Population Of Multipotent Progenitor Cells Research Articles

Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest.

The histone methyltransferase Polycomb repressive complex 2 (PRC2) is required for specification of the neural crest, and mis-regulation of neural crest development can cause severe congenital malformations. PRC2 is necessary for neural crest induction, but the embryonic, cellular, and molecular consequences of PRC2 activity after neural crest induction are incompletely understood. Here we show that Eed, a core subunit of PRC2, is required for craniofacial osteoblast differentiation and mesenchymal proliferation after induction of the neural crest. Integrating mouse genetics with single-cell RNA sequencing, our results reveal that conditional knockout of Eed after neural crest cell induction causes severe craniofacial hypoplasia, impaired craniofacial osteogenesis, and attenuated craniofacial mesenchymal cell proliferation that is first evident in post-migratory neural crest cell populations. We show that Eed drives mesenchymal differentiation and proliferation in vivo and in primary craniofacial cell cultures by regulating diverse transcription factor programs that are required for specification of post-migratory neural crest cells. These data enhance understanding of epigenetic mechanisms that underlie craniofacial development, and shed light on the embryonic, cellular, and molecular drivers of rare congenital syndromes in humans.

Safety and outcomes analysis: transcatheter implantation of autologous angiogenic cell precursors for the treatment of cardiomyopathy

BackgroundStem cell transplantation is an emerging therapy for severe cardiomyopathy, proffering stem cell recruitment, anti-apoptosis, and proangiogenic capabilities. Angiogenic cell precursors (ACP-01) are autologous, lineage-specific, cells derived from a multipotent progenitor cell population, with strong potential to effectively engraft, form blood vessels, and support tissue survival and regeneration.MethodsThis IRB approved outcome analysis reports upon 74 consecutive patients who failed medical management for severe cardiomyopathy, and were selected to undergo transcatheter intramyocardial or intracoronary implantation of ACP-01. Serious adverse events (SAEs) were reported. Cell analysis was conducted for each treatment. The left ventricular ejection fraction (LVEF) was measured by multi-gated acquisition scan (MUGA) or echocardiogram at 4 months ± 1.9 months and 12 months ± 5.5 months. Patients reported quality of life statements at 6 months (± 5.6 months).ResultsFifty-four of 74 patients met requirements for inclusion (48 males and five females; age 68.1 ± 11.3 years). The mean treatment cell number of 57 × 106 ACP-01 included 7.7 × 106 CD34 + and 21 × 106 CD31 + cells with 97.6% viability. SAEs included one death (previously unrecognized silent MI), ventricular tachycardia (n = 2) requiring cardioversion, and respiratory infection (n = 2). LVEF in the ischemic subgroup (n = 41) improved by 4.7% ± 9.7 from pre-procedure to the first follow-up (4 months ± 1.9 months) (p < 0.004) and by 7.2% ± 10.9 at final follow-up (n = 25) at average 12 months (p < 0.004). The non-ischemic dilated cardiomyopathy subgroup (n = 8) improved by 7.5% ± 6.0 at the first follow-up (p < 0.017) and by 12.2% ± 6.4 at final follow-up (p < 0.003, n = 6). Overall improvement in LVEF from pre-procedure to post-procedure was significant (Fisher’s exact test p < 0.004). LVEF improvement was most marked in the patients with the most severe cardiomyopathy (LVEF < 20%) improving from a mean 14.6% ± 3.4% pre-procedurally to 28.4% ± 8% at final follow-up. Quality of life statements reflected improvement in 33/50 (66%), no change in 14/50 (28%), and worse in 3/50 (6%).ConclusionTranscatheter implantation of ACP-01 for cardiomyopathy is safe and improves LVEF in the setting of ischemic and non-ischemic cardiomyopathy. The results warrant further investigation in a prospective, blinded, and controlled clinical study.Trial Registration: IRB from Genetic Alliance #APC01-001, approval date July 25, 2022.Condensed Cardiomyopathy is common and associated with high mortality. Stem cell transplantation is an emerging therapy. Angiogenic cell precursors (ACP-01) are lineage-specific endothelial progenitors, with strong potential for migration, engraftment, angiogenesis, and support of tissue survival and regeneration. A retrospective outcomes analysis of 53 patients with ischemic and non-ischemic dilated cardiomyopathy undergoing transcatheter implantation of ACP-01 demonstrated improvements in the left ventricular ejection fraction of 7.2% ± 10.9 (p < 0.004) and 12.2% ± 6.4, respectively, at 12 months (± 5) follow-up. Quality of life statements reflected improvement in 33/50 (66%) patients.

