Self-renewing Progenitors Research Articles

Generation of self-renewing neuromesodermal progenitors with neuronal and skeletal muscle bipotential from human embryonic stem cells.

Progress has been made in generating spinal cord and trunk derivatives from neuromesodermal progenitors (NMPs). However, maintaining the self-renewal of NMPs invitro remains a challenge. In this study, we developed a cocktail of small molecules and growth factors that induces human embryonic stem cells to produce self-renewing NMPs (srNMPs) under chemically defined conditions. These srNMPs maintain the state of neuromesodermal progenitors in prolonged culture and have the potential to generate mesodermal cells and neurons, even at the single-cell level. Additionally, suspended srNMP aggregates can spontaneously differentiate into all tissue types of early embryonic trunks. Furthermore, transplanted srNMP-derived muscle satellite cells or progenitors of motor neurons were integrated into skeletal muscle or the spinal cord, respectively, and contributed to regeneration in mouse models. In summary, srNMPs hold great promise for applications in developmental biology and as renewable cell sources for cell therapy for trunk and spinal cord injuries.

PHF6 suppresses self-renewal of leukemic stem cells in AML

Acute myeloid leukemia is characterized by uncontrolled proliferation of self-renewing myeloid progenitors accompanied by a differentiation arrest. PHF6 is a chromatin-binding protein mutated in myeloid leukemias, and its isolated loss increases mouse HSC self-renewal without malignant transformation. We report here that Phf6 knockout increases the aggressiveness of Hoxa9-driven AML over serial transplantation, and increases the frequency of leukemia initiating cells. We define the in vivo hierarchy of Hoxa9-driven AML and identify a population that we term the “LIC-e” (leukemia initiating cells enriched) population. We find that Phf6 loss expands the LIC-e population and skews its transcriptome to a more stem-like state; concordant transcriptome shifts are also observed on PHF6 knockout in a human AML cell line and in PHF6 mutant patient samples from the BEAT AML dataset. We demonstrate that LIC-e accumulation in Phf6 knockout AML occurs not due to effects on cell cycle or apoptosis, but due to an increase in the fraction of its progeny that retain LIC-e identity. Our work indicates that Phf6 loss increases AML self-renewal through context-specific effects on leukemia stem cells.

IPSC-Derived KitD816V/Jak2V817F Double-Mutant Kitjak Cell Lines Allow Inexpensive Large-Scale Production of Enucleated Red Blood Cells in the Absence of SCF and Erythropoietin

Cultured Red Blood Cells (cRBCs) have the potential to supplement transfusion needs and to be used as drug carriers or immune tolerization agents. Several methods have been devised to amplify primary Hematopoietic Progenitor cells (HPCs) into cRBCs. Although these protocols have the potential to relieve local blood shortages, they are not ideal for large scale cRBC production because the primary HPCs must constantly be replenished. Immortalized human erythroid cell lines have been developed by overexpressing the E6/E7 proteins of human papilloma virus 16, c-Myc, Bcl-XL, Spi1 or Bmi-1. Many of these lines can be cultured for months and in some cases indefinitely, and some retain their ability to differentiate into cRBCs with rates of enucleation varying between 0.1-30%. These lines are useful as research tools and are a potential source of cells for some translational applications. Nevertheless, karyotypic instability, the cost of the media and cytokines, and cumbersome culture procedure to maintain some of these lines remain major obstacles for their large-scale use. We previously developed the Pluripotent Stem Cell Robust Erythroid Differentiation (PSC-RED) protocol to differentiate induced-pluripotent stem cells (iPSCs) into cRBCs. The PSC-RED protocol yields a large number of enucleated RBCs, does not require any albumin, and allows transferrin recycling. Despite these advantages, the PSC-RED protocol is not well-adapted for large-scale cRBC production because differentiation of an iPSC into a RBC takes up to 45 days and because of cytokine cost. To eliminate the requirement for Stem Cell Factor (SCF) and Erythropoietin (Epo) which accounts for more than 95% of total cytokine cost in the PSC-RED protocol, we have used CRSPR/cas9 technology to introduce in iPSCs the kitD816V mutation that allows the SCF receptor (the kit gene) to constitutive signal in the absence of SCF, and the Jak2V617F mutation that allows constitutive signaling through the JAK/STAT pathway in the absence of Epo. We report here that kitD816V, Jak2V617F double mutants iPSCs readily differentiate into self-renewing erythroid progenitors cells that can grow in the absence of any cytokines for >60 population doubling ( >10 19 fold expansion for >5 months) in an inexpensive culture medium containing little more than two small molecules and low amounts of recombinant transferrin. Importantly, KitJak cell lines can be reproducibly generated from iPSCs by a two-week differentiation procedure without any time lag, suggesting that no other mutations are necessary to generate these self-renewing erythroid progenitors. Indeed, we have shown using a next-generation sequencing approach that KitJak cells appear to be remarkably karyotypically stable since multiple experiments fail to detect any chromosomal aneuploidy after long-term culture. KitJak cells have an immuno-phenotype of late CFU-E/pro-Erythroblast, can be cultured at high density and can be induced to differentiate into cRBCs using a 10- to 12-day differentiation protocol that leads to a 20 to 40-fold cell amplification. The rate of enucleation is 30-40%, providing an overall yield of 7-14 RBCs per self-renewing KitJak cell. The cRBCs produced express mostly fetal hemoglobin, are slightly larger that adult cells, and have normal hemoglobin content. The development of these KitJak cell lines greatly facilitates the production of cRBCs and will pave the way to large-scale erythroid cell production for transfusion and other translational applications. As a first application, we show that the RBCs produced are fully compatible with the Immucor red blood cell typing system and can be used as reagent red blood cells.

The zebrafish paralog six2b is required for early proximal pronephros morphogenesis

The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F0 crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule.

Toward an understanding of glucose metabolism in radial glial biology and brain development.

Decades of research have sought to determine the intrinsic and extrinsic mechanisms underpinning the regulation of neural progenitor maintenance and differentiation. A series of precise temporal transitions within progenitor cell populations generates all the appropriate neural cell types while maintaining a pool of self-renewing progenitors throughout embryogenesis. Recent technological advances have enabled us to gain new insights at the single-cell level, revealing an interplay between metabolic state and developmental progression that impacts the timing of proliferation and neurogenesis. This can have long-term consequences for the developing brain's neuronal specification, maturation state, and organization. Furthermore, these studies have highlighted the need to reassess the instructive role of glucose metabolism in determining progenitor cell division, differentiation, and fate. This review focuses on glucose metabolism (glycolysis) in cortical progenitor cells and the emerging focus on glycolysis during neurogenic transitions. Furthermore, we discuss how the field can learn from other biological systems to improve our understanding of the spatial and temporal changes in glycolysis in progenitors and evaluate functional neurological outcomes.

Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice.

Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months invivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung's facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.

MiR-6216 regulates neural stem cell proliferation by targeting RAB6B

Neural stem cells (NSCs) are a class of self-renewing, multipotent and undifferentiated progenitor cells that retain the capacity to both glial and neuronal lineages. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in stem cell fate determination and self-renewal. Our previous RNA-seq data indicated that the expression of miR-6216 was decreased in denervated hippocampal exosomes compared with normal. However, whether miR-6216 participates in regulating NSC function remains to be elucidated. In this study, we demonstrated that miR-6216 negatively regulates RAB6B expression. Forced overexpression of miR-6216 inhibited NSC proliferation, and overexpression of RAB6B promoted NSC proliferation. These findings suggest that miR-6216 played an important role in regulating NSC proliferation via targeting RAB6B, and improve the understanding of the miRNA-mRNA regulatory network that affects NSC proliferation.

Self-Renewing CD8+ T-cell Abundance in Blood Associates with Response to Immunotherapy.

Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non-small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7-) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7-CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7-CD8+ T cells. The observed correlation of TCF1 frequency in CCR7-CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.

Rapid induction and long-term self-renewal of neural crest-derived ectodermal chondrogenic cells from hPSCs

Articular cartilage is highly specific and has limited capacity for regeneration if damaged. Human pluripotent stem cells (hPSCs) have the potential to generate any cell type in the body. Here, we report the dual-phase induction of ectodermal chondrogenic cells (ECCs) from hPSCs through the neural crest (NC). ECCs were able to self-renew long-term (over numerous passages) in a cocktail of growth factors and small molecules. The cells stably expressed cranial neural crest-derived mandibular condylar cartilage markers, such as MSX1, FOXC1 and FOXC2. Compared with chondroprogenitors from iPSCs via the paraxial mesoderm, ECCs had single-cell transcriptome profiles similar to condylar chondrocytes. After the removal of the cocktail sustaining self-renewal, the cells stopped proliferating and differentiated into a homogenous chondrocyte population. Remarkably, after transplantation, this cell lineage was able to form cartilage-like structures resembling mandibular condylar cartilage in vivo. This finding provides a framework to generate self-renewing cranial chondrogenic progenitors, which could be useful for developing cell-based therapy for cranial cartilage injury.

Abstract 1240: Peripherally measured T cell self-renewal capacity associates with response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC)

Abstract Recent studies have suggested that ICB does not primarily restore function to terminally exhausted T cells, but instead drives proliferation and differentiation of progenitor T cells into fresh effector cells. T cell factor 1 (TCF1) marks and maintains T cell self-renewal while repressing differentiation and has been associated with increased antitumor activity. These findings suggest ICB may depend on the continued availability of self-renewing TCF1+ progenitor T cells as the source of fresh effector cells. We hypothesized that characterizing the immunophenotype of T cells in the peripheral blood, with a focus on markers of self-renewal, might be predictive of ICB response. Utilizing an institution-wide biospecimen repository, we evaluated peripheral blood samples using multi-parametric flow cytometry from patients with metastatic NSCLC treated with single-agent ICB who experienced durable clinical benefit (DCB; >1 year of treatment and/or clinical response >1 year) or primary resistance (RES; <6 months of treatment with evidence of disease progression). Our analysis focused on a CCR7- T cell subset, which is characterized by lower proportions of TCF1+ cells but increase during active immune responses. We analyzed samples collected from a cohort of 22 patients, 11 of whom had DCB and 11 with RES. In the pre-treatment samples (n=22), we determined that the frequency of TCF1+CCR7- CD8+ T cells was significantly higher in patients with DCB compared to patients with RES (32.6% v. 19.0%; P = 0.0452). Importantly, the median progression free survival was 17.0 months in patients with TCF1+ frequency above the median compared to 3.0 months in patients with TCF1+ frequency below the median (HR 0.43; P = 0.0185). Among the on-treatment samples (n=19), there was an increased frequency of PD1+ expression within the CCR7- CD8+ T cell subset compared to the paired baseline samples. Examination of those therapy-expanded PD1+ T cells revealed differences in the composition of TCF1+ versus TCF1- fractions in relation to treatment outcome. The mean ratio of TCF1+ to TCF1- amongst PD1+CCR7- T cells on-treatment was found to be significantly higher in the DCB group (1.483 [DCB] v. 0.4791 [RES], P=<0.0001). In this analysis, we identified a pattern of pre-and on-treatment T cell populations that correlates with durable benefit from ICB in patients with NSCLC. We observed that the frequency of TCF1 expression in the more differentiated effector memory T cell subsets prior to treatment correlates with DCB. While on treatment, both groups experienced expansion of PD1+ CD8 T cells, but the presence of greater fractions of TCF1+ T cells in the PD1+ CD8 T cell population correlates with DCB. This finding suggests that persistence and mobilization of self-renewing progenitor T cells is associated with long-lived ICB-induced anti-tumor activity. Citation Format: Rohan Maniar, Peter H. Wang, Robert S. Washburn, Naiyer A. Rizvi, Steven L. Reiner, Brian S. Henick. Peripherally measured T cell self-renewal capacity associates with response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1240.

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen.

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide–drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.

Mesenchymal Stem Cells Exposed to Persistently High Glucocorticoid Levels Develop Insulin-Resistance and Altered Lipolysis: A Promising In Vitro Model to Study Cushing's Syndrome.

BackgroundIn Cushing’s syndrome (CS), chronic glucocorticoid excess (GC) and disrupted circadian rhythm lead to insulin resistance (IR), diabetes mellitus, dyslipidaemia and cardiovascular comorbidities. As undifferentiated, self-renewing progenitors of adipocytes, mesenchymal stem cells (MSCs) may display the detrimental effects of excess GC, thus revealing a promising model to study the molecular mechanisms underlying the metabolic complications of CS.MethodsMSCs isolated from the abdominal skin of healthy subjects were treated thrice daily with GCs according to two different regimens: lower, circadian-decreasing (Lower, Decreasing Exposure, LDE) versus persistently higher doses (Higher, Constant Exposure, HCE), aimed at mimicking either the physiological condition or CS, respectively. Subsequently, MSCs were stimulated with insulin and glucose thrice daily, resembling food uptake and both glucose uptake/GLUT-4 translocation and the expression of LIPE, ATGL, IL-6 and TNF-α genes were analyzed at predefined timepoints over three days.ResultsLDE to GCs did not impair glucose uptake by MSCs, whereas HCE significantly decreased glucose uptake by MSCs only when prolonged. Persistent signs of IR occurred after 30 hours of HCE to GCs. Compared to LDE, MSCs experiencing HCE to GCs showed a downregulation of lipolysis-related genes in the acute period, followed by overexpression once IR was established.ConclusionsPreserving circadian GC rhythmicity is crucial to prevent the occurrence of metabolic alterations. Similar to mature adipocytes, MSCs suffer from IR and impaired lipolysis due to chronic GC excess: MSCs could represent a reliable model to track the mechanisms involved in GC-induced IR throughout cellular differentiation.

A Microfluidic Chip for Growth and Characterization of Adult Rat Hippocampal Progenitor Cell Neurospheroids

Adult hippocampal neural stem/progenitor cells (AHPCs), which are self-renewing multipotent progenitors that can differentiate into neurons, astrocytes, and oligodendrocytes, are suitable as a central nervous system (CNS) molecular model, and the formation of 3D AHPC neurospheroids are potentially suitable as an in vitro brain model. In this paper we report a new microfluidic chip to culture AHPC neurospheroids (NSs-AHPC) inside culture-chambers on chip. After cell fixation and immunostaining were conducted, the fluorescence images of NSs-AHPC were analyzed. It has been found that the AHPCs comprising neurospheroids remained highly viable. Cell proliferation and neuronal differentiation have also been observed, indicating the feasibility of NSs-AHPC as the in vitro brain model on chip. Given its simple-to-use, low-cost, and orderly arranged culture-chambers, this type of chip is particularly suitable for culturing and analyzing multiple in vitro brain models in an efficient manner. [2021-0199]

Chemical approach to generating long-term self-renewing pMN progenitors from human embryonic stem cells.

Spinal cord impairment involving motor neuron degeneration and demyelination can cause lifelong disabilities, but effective clinical interventions for restoring neurological functions have yet to be developed. In early spinal cord development, neural progenitors of the motor neuron (pMN) domain, defined by the expression of oligodendrocyte transcription factor 2 (OLIG2), in the ventral spinal cord first generate motor neurons and then switch the fate to produce myelin-forming oligodendrocytes. Given their differentiation potential, pMN progenitors could be a valuable cell source for cell therapy in relevant neurological conditions such as spinal cord injury. However, fast generation and expansion of pMN progenitors in vitro while conserving their differentiation potential has so far been technically challenging. In this study, based on chemical screening, we have developed a new recipe for efficient induction of pMN progenitors from human embryonic stem cells. More importantly, these OLIG2+ pMN progenitors can be stably maintained for multiple passages without losing their ability to produce spinal motor neurons and oligodendrocytes rapidly. Our results suggest that these self-renewing pMN progenitors could potentially be useful as a renewable source of cell transplants for spinal cord injury and demyelinating disorders.

Telomerase Inhibition Impairs Self-Renewal of ß-Catenin Activated Myeloproliferative Neoplasm Progenitors

IntroductionChronic phase myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML) and myelofibrosis (MF), arise from hematopoietic stem cells. However, they harbor varying propensities to undergo blast crisis (acute leukemic) transformation based on their capacity to give rise to tyrosine kinase inhibitor (TKI) resistant myeloid progenitors that activate self-renewal pathways, such as Wnt/b-catenin signaling. Because b-catenin has been reported to transcriptionally regulate human telomerase reverse transcriptase (hTERT), we investigated the capacity of a telomerase complex inhibitor, imetelstat, to prevent malignant progenitor self-renewal. The telomerase complex consists of hTERT, an RNA template subunit (TERC), and a protective shelterin scaffold. Imetelstat is a novel, first in class covalently lipidated 13-mer oligonucleotide telomerase inhibitor with clinical activity in myeloid malignancies. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms such as MF. However, the role of imetelstat in selectively eradicating self-renewing MPN progenitors has not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized MPN progenitor primagraft studies to investigate the ability of imetelstat to selectively inhibit malignant progenitor self-renewal at doses that spare normal progenitors as well as to determine the mechanism of action.Methods and ResultsWhen FACS-purified progenitors from human blast crisis (BC; n=9) were compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples, RNA-seq revealed upregulation of key genes in the TERT interactome suggesting a role for TERT activation in MPN progenitor blast crisis transformation. Human MPN progenitor supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 µM significantly inhibited (p <0.001, ANOVA) in vitro replating of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Also, imetelstat (5 µM) significantly reduced replating of MF (n=9) compared with normal progenitor (n=4) samples. Treatment of humanized mouse models, established with four different BC CML samples, with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in both bone marrow and spleen malignant progenitor burden compared with mismatch controls (p=0.003, and p=0.009, t test), and a significant reduction in human CD45+ cells in the splenic niche compared with mismatch controls (p=0.03, t test). In a MF model established in NSG-S mice, imetelstat also reduced human CD45+ cell engraftment (p=0.03, t test). Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat treated MF cells (p=0.18) and CML cells (p=0.4) compared with mismatch control. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human progenitors after imetelstat treatment (p=0.02) compared with vehicle control.ConclusionsNiche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated MPN progenitors to imetelstat in both in vitro and in vivo humanized pre-clinical MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat.Disclosures: This work was funded by the Moores Family Foundation, the Koman Family Foundation, the California Institute for Regenerative Medicine (DR1-01430), Janssen Research & Development, LLC, and by NIH NCI R21CA189705. DisclosuresHuang:Janssen Research & Development, LLC: Employment.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

An Update on Mesenchymal Stem Cell-Centered Therapies in Temporomandibular Joint Osteoarthritis.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by cartilage degeneration, disrupted subchondral bone remodeling, and synovitis, seriously affecting the quality of life of patients with chronic pain and functional disabilities. Current treatments for TMJOA are mainly symptomatic therapies without reliable long-term efficacy, due to the limited self-renewal capability of the condyle and the poorly elucidated pathogenesis of TMJOA. Recently, there has been increased interest in cellular therapies for osteoarthritis and TMJ regeneration. Mesenchymal stem cells (MSCs), self-renewing and multipotent progenitor cells, play a promising role in TMJOA treatment. Derived from a variety of tissues, MSCs exert therapeutic effects through diverse mechanisms, including chondrogenic differentiation; fibrocartilage regeneration; and trophic, immunomodulatory, and anti-inflammatory effects. Here, we provide an overview of the therapeutic roles of various tissue-specific MSCs in osteoarthritic TMJ or TMJ regenerative tissue engineering, with an additional focus on joint-resident stem cells and other cellular therapies, such as exosomes and adipose-derived stromal vascular fraction (SVF). Additionally, we summarized the updated pathogenesis of TMJOA to provide a better understanding of the pathological mechanisms of cellular therapies. Although limitations exist, MSC-centered therapies still provide novel, innovative approaches for TMJOA treatment.

Glucocorticoid agonists enhance retinal stem cell self-renewal and proliferation

BackgroundAdult mammalian retinal stem cells (RSCs) readily proliferate, self-renew, and generate progeny that differentiate into all retinal cell types in vitro. RSC-derived progeny can be induced to differentiate into photoreceptors, making them a potential source for retinal cell transplant therapies. Despite their proliferative propensity in vitro, RSCs in the adult mammalian eye do not proliferate and do not have a regenerative response to injury. Thus, identifying and modulating the mechanisms that regulate RSC proliferation may enhance the capacity to produce RSC-derived progeny in vitro and enable RSC activation in vivo.MethodsHere, we used medium-throughput screening to identify small molecules that can expand the number of RSCs and their progeny in culture. In vitro differentiation assays were used to assess the effects of synthetic glucocorticoid agonist dexamethasone on RSC-derived progenitor cell fate. Intravitreal injections of dexamethasone into adult mouse eyes were used to investigate the effects on endogenous RSCs.ResultsWe discovered that high-affinity synthetic glucocorticoid agonists increase RSC self-renewal and increase retinal progenitor proliferation up to 6-fold without influencing their differentiation in vitro. Intravitreal injection of synthetic glucocorticoid agonist dexamethasone induced in vivo proliferation in the ciliary epithelium—the niche in which adult RSCs reside.ConclusionsTogether, our results identify glucocorticoids as novel regulators of retinal stem and progenitor cell proliferation in culture and provide evidence that GCs may activate endogenous RSCs.

Derivation of Multipotent Neural Progenitors from Human Embryonic Stem Cells for Cell Therapy and Biomedical Applications.

Long-term neuroepithelial-like stem cells (lt-NES) derived from human embryonic stem cells are a stable self-renewing progenitor population with high neurogenic potential and phenotypic plasticity. Lt-NES are amenable to regional patterning toward neurons and glia subtypes and thus represent a valuable source of cells for many biomedical applications. For use in regenerative medicine and cell therapy, lt-NES and their progeny require derivation with high-quality culture conditions suitable for clinical use. In this chapter, we describe a robust method to derive multipotent and expandable lt-NES based on good manufacturing practice and cell therapy-grade reagents. We further describe fully defined protocols to terminally differentiate lt-NES toward GABA-ergic, dopaminergic, and motor neurons.

Long-term robustness of a T-cell system emerging from somatic rescue of a genetic block in T-cell development.

BackgoundThe potential of a single progenitor cell to establish and maintain long-term protective T-cell immunity in humans is unknown. For genetic disorders disabling T-cell immunity, somatic reversion was shown to support limited T-cell development attenuating the clinical phenotype. However, the cases reported so far deteriorated over time leaving unanswered the important question of long-term activity of revertant precursors and the robustness of the resulting T-cell system.MethodsWe applied TCRβ-CDR3 sequencing and mass cytometry on serial samples of a now 18 year-old SCIDX1 patient with somatic reversion to analyse the longitudinal diversification and stability of a T-cell system emerging from somatic gene rescue.FindingsWe detected close to 105 individual CDR3β sequences in the patient. Blood samples of equal size contained about 10-fold fewer unique CDR3β sequences compared to healthy donors, indicating a surprisingly broad repertoire. Despite dramatic expansions and contractions of individual clonotypes representing up to 30% of the repertoire, stable diversity indices revealed that these transient clonal distortions did not cause long-term repertoire imbalance. Phenotypically, the T-cell system did not show evidence for progressive exhaustion. Combined with immunoglobulin substitution, the limited T-cell system in this patient supported an unremarkable clinical course over 18 years.InterpretationGenetic correction in the appropriate cell type, in our patient most likely in a T-cell biased self-renewing hematopoietic progenitor, can yield a diverse T-cell system that provides long-term repertoire stability, does not show evidence for progressive exhaustion and is capable of providing protective and regulated T-cell immunity for at least two decades.FundingDFG EH 145/9-1, DFG SCHW 432/4-1 and the German Research Foundation under Germany's Excellence Strategy–EXC-2189–Project ID: 390939984.