchymal stromal cells (MSCs) capacity for anti-inflammation/antioxidant defense and paracrine actions on recipient hepatocytes. In MSCs, the master antioxidant regulator Nrf2 was found to bind directly to the antioxidant response element in the DKK1 promoter region. Activation of Nrf2 and DKK1 enhanced the anti-stress capacities of MSCs in vitro. In an acute-on-chronic liver failure (ACLF) murine model, transient co-overexpression of Nrf2 and DKK1 via plasmid transfection markedly improved MSC resilience against inflammatory and oxidative assaults, boosted MSC transplantation efficacy and promoted recipient liver regeneration because of a shift from the activation of the anti-regenerative IFN-{gamma}/STAT1 pathway to the pro-regenerative IL-6/STAT3 pathway in the liver. Moreover, specific ablation of DKK1 receptor CKAP4 but not LRP6 in recipient hepatocytes nullified therapeutic benefits from MSC transplantation. In long-term observations, tumorigenicity was undetected in mice following transplantation of such transiently preconditioned MCSs. In conclusion, co-stimulation of Nrf2/DKK1 signaling decisively and safely improves the efficacy of human MSC-based therapies in mouse ACLF models through apparently CKAP4-dependent paracrine mechanisms.

Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. In light of their unique expansion capabilities and their potency to be driven towards a chosen cell fate, pluripotent stem cells (PSC) are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failures and restore liver functions. In this study, we generated human PSC-derived immature hepatic progenitors (GStemHep) using current good manufacturing practice (cGMP) compliant conditions from PSC amplification, hepatic differentiation to cell cryopreservation. These GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor (HGF) and do not express MHC type I or II. The therapeutic potential of GStemHep was assessed in two clinically relevant models of acute liver failure. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute live failure, both in immunodeficient and immunocompetent animals in absence of immunosuppression. The mode of action was studied by several analytical methods including unbiased proteomic analyses. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation and a rapid cytoprotective effect. Therapeutic biological effects were observed as soon as 3 hours post-cell transplantation with reduction in serum transaminases and in liver necrosis. Mode of action of GStemHep relies on alleviation of inhibition factors of liver regeneration, increase in proliferationpromoting factors and decrease liver inflammation. In conclusion, we generated cGMP-compliant human PSC-derived immature hepatic progenitors that were highly effective in treating acute liver failure. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in absence of immunosuppression for a human PSC-based regenerative medicine of acute liver injuries.

Human fetal mesenchymal stem cells (hfMSCs) present advantageous characteristics compared to their adult counterparts and have emerged as potent cells in the field of regenerative medicine. In the context of skeletal regeneration, human amniotic fluid stem cells (AFSCs) have been shown to improve the quality and structure of the bone extracellular matrix in an experimental model of severe osteogenesis imperfecta. However, primary hfMSCs undergo replicative senescence during in vitro expansion, along with a progressive decrease in plasticity and tissue repair potential. To overcome this challenge, we rejuvenated AFSC to pluripotency using non-integrative episomal reprogramming and subsequently re-derived the cells towards the mesoderm to obtain induced MSCs (iMSCs). We found that iMSCs have a slower proliferation rate compared to their parental cell line (40h{+/-}2h vs. 29h{+/-}5h) but retain the multipotency and differentiation potential characteristic of MSCs. Comparative genomic analysis revealed that iMSCs express higher levels of genes involved in maintaining stemness, cell signaling, adhesion and migration, as well as promoting osteoblast differentiation, whilst AFSC expressed higher levels of genes involved in cell proliferation. In addition, iMSCs secrete small extracellular vesicles (iEVs) that have the potential to stimulate fibroblast migration, a key process in tissue repair and wound healing. Together, these data suggest that resetting the epigenetic clock of primary hfMSCs may represent a promising strategy to address the limitations associated with primary cell use and enhance their therapeutic potential.