Mesenchymal stem/stromal cells- a principal element for tumour microenvironment heterogeneity.

The heterogeneity of the tumor microenvironment (TME) is a major obstacle in cancer treatment, making most therapeutic interventions palliative rather than curative. Previous studies have suggested that the reason for the low efficacy of immunotherapy and the relapse of the original responders over time may be due to the complex network of mesenchymal stem/stromal cells (MSCs), a population of multipotent progenitor cells existing in a variety of tissues. Cancer-associated MSCs (CA-MSCs) have already been isolated from various types of tumors and are characterized by their vigorous pro-tumorigenic functions. Although the roles of CA-MSCs from different sources vary widely, their origins are still poorly understood. Current evidence suggests that when local resident or distally recruited MSCs interact with tumor cells and other components in the TME, "naïve" MSCs undergo genetic and functional changes to form CA-MSCs. In this review, we mainly focus on the multiple roles of CA-MSCs derived from different sources, which may help in elucidating the formation and function of the entire TME, as well as discover innovative targets for anti-cancer therapies.

Cell therapy for corneal regeneration: Current status and future perspectives

AbstractCurrently, the treatment of many serious ocular pathologies that affect the cornea focuses on the repair and regeneration of the affected structures—epithelium and endothelium—through cell therapy. On the ocular surface, there is a population of multipotent progenitor cells (limbal stem cells; LSC), located in the sclerocorneal limbus, which have a high proliferative, differentiating and regenerative capabilities. On the other hand, the corneal endothelium—the innermost corneal layer with little regenerative capacity in vivo—is responsible for controlling corneal homeostasis and transparency. Dysfunctions of both cell types—corneal epithelial and endothelial cells—are the leading cause of recoverable corneal blindness. Mesenchymal stem cells (MSCs) from adult tissue, obtained from adipose tissue and bone marrow aspirates, have the capacity for adipogenesis, osteogenesis, chondrogenesis, myogenesis and neurogenesis in vitro. However, its true potential towards epithelial differentiation seems to be limited, and its great clinical applicability seems to lie in its contribution to the homeostasis of the cellular microenvironment. Recent developments in the field of somatic cell reprogramming by induced pluripotent stem cells (IPSC) provide an exciting possibility for its future clinical application. Pluripotent stem cells can be obtained from different sources of adult tissue, among which IPSC (induced pluripotent stem cells) have excellent plasticity. Its ability to differentiate into corneal epithelial and endothelial phenotypes is still poorly understood. Likewise, in vitro organoid production methods are presented as an option for obtaining compromised and/or partially differentiated cell lines. In the meantime, their clinical use will require specific application and receptor safety so that they can correctly regenerate tissue, with added effort for adaptation of ‘clinical grade’ protocols. In this sense, the ocular tissue is characterized as an excellent candidate for a possible clinical application, since it constitutes a tissue with immune privilege, it is ‘isolated’ (intraocular spaces) from the other components of the body and also had a highly sophisticated avascular (cornea) and highly differentiated (retina and pigment epithelium) tissues. The possibility of establishing specific clinical‐grade protocols for cell differentiation and identifying specific cellular and molecular behaviour would contribute to a better understanding of the mechanisms involved in repair processes. One of the equally relevant aspects in advanced cell‐based therapies applied to corneal regeneration is related to the ideal composition of the extracellular matrix proteins and biocompatible/biomimetic substrates—artificial or natural—that may contribute to better cell differentiation and adaptation. All this would open innovative perspectives for a more rational and effective treatment of different pathologies associated with human corneal blindness, in the field of regenerative medicine through the application of advanced cell therapy.

Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34+ and CD34+CD38-CD45RA-CD90+ Cells

Hypoxic conditions preserve the multipotency and self-renewing capacity of murine bone marrow and human cord blood stem cells. Blood samples stored in sealed blood gas tubes become hypoxic as leukocytes metabolize and consume oxygen. Taken together, these observations suggest that peripheral blood stem cell (PBSC) samples stored under airtight conditions become hypoxic, and that the stem cells contained may undergo qualitative or quantitative changes. This study aimed to determine the effect of storage for 8 hours in a sealed system on PBSC samples. Granulocyte colony-stimulating factor-mobilized PBSC samples were collected prospectively from 9 patients with myeloma or amyloidosis prior to apheresis, followed by measurement of CO2, O2, hydrogen ion (pH), lactate, and glucose concentrations in the blood and immunophenotyping of stem cell and multipotent progenitor cell populations before and after 8 hours of storage in sealed blood collection tubes. Blood concentrations of O2 and glucose and pH measurements were significantly decreased, whereas concentrations of CO2 and lactate were significantly increased after storage. Significantly higher concentrations of CD34+ cells (552 ± 84 cells/106total nucleated cells [TNCs] versus 985 ± 143 cells/106TNCs; P=.03), CD34+CD38- cells (98 ± 32 cells/106TNCs versus 158 ± 52 cells/106TNCs; P=.03), CD34+CD38+ cells (444 ± 92 cells/106TNCs versus 789 ± 153 cells/106TNCs; P=.03), and CD34+CD38-CD45RA-CD90+ cells (55 ± 17 cells/106TNCs versus 89 ± 25 cells/106TNCs; P=.02) were detected after 8 hours of storage. The changes in concentrations of CD34+CD38+ cells and CD34+ cells were inversely associated with the change in glucose concentration (P=.003 and P < .001, respectively) and positively associated with the change in lactate concentration (P=.01 and P <.001, respectively) after 8 hours of airtight storage. Storage of PBSC samples in a sealed, airtight environment is associated with microenvironmental changes consistent with hypoxia and increased concentrations of immunophenotypically defined stem cells. These results may have clinical implications with regard to the collection and processing of stem cell products and warrant confirmation with functional and mechanistic studies.

Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states

Notch signaling is required to repress the formation of vertebrate cone photoreceptors and to maintain the proliferative potential of multipotent retinal progenitor cells. However, the mechanism by which Notch signaling controls these processes is unknown. Recently, restricted retinal progenitor cells with limited proliferation capacity and that preferentially generate cone photoreceptors have been identified. Thus, there are several potential steps during cone genesis that Notch signaling could act. Here we use cell type specific cis-regulatory elements to localize the primary role of Notch signaling in cone genesis to the formation of restricted retinal progenitor cells from multipotent retinal progenitor cells. Localized inhibition of Notch signaling in restricted progenitor cells does not alter the number of cones derived from these cells. Cell cycle promotion is not a primary effect of Notch signaling but an indirect effect on progenitor cell state transitions that leads to depletion of the multipotent progenitor cell population. Taken together, this suggests that the role of Notch signaling in cone photoreceptor formation and proliferation are both mediated by a localized function of Notch in multipotent retinal progenitor cells to repress the formation of restricted progenitor cells.

Sulforaphane Protects Against Ethanol-Induced Apoptosis in Human Neural Crest Cells Through Diminishing Ethanol-Induced Hypermethylation at the Promoters of the Genes Encoding the Inhibitor of Apoptosis Proteins.

The neural crest cell (NCC) is a multipotent progenitor cell population that is sensitive to ethanol and is implicated in the Fetal Alcohol Spectrum Disorders (FASD). Studies have shown that sulforaphane (SFN) can prevent ethanol-induced apoptosis in NCCs. This study aims to investigate whether ethanol exposure can induce apoptosis in human NCCs (hNCCs) through epigenetically suppressing the expression of anti-apoptotic genes and whether SFN can restore the expression of anti-apoptotic genes and prevent apoptosis in ethanol-exposed hNCCs. We found that ethanol exposure resulted in a significant increase in the expression of DNMT3a and the activity of DNMTs. SFN treatment diminished the ethanol-induced upregulation of DNMT3a and dramatically reduced the activity of DNMTs in ethanol-exposed hNCCs. We also found that ethanol exposure induced hypermethylation at the promoter regions of two inhibitor of apoptosis proteins (IAP), NAIP and XIAP, in hNCCs, which were prevented by co-treatment with SFN. SFN treatment also significantly diminished ethanol-induced downregulation of NAIP and XIAP in hNCCs. The knockdown of DNMT3a significantly enhanced the effects of SFN on preventing the ethanol-induced repression of NAIP and XIAP and apoptosis in hNCCs. These results demonstrate that SFN can prevent ethanol-induced apoptosis in hNCCs by preventing ethanol-induced hypermethylation at the promoter regions of the genes encoding the IAP proteins and diminishing ethanol-induced repression of NAIP and XIAP through modulating DNMT3a expression and DNMT activity.

