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Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel Hprt DsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity. [ABSTRACT FROM AUTHOR]

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro , suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product. [ABSTRACT FROM AUTHOR]

Abstract Background Serum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair. Methods Heart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility. Results Transitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs. Conclusions Continuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction.

Summary: When in certain culture conditions, organotypic cultures are able to mimic developmental stages of an organ, generating higher-order structures containing functional subunits and progenitor niches. Despite the major advances in the area, researchers have not been able to fully recapitulate the complexity of kidney tissue. Pluripotent stem cells are extensively used in the field, but very few studies make use of adult stem cells. Herein, we describe a simple and feasible method for achieving glomerular epithelial differentiation on an organotypic model comprising human renal progenitor cells from adult kidney (hRPCs). Their glomerular differentiative potential was studied using retinoic acid (RA), a fundamental molecule for intermediate mesoderm induction on early embryogenesis. Immunofluorescence, specific cell surface markers expression and gene expression analysis confirm the glomerular differentiative potential of RA in a short-term culture. We also compared the potential of RA with a potent WNT agonist, CHIR99021, on the differentiative capacity of hRPCs. Gene expression and immunofluorescence analysis confirmed that hRPCs are more sensitive to RA stimulation when compared to CHIR9901. Endothelial cells were also included on the spheroids, resulting in a higher organizational level. The assembly potential of these cells and their selective stimulation will give new insights on adult organotypic cell culture studies and will hopefully guide more works in this important area of research. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a degenerative disorder characterized by chondrocyte apoptosis and degeneration of articular cartilage resulting in loss of mobility and pain. Inflammation plays a key role in the development and progression of OA both on the side of apoptosis and repair, while its exact role in pathogenesis has yet to be fully elucidated. Few studies have examined the cellular composition (inflammatory cells and/or progenitor cells) in the synovium of patients with pre-OA (asymptomatic with cartilage damage). Therefore, in the current study, mesenchymal progenitor cells (MPCs) and macrophages were enumerated within normal, pre-OA and OA synovium. No differences were observed between MPCs in normal vs. pre-OA, however, fewer macrophages were observed in pre-OA vs. normal synovium. Osteoarthritic synovium contained greater numbers of both MPCs and macrophages. Interestingly, the localization of MPCs and macrophages was affected by disease severity. In normal and pre-OA synovium, MPCs and macrophages co-localized, while in OA synovium, MPCs and macrophage populations were spatially distinct. Examining the cellular interactions between MPCs and macrophages in synovium may be essential for understanding the role of these cells in the onset and/or pathogenesis of the disease. This study has provided a first step by examining these cell types both spatially and temporally (e.g., disease severity). Further cellular and molecular studies will be needed to determine the functions of these cells in the context of disease and in relation to each other and the joint as a whole. [ABSTRACT FROM AUTHOR]

Abstract: Degeneration of the intervertebral disc (IVD) is a major spinal disorder that associates with neck and back pain. Recent studies of clinical samples and animal models for IVD degeneration have identified cells with multi-potency in the IVD. However, IVD tissue-specific progenitor cells and their niche components are not clear, although degenerated IVD-derived cells possess in vitro characteristics of mesenchymal stromal cell (MSCs). Here, we firstly identified the tissue-specific intervertebral disc progenitor cells (DPCs) from healthy Rhesus monkey and report the niche components modulated the survival of DPCs under hypoxia. DPCs possess clonogenicity, multipotency and retain differentiation potential after extended expansion in vitro and in vivo. In particular, the nucleus pulposus-derived DPCs are sensitive to low oxygen tension and undergo apoptosis under hypoxic conditions due to their inability to induce/stabilize hypoxia-inducible factors (HIF). The presence of small leucine-rich proteoglycans (SLRP), biglycan or decorin, can reduce the susceptibility of DPCs to hypoxia-induced apoptosis via promoting the activation/stabilization of HIF-1α and HIF-2α. As IVD is avascular, we propose SLRPs are niche components of DPCs in IVD homeostasis, providing new insights in progenitor cell biology and niche factors under a hypoxic microenvironment. [Copyright &y& Elsevier]

Cell therapy, the transplantation of progenitor cells into the myocardium, has been proposed as a possible treatment strategy for heart failure. Despite the lack of repopulation of the heart with progenitor cells, cell therapy induces a modest but well-documented functional improvement in patients. It is thought that paracrine mechanisms may account for the observed changes in heart function. However, there is little evidence that directly supports this hypothesis. We discuss the current views in the literature and present some preliminary data proposing that adult progenitor cells influence contractility and Ca2+ handling in neighboring failing cardiomyocytes by soluble mediators. This can be tested using a co-culture system. Our results suggest that soluble mediators from adult progenitor cells can enhance failing cardiomyocyte function, supporting the paracrine hypothesis. This co-culture strategy can be employed to identify cell-specific soluble mediators and their cellular targets during cell therapy for the treatment of heart disease. [ABSTRACT FROM AUTHOR]

Background: Adult progenitor cells presumably demonstrate clonogenicity, self-renewal, and multipotentiality, and can regenerate cells under various conditions. Definitive evidence demonstrating the existence of such progenitor cells in adult mammalian kidneys is lacking., Method: We performed in vivo lineage tracing and thymidine analogue labeling using adult tamoxifen-inducible ( Aqp2 ECE/+ RFP/+ , Aqp2 ECE/+ Brainbow/+ , and Aqp2 ECE/+ Brainbow/Brainbow ) and WT mice. The tamoxifen-inducible mice were analyzed between 1 and 300 days postinduction. Alternatively, WT and tamoxifen-induced mice were subjected to unilateral ureteral obstruction and thymidine analogue labeling and analyzed 2-14 days post-surgery. Multiple cell-specific markers were used for high-resolution immunofluorescence confocal microscopy to identify the cell types derived from Aqp2 + cells., Results: Like their embryonic counterparts, adult cells expressing Aqp2 and V-ATPase subunits B1 and B2 (Aqp2 + B1B2 + ) are the potential Aqp2 + progenitor cells (APs). Adult APs rarely divide to generate daughter cells, either maintaining the property of the AP (self-renewal) or differentiating into DCT2/CNT/CD cells (multipotentiality), forming single cell-derived, multiple-cell clones (clonogenicity) during tissue maintenance. APs selectively and continuously regenerate DCT2/CNT/CD cells in response to injury resulting from ureteral ligation. AP proliferation demonstrated direct correlation with Notch activation and was inversely correlated with development of kidney fibrosis. Derivation of both intercalated and DCT2 cells was found to be cell division-dependent and -independent, most likely through AP differentiation which requires cell division and through direct conversion of APs and/or regular principal cells without cell division, respectively., Conclusion: Our study demonstrates that Aqp2 + B1B2 + cells behave as adult APs to maintain and repair DCT2/CNT1/CNT2/CD segments., (Copyright © 2022 by the American Society of Nephrology.)

Background: Serum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair. Methods: Heart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility. Results: Transitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs. Conclusions: Continuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction. [ABSTRACT FROM AUTHOR]