BackgroundMesenchymal stem cells (MSCs) offer clinical promise for use in cell therapy approaches for regenerative medicine. A therapeutic challenge is that MSCs from different tissues are phenotypically and functionally distinct. Therefore, this study aims to molecularly characterize oral-derived MSCs by defining one of the three hallmarks of MSCs, differentiation potential, to discern their true molecular identities. MethodsThree different populations of oral tissue MSCs (from alveolar bone-aBMSCs; from dental pulp-DPSCs; and from gingiva-GMSCs) from three different patients were isolated and cultured. These MSCs were characterized for their stemness by flow cytometry and multi-differentiation potential, and their RNA was also isolated and analyzed quantitatively with RNA sequencing. Total mRNA-seq was performed and differentially expressed genes (DEGs) were identified in pairwise (DPSCs vs. aBMSCs, GMSCs vs. aBMSCs, and GMSCs vs. DPSCs) and tissue-specific comparisons (aBMSCs vs. Others, DPSCs vs. Others, GMSCs vs. Others) (FDR, p<0.05). Further, these DEGs, either common between MSC populations or unique to a specific MSC population, were evaluated for pathways and biological processes ResultsaBMSCs, DPSCs, and GMSCs were successfully isolated and characterized. The tissue-specific comparison revealed that DEGs were most numerous in DPSCs (693 genes) as compared to aBMSCs (103 genes) or DPSCs (232 genes). Statistically significant DEGs through pairwise comparisons present higher numbers in GMSCs vs. DPSCs (627) as compared to either DPSCs vs aBMSCs (286) or GMSCs vs. aBMSCs (82). Further analysis found that RUNX2, IBSP, SOX6, ACAN, and VCAM1 were significantly upregulated in aBMSCs. In DPSCs, BMP4 and IL6 were significantly downregulated, whereas AXL and NES were significantly upregulated. In GMSCs, AGPT1, SEMA4D, and PGDFA were significantly downregulated. Additionally, MAPK, PI3-AKT, and RAS signaling pathways were significantly regulated in GMSCs. Interestingly, aBMSCs and DPSCs revealed positive regulation of osteoblast differentiation, whereas GMSCs revealed negative regulation of osteoblast differentiation. DPSCs also revealed negative regulation of angiogenesis. ConclusionsOral-derived MSCs have an inherent "landscape" of differentiation defined by their tissue of origin; yet this differentiation potential can be modulated by their microenvironment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/606413v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@60aeceorg.highwire.dtl.DTLVardef@17480a2org.highwire.dtl.DTLVardef@1a9315aorg.highwire.dtl.DTLVardef@e74d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundVascular mural cells (MC) are essential components of vasculature, playing critical roles in tissue regeneration and cell therapy. The use of animal derived ancillary materials, like fetal bovine serum (FBS), in the induction of MC from human pluripotent stem cells (hPSCs), represents one of the biggest limitations to guarantee preclinical safety standards required to use this products in clinical settings. This study aimed to validate human platelet lysate (hPL) as a serum-free alternative for MC differentiation from hPSCs. MethodsComparison of MC differentiation efficiency from hiPSC using FBS vs hPL supplemented cultures was performed, along with functionality and gene expression assessment through bulk RNA sequencing. ResultsOptimization of hPL concentration identified hPL1% as the most effective condition, yielding PDGFR-{beta}+/CNN1+ MC, with a comparable efficiency to FBS10% and similar interaction with endothelial cells in vascular formation assays. However, distinct transcriptional profiles revealed that FBS10% and hPL1% drive differentiation toward different MC subphenotypes; hPL1% promoted contractile gene expression, while FBS10% enriched extracellular matrix pathways. Higher hPL concentrations further shifted differentiation toward cardiomyocytes. ConclusionIn monolayer in vitro differentiation of MC from hiPSC, the differentiation efficiency using hPL 1% supplementation is equivalent to FBS 10%, while supporting a more contractile phenotype. These findings establish hPL as a xeno-minimized, clinically compliant substitute for FBS for hPSC-derived MC differentiation, an important breakthrough for regenerative medicine.

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Volumetric muscle loss (VML) injuries result in chronic fibrosis, inflammation, and persistent functional deficits. Fibro-adipogenic progenitor (FAP) cells are a heterogeneous, muscle-resident stromal cell population that play a crucial role in muscle regeneration, but also contribute to fibrosis in muscle disease. The role of FAPs in VML is not well established and may be critical target to ensure functional muscle regeneration after VML. We utilized a VML model in the mouse quadriceps to study the location, secretome, surface marker distribution, gene expression, and single-cell transcriptional profile of FAPs after VML. After VML, a subpopulation of FAPs highly expressed {beta}1-integrin and were elevated in the post-VML muscle tissue; these FAPs had increased fibrotic gene expression and increased myofibroblast differentiation potential. Transforming growth factor-{beta}1 (TGF-{beta}1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) were identified as secreted proteins from VML derived FAPs that produced both pro-fibrotic and anti-myogenic signaling. These data establish an aberrant FAP sub-population that are elevated in VML injury and provides novel targets for future scarless muscle regeneration in VML.