Mesenchymal Stromal Cells, a New Player in Reducing Complications From Liver Transplantation?

In response to the global burden of liver disease there has been a commensurate increase in the demand for liver transplantation. However, due to a paucity of donor organs many centers have moved toward the routine use of marginal allografts, which can be associated with a greater risk of complications and poorer clinical outcomes. Mesenchymal stromal cells (MSC) are a multi-potent progenitor cell population that have been utilized to modulate aberrant immune responses in acute and chronic inflammatory conditions. MSC exert an immunomodulatory effect on innate and adaptive immune systems through the release of both paracrine soluble factors and extracellular vesicles. Through these routes MSC can switch the regulatory function of the immune system through effects on macrophages and T regulatory cells enabling a switch of phenotype from injury to restoration. A key benefit seems to be their ability to tailor their response to the inflammatory environment without compromising the host ability to fight infection. With over 200 clinical trials registered to examine MSC therapy in liver disease and an increasing number of trials of MSC therapy in solid organ transplant recipients, there is increasing consideration for their use in liver transplantation. In this review we critically appraise the potential role of MSC therapy in the context of liver transplantation, including their ability to modulate reperfusion injury, their role in the reduction of medium term complications in the biliary tree and their potential to enhance tolerance in transplanted organs.

Skeletogenic Capacity of Human Perivascular Stem Cells Obtained Via Magnetic-Activated Cell Sorting.

Human perivascular stem/stromal cells (PSC) are a multipotent mesenchymal progenitor cell population defined by their perivascular residence. PSC are increasingly studied for their application in skeletal regenerative medicine. PSC from subcutaneous white adipose tissue are most commonly isolated via fluorescence-activated cell sorting (FACS), and defined as a bipartite population of CD146+CD34-CD31-CD45- pericytes and CD34+CD146-CD31-CD45- adventitial cells. FACS poses several challenges for clinical translation, including requirements for facilities, equipment, and personnel. The purpose of this study is to identify if magnetic-activated cell sorting (MACS) is a feasible method to derive PSC, and to determine if MACS-derived PSC are comparable to our previous experience with FACS-derived PSC. In brief, CD146+ pericytes and CD34+ adventitial cells were enriched from human lipoaspirate using a multistep column approach. Next, cell identity and purity were analyzed by flow cytometry. In vitro multilineage differentiation studies were performed with MACS-defined PSC subsets. Finally, in vivo application was performed in nonhealing calvarial bone defects in Scid mice. Results showed that human CD146+ pericytes and CD34+ adventitial cells may be enriched by MACS, with defined purity, anticipated cell surface marker expression, and capacity for multilineage differentiation. In vivo, MACS-derived PSC induce ossification of bone defects. These data document the feasibility of a MACS approach for the enrichment and application of PSC in the field of tissue engineering and regenerative medicine. Impact Statement Our findings suggest that perivascular stem/stromal cells, and in particular adventitial cells, may be isolated by magnetic-activated cell sorting and applied as an uncultured autologous stem cell therapy in a same-day setting for bone defect repair.

Sulforaphane protects against ethanol-induced apoptosis in neural crest cells through restoring epithelial-mesenchymal transition by epigenetically modulating the expression of Snail1

Ethanol-induced apoptosis in neural crest cells (NCCs), a multipotent progenitor cell population, is implicated in the Fetal Alcohol Spectrum Disorders (FASD). Studies have demonstrated that sulforaphane (SFN) can prevent ethanol-induced apoptosis in NCCs. The objective of this study is to investigate whether ethanol exposure can induce apoptosis in NCCs by inhibiting epithelial-mesenchymal transition (EMT) and whether SFN can prevent ethanol-induced apoptosis by epigenetically modulating the expression of Snail1, a key transcriptional factor that promotes EMT. We found that ethanol exposure resulted in a significant increase in apoptosis in NCCs. Co-treatment with SFN significantly reduced ethanol-induced apoptosis. Treatment with SFN also dramatically diminished ethanol-induced changes in the expression of E-cadherin and vimentin, and restored EMT in ethanol-exposed NCCs. In addition, ethanol exposure reduced the levels of trimethylation of histone H3 lysine 4 (H3K4me3) at the promoters of Snail1. SFN treatment diminished the ethanol-induced reduction of H3K4me3 at the promoter regions of the Snail1 gene, restored the expression of Snail1 and down-regulated Snail1 target gene E-cadherin. Knockdown of Snail1 significantly reduced the protective effects of SFN on ethanol-induced apoptosis. These results demonstrate that SFN can protect against ethanol-induced apoptosis by preventing ethanol-induced reduction in the levels of H3K4me3 at the promoters of Snail1, restoring the expression of Snail1 and EMT in ethanol-exposed NCCs.

Ras activation in retinal progenitor cells induces tumor formation in the eye

The RAS gene family members, H-RAS, K-RAS, and N-RAS, are frequently mutated in human cancer. A subset of retinal tumors displays K-RAS mutations; however, the specific role of RAS activation on retinal tumor formation is unclear. To examine the role of RAS in retinal development, we overexpressed the mutant H-RAS gene (G12V) in retinal progenitor cells (RPCs), a multipotent progenitor cell population that gives rise to all six neuron types in the retina and to the Muller glia. The Msi1CreER mouse strain was used to induce mosaic activation of Ras (RasV12) in the RPCs of the postnatal retina. RAS-activated RPCs translocated to the basal part of the retina, differentiated into cells with glial characteristics, and underwent apoptosis. We next induced RAS activation in a large population of RPCs in the embryonic retina using the Pax6Cre mouse strain. In contrast to the phenotype observed in Msi1CreER;RasV12 mice, Ras-activated cells retained their apical attachment. Basal translocation was partially suppressed in the retina of Pax6Cre;RasV12 mice, indicating that basal translocation of Ras-activated cells was not cell autonomous. Notably, RAS-activated retinal cells were highly proliferative and promoted the formation of eye tumors in Pax6Cre;RasV12 mice. Together, our data indicate that the tumorigenicity of RAS activation in RPCs is context dependent, with tumor formation occurring when RAS activity is present in a large cluster of embryonic RPCs.

Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans.

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

The ferroptosis inducer erastin promotes proliferation and differentiation in human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) contain multipotent progenitor cell populations and possess the potential to differentiate into various types of immune cells under both physiological and pathological conditions. Ferroptosis is a type of oxidative stress-associated cell death that is mainly mediated by lipid peroxidation. However, the function of ferroptosis in cell differentiation remains unknown. Here, we showed that the ferroptosis inducer erastin did not cause cell death in human PBMCs. In contrast, erastin-induced lipid peroxidation promoted human PBMC proliferation and differentiation into B cells and natural killer cells through inhibition of bone morphogenetic protein family expression. These findings uncover a new immune modulation function of erastin in promoting PBMC proliferation and differentiation.

C-Kit Cardiac Progenitor Cell Based Cell Sheet Improves Vascularization and Attenuates Cardiac Remodeling following Myocardial Infarction in Rats.

The adult heart contains small populations of multipotent cardiac progenitor cells (CPC) that present a convenient and efficient resource for treatment of myocardial infarction. Several clinical studies of direct CPC delivery by injection have already been performed but showed low engraftment rate that limited beneficial effects of procedure. «Cell sheet» technology has been developed to facilitate longer retention of grafted cells and show new directions for cell-based therapy using this strategy. In this study we hypothesized that СPC-based cell sheet transplantation could improve regeneration after myocardial infarction. We demonstrated that c-kit+ CPC were able to form cell sheets on temperature-responsive surfaces. Cell sheet represented a well-organized structure, in which CPC survived, retained ability to proliferate, expressed progenitor cell marker Gata-4 formed connexin-43+ gap junctions, and were surrounded by significant amount of extracellular matrix proteins. Transplantation of cell sheets after myocardial infarction resulted in CPC engraftment as well as their proliferation, migration, and differentiation; cell sheets also stimulated neovascularization and cardiomyocyte proliferation in underlining myocardium and ameliorated left ventricular remodeling. Obtained data strongly supported potential use of CPC sheet transplantation for repair of damaged heart.

Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors

BackgroundThe expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional β cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1−/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development.MethodsThe hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques.ResultsBased on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional β cells, and an additional novel population did not express PDX1 (PDX1−/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures.ConclusionsThese data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1−/NKX6.1+ population may be involved in a unique trajectory to generate β cells in humans.

Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1α, IL-1β, IFN-γ, and TNF-α gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.