Human iPSC-derived neuronal networks are increasingly being employed in basic and applied research to enhance translation. Astrocytes are essential for neuronal network function, but are often not included, or replaced with mouse astrocytes, which compromises translation. Current protocols produce hiPSC-derived astrocytes by stepwise differentiation using small molecules and cytokines, or by forward programming by inducing transcription factors introduced by lentiviral transduction. Here we created a stable, inducible hiPSC line capable of producing iAstrocytes by introducing the transcription factors NFIB and SOX9 into the AAVS1 locus of the BIHi005-A hiPSC line. iAstrocytes induced from this line upregulated astrocytic genes over four weeks in culture, expressed GFAP and S100B and exhibited spontaneous calcium waves and responses to ATP and CPA. In co-cultures, iAstrocytes supported the growth and function of mature iNeuron networks. Pre- and post-synaptic markers and synchronous neuronal activity measured by high-density multi-electrode array recordings and neuronal calcium imaging, appeared by four weeks. The use of iAstrocytes will help to standardize the use of human astrocytes to support human neural networks and enhance translation.

It is still unclear if immune responses will compromise the large scale utilization of cell therapies derived from human induced pluripotent stem cells (hiPSCs). To answer this question, we used humanized mouse models and evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were rejected, engraftment of autologous cells was tolerated, indicating reprogramming and differentiation procedures are not immunogenic. We also demonstrated that hiPSC-derived myogenic progenitors, in opposition to hiPSCs, are not targeted by natural killer (NK) cells both in vitro and in vivo. Yet, adoptive transfer of NK cells can prevent the formation of hiPSC-derived teratoma. Overall, our findings suggest that hiPSC-derived muscular therapies will be tolerated in presence of a competent human immune system and highlight the risk of forming a teratoma if using partially differentiated autologous human cells. HighlightsO_LIhiPSC-derived myofibers are tolerated in autologous humanized mouse models C_LIO_LIInfiltration of autologous T cells is not predictive of successful skeletal muscle engraftment C_LIO_LIAdoptive transfer of NK cells prevents the formation of hiPSCs derived teratomas C_LIO_LINK cells are unable to reject established teratomas C_LI

BackgroundTwo types of mammalian pluripotent stem cells (PSC), i.e. naive and primed possess distinct cellular characteristics. It is largely unknown how these differences are generated during naive-to-primed transition process. We have established a robust in vitro transition system using a Wnt inhibitor for the first time and analyzed dynamic changes in cellular status via single-cell RNA-sequencing and C1 CAGE analyses. ResultsAnalysis of known marker genes suggested that the cell transition process progresses as expected. However, cluster analyses revealed a sudden increase in expression profile diversities three and four days after induction of the transition. These expression diversities can be reconciled by the presence of two subpopulations with distinct transcription profiles emerging at these time points. One of the subpopulations appears transiently, and surprisingly these cells showed a global downregulation of gene expression. Moreover, initiation of random X chromosome inactivation (XCI) coincides with the appearance of these transient cells. The other subpopulation can be maintained as a stem cell line and possesses expression profiles more similar to those of primed epiblast stem cells (EpiSC) than embryonic stem cells (ESC). However, there are important differences in gene expression related to epithelial-mesenchymal transition (EMT), suggesting that this subpopulation may represent a novel pluripotent state that has an intermediate cellular phenotype between ESC and EpiSC. ConclusionsThese findings should contribute to our understanding of the establishment and maintenance of distinct differentiation statuses of mammalian PSCs and provide new insights into the pluripotency spectrum in general.

ABSTRACT/ SUMMARYOligodendrocytes (OLs) are the only myelinating glia in the central nervous system (CNS). In congenital myelin disorders, OL dysfunction or death results in loss of myelin. This causes progressive and irreversible impairment to motor and cognitive functions, and is amongst the most disabling neurological disorder. Neonatal engraftment by glial progenitor cells (GPCs) allows the robust myelination of congenitally dysmyelinated brain, thereby preserving brain function and quality of life of patients. However, endogenous sources of glial progenitors are hard to obtain without causing secondary injury, while use of exogenous sources such as embryonic stem cells and induced-pluripotent stem cells face considerable ethical and safety issues. To circumvent such hurdles, we asked whether NG2+ cells in the bone marrow could be a potential cell source for GPCs. We successfully generated glial progenitor cells (GPCs) from human bone marrow stromal cells (hBMSCs) from 3 donors using a 14- day induction protocol. The generated hBMSC-GPCs were highly enriched in OPC marker expression, including OLIG2, PDGFR, NG2, SOX10 and O4, and showed efficient differentiation into myelinogenic oligodendrocytes when transplanted into postnatal day 7 (P7) myelin-deficient shiverer mice. Remyelination of the shiverer mouse brain significantly extended lifespan and improved motor function. The novel induction protocol described here provides a method for fast, simple and effective glial therapy for myelin disorders, overcoming existent hurdles of cell source restriction and time frame requirement. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/658997v8_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@104a62corg.highwire.dtl.DTLVardef@399a67org.highwire.dtl.DTLVardef@1c81ee1org.highwire.dtl.DTLVardef@16374da_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Human bone marrow stromal cells (BMSCs) can be programmed to myelinating glia (GPCs, glial progenitor cells), via a novel 14-day in vitro induction protocol - Transplantation of these hBM-GPCs robustly remyelinated myelin deficient shiverer mice. - hBM-GPC transplant significantly extended lifespan, increased body weight and improved motor function

BackgroundThe colonic mucosa consists of cell populations derived from multiple lineages. Induced pluripotent stem cells (iPSCs) are capable of generating large numbers of differentiated cells from any lineage. Thus, iPSCs are highly versatile for derivation of intestinal cells for generation of colonic mucosal tissue for clinical and biological applications. ObjectiveWe set out to create a human iPSC (hiPSC) multi-lineage co-differentiation platform capable of generating colonic mucosal tissue in vitro. DesignWe used hiPSCs and designed a differentiation protocol consisting of small molecules and recombinant growth factors to generate multiple cell lineages. Cells were seeded onto collagen hydrogels (forming colonic patches - CoPs) and modulated with multiple growth factors important in intestinal biology. CoPs were transplanted into immunosuppressed mice. Generated cells and tissues were profiled with transcriptomic analysis. ResultshiPSC co-differentiation led to multiple intestinal epithelial, mesenchymal and endothelial cell populations. Seeded onto collagen scaffolds these cells created CoPs, which were transplanted into mouse subcutis. Engrafted CoPs developed into normal-looking colonic mucosa containing epithelial crypts (with enterocytes, goblet cells and neuroendocrine cells), multiple lamina propria-resident stromal populations and muscularis mucosae smooth muscle. They anastomosed to murine vasculature and maintained in-vitro for several weeks. We demonstrated that CoPs respond to known signalling pathways important in colonic mucosal biology and fibrogenesis, showing potential to provide a complex model of colonic pathobiology. ConclusionThis platform could offer an accurate model of intestinal pathobiology, supply cells for regenerative cell therapies to treat intestinal disease, and provide therapeutic autologous grafts to repair damaged colon.

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.

Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) are a promising source for cell therapy approaches to treat neurodegenerative and demyelinating disorders. Despite ongoing efforts to characterize hiPSC-derived cells in vitro and in vivo, we lack comprehensive genome- and transcriptome-wide studies addressing hiPSC-NSC identity and safety, which are critical for establishing accepted criteria for prospective clinical applications. Here, we evaluated the transcriptional and epigenetic signatures of hiPSCs and differentiated hiPSC-NSC progeny, finding that the hiPSC-to-NSC transition results in a complete loss of pluripotency and the acquisition of a radial glia-associated transcriptional signature. Importantly, hiPSC-NSCs share with somatic human fetal NSCs (hfNSCs) the main transcriptional and epigenetic patterns associated with NSC-specific biology. In vivo, long-term observation (up to 10 months) of mice intracerebrally transplanted as neonates with hiPSC-NSCs showed robust engraftment and widespread distribution of human cells in the host brain parenchyma. Engrafted hiPSC-NSCs displayed multilineage potential and preferentially generated glial cells. No hyperproliferation, tumor formation, or expression of pluripotency markers was observed. Finally, we identified a novel role of the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1) in the regulation of astroglial commitment of hiPSC-NSCs. Overall, these comprehensive in vitro and in vivo analyses provide transcriptional and epigenetic reference datasets to define the maturation stage of NSCs derived from different hiPSC sources, and to clarify the safety profile of hiPSC-NSCs, supporting their continuing development as an alternative to somatic hfNSCs in treating neurodegenerative and demyelinating disorders.