Analysis of M\xfcller glia specific genes and their histone modification using Hes1-promoter driven EGFP expressing mouse

Retinal neurons and Müller glia are generated from a common population of multipotent retinal progenitor cells. We purposed to identify Müller glia-specific molecular signatures during retinal development. Using transgenic mice carrying the Hes1 promoter (pHes1) followed by EGFP, we purified EGFP-positive Müller glia and other EGFP-negative retinal cells from developing retinas and subjected them to RNA sequencing analysis. Gene expression pattern of EGFP-positive cell was similar to genes expressed in retinal progenitors, and they were downregulated in other cell lineages. Then, we examined the modification profiles of H3K27me3 and H3K4me3 by referring to chromatin immunoprecipitation-sequencing data of rods and other cells. Clustering of the H3K4me3 and H3K27me3 values followed by ontology analysis revealed a high incidence of transcription factors including Hes1 in clusters with high H3K27me3 levels. Hes1 expression level decreased dramatically, and the H3K27me3 level at the Hes1-locus was upregulated strongly during retinal development. Furthermore, the Hes1 expression level was upregulated in an Ezh2-knockout retina. These results suggest that downregulation of Müller glia-related genes in other lineage rather than upregulation of them in Müller glia contributed Müller-specific molecular features, and a role for modified H3K27me3 in suppressing Müller glia-related genes in other retinal cell lineages to avoid unfavorable expression.

Mechanosensory organ regeneration in zebrafish depends on a population of multipotent progenitor cells kept latent by Schwann cells.

BackgroundRegenerating damaged tissue is a complex process, requiring progenitor cells that must be stimulated to undergo proliferation, differentiation and, often, migratory behaviors and morphological changes. Multiple cell types, both resident within the damaged tissue and recruited to the lesion site, have been shown to participate. However, the cellular and molecular mechanisms involved in the activation of progenitor cell proliferation and differentiation after injury, and their regulation by different cells types, are not fully understood. The zebrafish lateral line is a suitable system to study regeneration because most of its components are fully restored after damage. The posterior lateral line (PLL) is a mechanosensory system that develops embryonically and is initially composed of seven to eight neuromasts distributed along the trunk and tail, connected by a continuous stripe of interneuromastic cells (INCs). The INCs remain in a quiescent state owing to the presence of underlying Schwann cells. They become activated during development to form intercalary neuromasts. However, no studies have described if INCs can participate in a regenerative event, for example, after the total loss of a neuromast.ResultsWe used electroablation in transgenic larvae expressing fluorescent proteins in PLL components to completely ablate single neuromasts in larvae and adult fish. This injury results in discontinuity of the INCs, Schwann cells, and the PLL nerve. In vivo imaging showed that the INCs fill the gap left after the injury and can regenerate a new neuromast in the injury zone. Further, a single INC is able to divide and form all cell types in a regenerated neuromast and, during this process, it transiently expresses the sox2 gene, a neural progenitor cell marker. We demonstrate a critical role for Schwann cells as negative regulators of INC proliferation and neuromast regeneration, and that this inhibitory property is completely dependent on active ErbB signaling.ConclusionsThe potential to regenerate a neuromast after damage requires that progenitor cells (INCs) be temporarily released from an inhibitory signal produced by nearby Schwann cells. This simple yet highly effective two-component niche offers the animal robust mechanisms for organ growth and regeneration, which can be sustained throughout life.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0249-2) contains supplementary material, which is available to authorized users.

Prevention of Treacher Collins syndrome craniofacial anomalies in mouse models via maternal antioxidant supplementation.

Craniofacial anomalies account for approximately one-third of all birth defects and are a significant cause of infant mortality. Since the majority of the bones, cartilage and connective tissues that comprise the head and face are derived from a multipotent migratory progenitor cell population called the neural crest, craniofacial disorders are typically attributed to defects in neural crest cell development. Treacher Collins syndrome (TCS) is a disorder of craniofacial development and although TCS arises primarily through autosomal dominant mutations in TCOF1, no clear genotype–phenotype correlation has been documented. Here we show that Tcof1 haploinsufficiency results in oxidative stress-induced DNA damage and neuroepithelial cell death. Consistent with this discovery, maternal treatment with antioxidants minimizes cell death in the neuroepithelium and substantially ameliorates or prevents the pathogenesis of craniofacial anomalies in Tcof1+/− mice. Thus maternal antioxidant dietary supplementation may provide an avenue for protection against the pathogenesis of TCS and similar neurocristopathies.

Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1−/− MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1−/− MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1−/− MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs.