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The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue‐and bone marrow‐derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle‐ and small‐sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC‐EVs overexpressed pro‐angiogenic factors in comparison to the BMSC‐counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC‐EVs contained a higher amount of pro‐differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications., This study shows how the differences among bone marrow‐ and adipose tissue‐derived mesenchymal stromal cells (BMSCs and ADSCs, respectively) are mirrored in the corresponding extracellular vesicles (EVs). The role of ADSCs‐ and BMSCs‐derived EVs in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics.

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue‐specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide‐label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long‐ and short‐lived TSC and showed that short‐lived TSC clones had significantly shorter telomeres than their long‐lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation., Biological aging of tracheobronchial tissue specific stem cells (TSC) and their trophic unit, the pseudostratified conducting airway epithelium. Each injury activates a subset of TSC which proliferate and undergo terminal differentiation. Thus, each injury cycle depletes the TSC pool and many injuries compromise epithelial regeneration. Over time, biological age exceeds chronological age and increases the risk of chronic lung disease.

The reconstruction of irradiated bone defects after settlement of skeletal tumors remains a significant challenge in clinical applications. In this study, we explored radiation‐induced skeletal stem cell (SSC) stemness impairments and rescuing effects of ferulic acid (FA) on SSCs in vitro and in vivo. The immunophenotype, cell renewal, cell proliferation, and differentiation of SSCs in vitro after irradiation were investigated. Mechanistically, the changes in tissue regeneration‐associated gene expression and MAPK pathway activation in irradiated SSCs were evaluated. The regenerative capacity of SSCs in the presence of FA in an irradiated bone defect mouse model was also investigated. We found that irradiation reduced CD140a‐ and CD105‐positive cells in skeletal tissues and mouse‐derived SSCs. Additionally, irradiation suppressed cell proliferation, colony formation, and osteogenic differentiation of SSCs. The RNA‐Seq results showed that tissue regeneration‐associated gene expression decreased, and the Western blotting results demonstrated the suppression of phosphorylated p38/MAPK and ERK/MAPK in irradiated SSCs. Notably, FA significantly rescued the radiation‐induced impairment of SSCs by activating the p38/MAPK and ERK/MAPK pathways. Moreover, the results of imaging and pathological analyses demonstrated that FA enhanced the bone repair effects of SSCs in an irradiated bone defect mouse model substantially. Importantly, inhibition of the p38/MAPK and ERK/MAPK pathways in SSCs by specific chemical inhibitors partially abolished the promotive effect of FA on SSC‐mediated bone regeneration. In summary, our findings reveal a novel function of FA in repairing irradiated bone defects by maintaining SSC stemness and suggest that the p38/MAPK and ERK/MAPK pathways contribute to SSC‐mediated tissue regeneration postradiation., Irradiation caused significant impairment of skeletal stem cell (SSC) stemness via inactivation of MAPK pathways. Ferulic acid (FA) rescued the irradiated SSC partially via reactivated MAPK signaling.

Basic mechanism of spine development is poorly understood. Type II collagen positive (Col2+) cells have been reported to encompass early mesenchymal progenitors that continue to become chondrocytes, osteoblasts, stromal cells, and adipocytes in long bone. However, the function of Col2+ cells in spine and intervertebral disc (IVD) development is largely unknown. To further elucidate the function of Col2+ progenitors in spine, we generated the mice with ablation of Col2+ cells either at embryonic or at postnatal stage. Embryonic ablation of Col2+ progenitors caused the mouse die at newborn with the absence of all spine and IVD. Moreover, postnatal deletion Col2+ cells in spine resulted in a shorter growth plate and endplate cartilage, defected inner annulus fibrosus, a less compact and markedly decreased gel‐like matrix in the nucleus pulposus and disorganized cell alignment in each compartment of IVD. Genetic lineage tracing IVD cell populations by using inducible Col2‐creERT;tdTomato reporter mice and non‐inducible Col2‐cre;tdTomato reporter mice revealed that the numbers and differentiation ability of Col2+ progenitors decreased with age. Moreover, immunofluorescence staining showed type II collagen expression changed from extracellular matrix to cytoplasm in nucleus pulposus between 6 month and 1‐year‐old mice. Finally, fate‐mapping studies revealed that Col2+ progenitors are essential for IVD repair in IVD injured model. In summary, embryonic Col2+ cells are the major source of spine development and Col2+ progenitors are the important contributors for IVD repair and regeneration., Type II collagen‐positive embryonic progenitors are the major contributors to spine and intervertebral disc development and repair.

Endothelial progenitor cells (EPCs) promote the maintenance of the endothelium by secreting vasoreparative factors. A population of EPCs known as early outgrowth cells (EOCs) is being investigated as novel cell‐based therapies for the treatment of cardiovascular disease. We previously demonstrated that the absence of liver X receptors (LXRs) is detrimental to the formation and function of EOCs under hypercholesterolemic conditions. Here, we investigate whether LXR activation in EOCs is beneficial for the treatment of atherosclerosis. EOCs were differentiated from the bone marrow of wild‐type (WT) and LXR‐knockout (Lxrαβ−/−) mice in the presence of vehicle or LXR agonist (GW3965). WT EOCs treated with GW3965 throughout differentiation showed reduced mRNA expression of endothelial lineage markers (Cd144, Vegfr2) compared with WT vehicle and Lxrαβ−/− EOCs. GW3965‐treated EOCs produced secreted factors that reduced monocyte adhesion to activated endothelial cells in culture. When injected into atherosclerosis‐prone Ldlr−/− mice, GW3965‐treated EOCs, or their corresponding conditioned media (CM) were both able to reduce aortic sinus plaque burden compared with controls. Furthermore, when human EOCs (obtained from patients with established CAD) were treated with GW3965 and the CM applied to endothelial cells, monocyte adhesion was decreased, indicating that our results in mice could be translated to patients. Ex vivo LXR agonist treatment of EOCs therefore produces a secretome that decreases early atherosclerosis in Ldlr−/− mice, and additionally, CM from human EOCs significantly inhibits monocyte to endothelial adhesion. Thus, active factor(s) within the GW3965‐treated EOC secretome may have the potential to be useful for the treatment of atherosclerosis., Bone marrow cells differentiated with liver x receptors agonist (GW) produce a population of early outgrowth cells (EOCs) with an alternative secretome, capable of decreasing monocyte‐endothelial adhesion in culture, and reducing atherosclerosis in vivo. GW‐treated EOCs derived from peripheral blood mononuclear cells (MNCs) of patients with coronary artery disease also produced a secretome that reduced monocyte‐endothelial adhesion in culture.

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair., Bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) were obtained from human bone marrow. Induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) were generated from induced pluripotent stem cell‐derived iNCCs. Cells were luciferase transfected and seeded onto decellularized allografts, which were implanted into 5 mm circular calvarial defects in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Allograft only was used as controls. Mice were followed for a duration of 8 weeks. Cell survival, allograft integration, and tissue morphology and transplanted cell contribution were monitored via bioluminescence imaging (BLI), micro‐computed tomography (μCT), and histology and immunofluorescence (IF). Green arrow: BLI; yellow arrow: μCT; Orange arrow: histology and IF; Blue arrow: biomechanical test.

Bone morphogenetic protein 2 (BMP2)‐induced heterotopic bone formation (HBF) starts synchronously from zero upon BMP2 induction, which is advantageous for lineage tracking. The studies reported here in GLAST‐CreErt2:tdTomato red (TR)floxSTOPflox mice during BMP2‐induced HBF show 78.8 ± 11.6% of chondrocytes and 86.5 ± 1.9% of osteoblasts are TR+ after approximately 1 week. Clustering after single‐cell RNAseq resulted in nine cell types, and analysis revealed one as a highly replicating stem‐like cell (RSC). Pseudotiming suggested that the RSC transitions to a mesenchymal stem‐like cell that simultaneously expresses multiple osteoblast and chondrocyte transcripts (chondro‐osseous progenitor [COP]). RSCs and COPs were isolated using flow cytometry for unique surface markers. Isolated RSCs (GLAST‐TR+ Hmmr+ Cd200−) and COPs (GLAST‐TR+ Cd200+ Hmmr−) were injected into the muscle of mice undergoing HBF. Approximately 9% of the cells in heterotopic bone (HB) in mice receiving RSCs were GLAST‐TR+, compared with less than 0.5% of the cells in mice receiving COPs, suggesting that RSCs are many times more potent than COPs. Analysis of donor‐derived TR+ RSCs isolated from the engrafted HB showed approximately 50% were COPs and 45% were other cells, presumably mature bone cells, confirming the early nature of the RSCs. We next isolated RSCs from these mice (approximately 300) and injected them into a second animal, with similar findings upon analysis of HBF. Unlike other methodology, single cell RNAseq has the ability to detect rare cell populations such as RSCs. The fact that RSCs can be injected into mice and differentiate suggests their potential utility for tissue regeneration., Blue arrows show the trajectory of heterotopic bone formation after bone morphogenetic protein 2 induction. The replicating stem‐like cell (RSC) is an epithelial cell that undergoes an epithelial‐mesenchymal transition in forming the chondro‐osseous progenitor (COP) that expresses both osteoblast and chondrocyte transcripts. Red arrows show the fluorescence‐activated cell sorting isolation of either TR+ RSCs or TR+ COPs and the injection of 3000 of each TR+ cell into wild‐type mice undergoing heterotopic ossification.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs., p75++ CjECs(conjunctival epithelial cells) were enriched by immuno‐magnetic beads and their stem cell properties were detected. After 10 days of culture on amniotic membrane, conjunctiva constructed by p75++/− CjECs was transplanted to a rabbit model of conjunctival defect to test the repair effect. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs.

Hematopoietic stem cell transplantation (HSCT) is a treatment for many malignant, congenital, and acquired hematologic diseases. Some outstanding challenges in the HSCT field include the paucity of immunologically‐matched donors, our inability to effectively expand hematopoeitic stem cells (HSCs) ex vivo, and the high infection risk during engraftment. Scientists are striving to develop protocols to generate, expand, and maintain HSCs ex vivo, however these are not yet ready for clinical application. Given these problems, advancing our understanding of HSC specification, regulation, and differentiation in preclinical models is essential to improve the therapeutic utility of HSCT. In this review, we link biomedical researchers and transplantation clinicians by discussing the potential therapeutic implications of recent fundamental HSC research in model organisms. We consider deficiencies in current HSCT practice, such as problems achieving adequate cell dose for successful and rapid engraftment, immense inflammatory cascade activation after myeloablation, and graft‐vs‐host disease. Furthermore, we discuss recent advances in the field of HSC biology and transplantation made in preclinical models of zebrafish, mouse, and nonhuman primates that could inform emerging practice for clinical application., Clinical (established) and preclinical (theoretical) advances are informing treatment options for circumventing current hematopoietic stem cell transplantation (HSCT) limitations including post‐transplant infection, low HSC donor supply, inflammation, and graft‐vs‐host disease (GVHD). This review covers the advances in preclinical hematopoietic stem cell models and possible implications for improving therapeutic transplantation.

Niches are specialized tissue microenvironments that control stem cells functioning. The bone marrow mesenchymal stem cell niche defines a location within the marrow in which mesenchymal stem cells are retained and produce new cells throughout life. Deciphering the signaling mechanisms by which the niche regulates stem cell fate will facilitate the use of these cells for therapy. Recent studies, by using state‐of‐the‐art methodologies, including sophisticated in vivo inducible genetic techniques, such as lineage‐tracing Cre/loxP mediated systems, in combination with pharmacological inhibition, provide evidence that sensory neuron is an important component of the bone marrow mesenchymal stem cell niche. Strikingly, knockout of a specific receptor in sensory neurons blocked stem cell function in the bone marrow. The knowledge arising from these discoveries will be crucial for stem cell manipulation in the future. Here, we review recent progress in our understanding of sensory nerves biology in the stem cell niche., Schematic illustration summarizing the findings from genetic deletion of EP4 specifically from sensory nerve fibers in the bone marrow mesenchymal stem cell niche.

Cellular replacement in the heart is restricted to postnatal stages with the adult heart largely postmitotic. Studies show that loss of regenerative properties in cardiac cells seems to coincide with alterations in metabolism during postnatal development and maturation. Nevertheless, whether changes in cellular metabolism are linked to functional alternations in cardiac cells is not well studied. We report here a novel role for uncoupling protein 2 (UCP2) in regulation of functional properties in cardiac tissue derived stem‐like cells (CTSCs). CTSC were isolated from C57BL/6 mice aged 2 days (nCTSC), 2 month (CTSC), and 2 years old (aCTSC), subjected to bulk‐RNA sequencing that identifies unique transcriptome significantly different between CTSC populations from young and old heart. Moreover, results show that UCP2 is highly expressed in CTSCs from the neonatal heart and is linked to maintenance of glycolysis, proliferation, and survival. With age, UCP2 is reduced shifting energy metabolism to oxidative phosphorylation inversely affecting cellular proliferation and survival in aged CTSCs. Loss of UCP2 in neonatal CTSCs reduces extracellular acidification rate and glycolysis together with reduced cellular proliferation and survival. Mechanistically, UCP2 silencing is linked to significant alteration of mitochondrial genes together with cell cycle and survival signaling pathways as identified by RNA‐sequencing and STRING bioinformatic analysis. Hence, our study shows UCP2‐mediated metabolic profile regulates functional properties of cardiac cells during transition from neonatal to aging cardiac states., Loss in cardiac regeneration coincides with metabolic alterations. Whether cellular metabolism regulates cardiac cells function from young to old age remains unknown. Here we demonstrate that uncoupling protein 2 (UCP2) promotes increased glycolysis, proliferation, and survival in cardiac tissue derived stem‐like cells in neonatal heart. In aging, UCP2 is reduced increasing oxidative phosphorylation, reducing glycolysis parallel with reduced proliferation and survival.

Although mesenchymal stem/stromal cells (MSCs) are being explored in numerous clinical trials as proangiogenic and proregenerative agents, the influence of tissue origin on the therapeutic qualities of these cells is poorly understood. Complicating the functional comparison of different types of MSCs are the confounding effects of donor age, genetic background, and health status of the donor. Leveraging a clinical setting where MSCs can be simultaneously isolated from discarded but healthy bone and thymus tissues from the same neonatal patients, thereby controlling for these confounding factors, we performed an in vitro and in vivo paired comparison of these cells. We found that both neonatal thymus (nt)MSCs and neonatal bone (nb)MSCs expressed different pericytic surface marker profiles. Further, ntMSCs were more potent in promoting angiogenesis in vitro and in vivo and they were also more motile and efficient at invading ECM in vitro. These functional differences were in part mediated by an increased ntMSC expression of SLIT3, a factor known to activate endothelial cells. Further, we discovered that SLIT3 stimulated MSC motility and fibrin gel invasion via ROBO1 in an autocrine fashion. Consistent with our findings in human MSCs, we found that SLIT3 and ROBO1 were expressed in the perivascular cells of the neonatal murine thymus gland and that global SLIT3 or ROBO1 deficiency resulted in decreased neonatal murine thymus gland vascular density. In conclusion, ntMSCs possess increased proangiogenic and invasive behaviors, which are in part mediated by the paracrine and autocrine effects of SLIT3., Comparison of mesenchymal stem/stromal cells (MSCs) from the human neonatal thymus and bone revealed that the axon guidance molecule SLIT3 is important for MSC proangiogenic effects. Not only is SLIT3 an endothelial cell stimulatory factor, but it also promotes MSC migration and invasion in an autocrine fashion via the ROBO1 receptor. Deficiency of either SLIT3 or ROBO1 can decrease the vascularization of the neonatal thymus.

While human adipose‐derived stem cells (hADSCs) are known to possess osteogenic differentiation potential, the bone tissues formed are generally considered rudimentary and immature compared with those made by bone‐derived precursor cells such as human bone marrow‐derived mesenchymal stem cells (hBMSCs) and less commonly studied human calvarium osteoprogenitor cells (hOPs). Traditional differentiation protocols have tended to focus on osteoinduction of hADSCs through the addition of osteogenic differentiation media or use of stimulatory bioactive scaffolds which have not resulted in mature bone formation. Here, we tested the hypothesis that by reproducing the physical as well as biochemical bone microenvironment through the use of three‐dimensional (3D) culture and vascularization we could enhance osteogenic maturation in hADSCs. In addition to biomolecular characterization, we performed structural analysis through extracellular collagen alignment and mineral density in our bone tissue engineered samples to evaluate osteogenic maturation. We further compared bone formed by hADSCs, hBMSCs, and hOPs against mature human pediatric calvarial bone, yet not extensively investigated. Although bone generated by all three cell types was still less mature than native pediatric bone, a fibrin‐based 3D microenvironment together with vascularization boosted osteogenic maturation of hADSC making it similar to that of bone‐derived osteoprogenitors. This demonstrates the important role of vascularization and 3D culture in driving osteogenic maturation of cells easily available but constitutively less committed to this lineage and suggests a crucial avenue for recreating the bone microenvironment for tissue engineering of mature craniofacial bone tissues from pediatric hADSCs, as well as hBMSCs and hOPs., Human adipose‐derived stem cells (hADSCs) are a more accessible source of cells for bone tissue engineering but limited by immature osteogenic differentiation capacity. This study demonstrates that three‐dimensional microenvironment and early vascularization promote osteogenic maturation achieved by hADSC comparable to calvarial bone‐derived precursor cells such as human bone marrow‐derived mesenchymal stem cells (hBMSCs) and human osteoprogenitor cells (hOPs).

Glial cells are crucial for the development of the central nervous system and the maintenance of chemical homeostasis. The process of gliogenesis has been well studied in the rodent brain, but it remains less well studied in the human brain. In addition, rodent glial cells differ from human counterparts in terms of morphologies, functions, and anatomical locations. Cerebral organoids (also referred to as spheroids) derived from human pluripotent stem cells (hPSCs) have been developed and are suitable cell‐based models for researching developmental and neurodegenerative diseases. The in vitro generation of glia, including astrocytes and oligodendrocytes, from such organoids represents a promising tool to model neuronal diseases. Here, we showed that three‐dimensional (3D) culture of OLIG2‐ and NKX2.2‐expressing neurospheres produced efficiently mature astrocytes and oligodendrocytes in terms of morphologies and expression pattern recapitulating native 3D environment. Our findings provide important insights for developmental research of the human brain and glial specification that may facilitate patient‐specific disease modeling., Human pluripotent stem cell‐derived cerebral organoids can mimic development of the mammalian central nervous system. In this study, we demonstrate that pre‐patterned stem/progenitor cells, especially OLIG2/NKX2.2‐expressing pre‐oligodendrocyte progenitor cells, facilitate glial specification during organoid development. The resulting spheroids can serve as in vitro model for myelination to evaluate promyelination drugs and a source for cell therapy in demyelinating disease.

Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy. Significance statement Radiation-induced lung injury is a dose-limiting toxicity that limits the effective dose that should be administered and forces the interruption of the treatment. The NOTCH signaling pathway is a potential therapeutic target for lung cancer because its inhibition reduces tumor growth and synergizes with radiotherapy and chemotherapy in preclinical models. However, the effect of inhibiting NOTCH in irradiated normal lung tissue is not known and could impact the therapeutic benefit of combination treatments. This study demonstrates that small-molecule inhibitors of the NOTCH pathway enhance the survival of irradiated primary human and murine bronchial epithelial lung stem cells. This finding may be beneficial in lung cancer treatment with radiotherapy and NOTCH inhibitors by protecting normal lung tissue while increasing tumor control.

The potential of obtaining cell cultures with neural crest resemblance (neural crest‐derived stem cells [NCSCs]) from dental‐related tissues, including human dental pulp cells (hDPCs), has been discussed in the literature. However, most reports include the use of serum‐rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum‐free, and also under sphere‐aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest‐related markers. Culturing hDPCs as neurospheres (hDPC‐NCSCs) resulted in an increased expression of neural crest‐related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural‐like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1, and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum‐free medium, as NCSC that differentiate into neural‐like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs., Human dental pulp tissue collected from a varied cohort of samples was used to directly establish monolayer cultures in defined serum‐free conditions. In monolayer, the cultures maintained an mesenchymal stem cell (MSC) phenotype regardless of the initial serum‐free conditions. A Neural crest‐derived stem cell phenotype was favored by adding BMP4 before or after transferring the cells to a 3D culture.

Adipogenesis participates in many physiological and pathological processes, such as obesity and diabetes, and is regulated by a series of precise molecular events. However, the molecules involved in this regulation have not been fully characterized. In this study, we identified a long noncoding (lnc)RNA, lncRNA‐Adi, which is highly expressed in adipose tissue‐derived stromal cells (ADSCs) that are differentiating into adipocytes. Knockdown of lncRNA‐Adi impaired the adipogenic differentiation ability of ADSCs. Moreover, lncRNA‐Adi was found to interact with microRNA (miR)‐449a to enhance the expression of cyclin‐dependent kinase (CDK)6 during adipogenesis. The mechanism by which lncRNA‐Adi regulates adipogenesis was determined to involve an lncRNA‐Adi‐miR‐449a interaction that competes with the CDK6 3′ untranslated region to increase CDK6 translation and activate the pRb‐E2F1 pathway to promote adipogenesis. These findings provide valuable information and a new study angle to search for therapeutic targets against metabolic disorders such as obesity and diabetes., Here, we screened out a novel lncRNA, which was named lncRNA‐Adi, that highly expressed in adipogenic‐induced adipose tissue‐derived stromal cells (ADSCs). Then, knockdown of lncRNA‐Adi impaired the adipogenic differentiation ability of ADSCs. Finally, we verified that lncRNA‐Adi could protect CDK6 from degradation by competitively interacting with miR‐449a, to improve CDK6 translation level and activate pRb‐E2F1 pathway, which is crucial to promoting adipogenesis.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum‐free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA‐CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA‐treated cultures were able to engraft in immune‐deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA‐mediated ex vivo HSC expansion for gene modification of adult HSCs., Valproic acid (VPA)‐mediated ex vivo expansion of adult bone marrow and mobilized peripheral blood CD34+ cells resulted in a cellular product characterized by high viability, enrichment with CD34+CD45RA‐CD90+ cells, increased aldehyde dehydrogenase (ALDH) activity, robust multipotent clonogenic potential, and decreased mitochondrial potential. VPA‐expanded grafts were able to establish unbiased multilineage human hematopoietic‐cell chimerism in NSG mice at 16 weeks post‐transplantation.

There is increasing interest in the use of adipose‐derived mesenchymal stromal cells (ASCs) for wound repair. As the fate of administered cells is still poorly defined, we aimed to establish the location, survival, and effect of ASCs when administered either systemically or locally during wound repair under physiological conditions. To determine the behavior of ASCs, a rat model with wounds on the dorsal aspect of the hind paws was used and two treatment modes were assessed: ASCs administered systemically into the tail vein or locally around the wound. ASCs were transduced to express both firefly luciferase (Fluc) and green fluorescent protein to enable tracking by bioluminescence imaging and immunohistological analysis. Systemically administered ASCs were detected in the lungs 3 hours after injection with a decrease in luminescent signal at 48 hours and signal disappearance from 72 hours. No ASCs were detected in the wound. Locally administered ASCs remained strongly detectable for 7 days at the injection site and became distributed within the wound bed as early as 24 hours post injection with a significant increase observed at 72 hours. Systemically administered ASCs were filtered out in the lungs, whereas ASCs administered locally remained and survived not only at the injection site but were also detected within the wound bed. Both treatments led to enhanced wound closure. It appears that systemically administered ASCs have the potential to enhance wound repair distally from their site of entrapment in the lungs whereas locally administered ASCs enhanced wound repair as they became redistributed within the wound bed., Adipose‐derived mesenchymal stromal cells were administered either systemically or locally in a rat model of wound repair under physiological conditions. It appears that systemically administered adipose‐derived mesenchymal stromal cells have the potential to enhance wound repair distally from their site of entrapment in the lungs whereas locally administered adipose‐derived mesenchymal stromal cells enhanced wound repair as they became redistributed within the wound bed.

Adipose‐derived mesenchymal stem cells (ASCs) exhibit great potential in regenerative medicine, and in vitro expansion is frequently necessary to obtain a sufficient number of ASCs for clinical use. Fibroblast growth factor 2 (FGF2) is a common supplement in the ASC culture medium to enhance cell proliferation. To achieve clinical applicability of ASC‐based products, prolonged culture of ASCs is sometimes required to obtain sufficient quantity of ASCs. However, the effect of FGF2 on ASCs during prolonged culture has not been previously determined. In this study, ASCs were subjected to prolonged in vitro culture with or without FGF2. FGF2 maintained the small cell morphology and expedited proliferation kinetics in early ASC passages. After prolonged in vitro expansion, FGF2‐treated ASCs exhibited increased cell size, arrested cell proliferation, and increased cellular senescence relative to the control ASCs. We observed an upregulation of FGFR1c and enhanced expression of downstream STAT3 in the initial passages of FGF2‐treated ASCs. The application of an FGFR1 or STAT3 inhibitor effectively blocked the enhanced proliferation of ASCs induced by FGF2 treatment. FGFR1c upregulation and enhanced STAT3 expression were lost in the later passages of FGF2‐treated ASCs, suggesting that the continuous stimulation of FGF2 becomes ineffective because of the refractory downstream FGFR1 and the STAT3 signaling pathway. In addition, no evidence of tumorigenicity was noted in vitro and in vivo after prolonged expansion of FGF2‐cultured ASCs. Our data indicate that ASCs have evolved a STAT3‐dependent response to continuous FGF2 stimulation which promotes the initial expansion but limits their long‐term proliferation., Fibroblast growth factor 2 (FGF2) exerts positive effects in the early passages of adipose‐derived mesenchymal stem cells (ASCs), including maintenance of cellular morphology, less senescent cells, and expedition of proliferative kinetics. However, after prolonged in vitro expansion with FGF2, decreased proliferation with more senescent cells was noted, which was probably mediated through FGFR1 and the downstream STAT3 signaling pathway.

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c‐Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast‐like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast‐like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra‐embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra‐embryonic tissue formation., Adult c‐Kit+cardiac interstitial cells (cCICs) were assessed for retention, persistence, and phenotypic characteristics at three developmental stages: embryonic (E3.5), fetal (E15.5), and neonatal hearts (P3). cCICs delivered into E3.5 subsequently integrated into extra‐embryonic membranes. cCICs delivered into E15.5 and P3 exhibited a fibroblast‐like phenotype, with persistence and cell cycle activity up to 28 days in P3 hearts.

Significant progress has been made in recent years in characterizing human multipotent progenitor cells (hMPCs) of the early pancreas; however, the identity and persistence of these cells during the second trimester, after the initiation of branching morphogenesis, remain elusive. Additionally, studies on hMPCs have been hindered by few isolation methods that allow for the recovery of live cells. Here, we investigated the tip progenitor domain in the branched epithelium of human fetal pancreas between 13.5 and 17.5 gestational weeks by immunohistological staining. We also used a novel RNA‐based technology to isolate live cells followed by gene expression analyses. We identified cells co‐expressing SOX9 and PTF1A, two transcription factors known to be important for pancreatic MPCs, within the tips of the epithelium and observed a decrease in their proportions over time. Pancreatic SOX9+/PTF1A+ cells were enriched for MPC markers, including MYC and GATA6. These cells were proliferative and appeared active in branching morphogenesis and matrix remodeling, as evidenced by gene set enrichment analysis. We identified a hub of genes pertaining to the expanding tip progenitor niche, such as FOXF1, GLI3, TBX3, FGFR1, TGFBR2, ITGAV, ITGA2, and ITGB3. YAP1 of the Hippo pathway emerged as a highly enriched component within the SOX9+/PTF1A+ cells. Single‐cell RNA‐sequencing further corroborated the findings by identifying a cluster of SOX9+/PTF1A+ cells with multipotent characteristics. Based on these results, we propose that the SOX9+/PTF1A+ cells in the human pancreas are uncommitted MPC‐like cells that reside at the tips of the expanding pancreatic epithelium, directing self‐renewal and inducing pancreatic organogenesis. stem cells translational medicine 2019;8:1249&1264, Schematic representation of SOX9+/PTF1A+ cell isolation and characterization.

Cell‐based therapy for the treatment of inflammatory disorders has focused on the application of mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs). Despite the recent positive findings in industry‐sponsored clinical trials of MSCs and MAPCs for graft vs host disease (GvHD), cell therapy is efficacious in some but not all patients, highlighting the need to identify strategies to enhance cell‐based therapeutic efficacy. Here, we demonstrate the capacity for interferon (IFN)‐γ licensing to enhance human MAPC efficacy and retention following early administration in a humanized mouse model of acute GvHD (aGvHD). Activation of the nuclear receptor peroxisome proliferator‐activated receptor delta (PPARδ) negatively influenced the retention and efficacy of human MAPCs as well as IFN‐γ‐licensed MAPCs in the aGvHD model. PPARδ antagonism significantly enhanced the efficacy of human MAPCs when administered early in the humanized aGvHD model. COX‐2 expression in human MAPC was significantly decreased in IFN‐γ licensed MAPCs exposed to a PPARδ agonist. Importantly, MAPC exposure to the PPARδ antagonist in the presence of a COX‐2 inhibitor indomethacin before administration significantly reduced the efficacy of PPARδ antagonized MAPCs in the aGvHD humanized mouse model. This is the first study to demonstrate the importance of PPARδ in human MAPC efficacy in vivo and highlights the importance of understanding the disease microenvironment in which cell‐based therapies are to be administered. In particular, the presence of PPARδ ligands may negatively influence MAPC or MSC therapeutic efficacy., Interferon‐γ (IFN‐γ) licensing enhances the therapeutic efficacy of human multipotent adult progenitor cells (MAPCs) in a mouse model of acute graft vs host disease (aGvHD). Activation of peroxisome proliferator‐activated receptor (PPAR) δ reduces, whereas antagonism of PPARδ(−) enhances MAPC efficacy in aGvHD. PPARδ agonism reduces COX‐2 expression in IFN‐γ licensed MAPC. Figure created with BioRender.com.

Hematopoietic stem progenitor cells (HSPCs) mobilized to peripheral blood, rather than those remaining in the bone marrow (BM), are commonly used as stem cell source in the clinic. As reactive oxygen species (ROS) are suggested as mediator of HSPC mobilization, we examined the impacts of glucose oxidase (GO) on peripheral mobilization of BM HSPCs and the associated mechanisms. Intravenous injection of GO induced HSPC mobilization even by single treatment, and the GO‐mobilized cells maintained their long‐term reconstituting and differentiating potentials in conditioned recipients. GO‐injected mice lived a normal life without adverse effects such as stem cell senescence, hematopoietic disorders, and blood parameter alteration. The mobilization effect of GO was even evident in animal models showing poor mobilization, such as old, 5‐fluorouracil‐treated, or alendronate‐treated mice. Importantly, combined injection of GO with granulocyte colony‐stimulating factor (G‐CSF) and/or AMD3100 enhanced more greatly HSPC mobilization than did G‐CSF, AMD3100, or both. The GO‐stimulated HSPC mobilization was almost completely attenuated by n‐acetyl‐L‐cysteine treatment. Collectively, our results not only highlight the potential role of GO in HSPC mobilization via ROS signaling, but also provide a GO‐based new strategy to improve HSPC mobilization in poorly mobilizing allogeneic or autologous donors via combination with G‐CSF and/or AMD3100., Single injection of glucose oxidase (GO) induces peripheral mobilization of hematopoietic stem progenitor cells (HSPCs) that maintain colony forming, repopulating, and multilineage differentiating potentials without hematopoietic disorders. Single GO injection not only mobilizes HPSCs even in the mice exhibiting poor HSPC mobilization, but also enhances synergistically mobilizer‐induced HSPC mobilization. Collectively this study provides an evidence that combined treatment of GO with commonly used mobilizers augments therapeutic efficacy in HSPC transplantation without adverse events.

The renal mesenchyme contains heterogeneous cells, including interstitial fibroblasts and pericytes, with key roles in wound healing. Although healing is impaired in aged kidneys, the effect of age and injury on the mesenchyme remains poorly understood. We characterized renal mesenchymal cell heterogeneity in young vs old animals and after ischemia‐reperfusion‐injury (IRI) using multiplex immunolabeling and single cell transcriptomics. Expression patterns of perivascular cell markers (α‐SMA, CD146, NG2, PDGFR‐α, and PDGFR‐β) correlated with their interstitial location. PDGFR‐α and PDGFR‐β co‐expression labeled renal myofibroblasts more efficiently than the current standard marker α‐SMA, and CD146 was a superior murine renal pericyte marker. Three renal mesenchymal subtypes; pericytes, fibroblasts, and myofibroblasts, were recapitulated with data from two independently performed single cell transcriptomic analyzes of murine kidneys, the first dataset an aging cohort and the second dataset injured kidneys following IRI. Mesenchymal cells segregated into subtypes with distinct patterns of expression with aging and following injury. Baseline uninjured old kidneys resembled post‐ischemic young kidneys, with this phenotype further exaggerated following IRI. These studies demonstrate that age modulates renal perivascular/interstitial cell marker expression and transcriptome at baseline and in response to injury and provide tools for the histological and transcriptomic analysis of renal mesenchymal cells, paving the way for more accurate classification of renal mesenchymal cell heterogeneity and identification of age‐specific pathways and targets., Kidney mesenchyme contains progenitor cells important in disease aetiology yet is poorly characterized with age and lacks research tools. Healthy and ischemia reperfusion injured (IRI) kidneys from old and young mice, analyzed using immunohistochemistry (IHC) and single cell RNA sequencing (scRNAseq), uncovered differences in aged injury response and provided more detailed characterization of kidney mesenchyme. BV = blood vessel.

The Notch signaling pathway is a fundamental regulator of cell fate determination in homeostasis and regeneration. In this work, we aimed to determine how Notch signaling mediates the interactions between perivascular stem cells and their niches in human dental mesenchymal tissues, both in homeostatic and regenerative conditions. By single cell RNA sequencing analysis, we showed that perivascular cells across the dental pulp and periodontal human tissues all express NOTCH3, and that these cells are important for the response to traumatic injuries in vivo in a transgenic mouse model. We further showed that the behavior of perivascular NOTCH3‐expressing stem cells could be modulated by cellular and molecular cues deriving from their microenvironments. Taken together, the present studies, reinforced by single‐cell analysis, reveal the pivotal importance of Notch signaling in the crosstalk between perivascular stem cells and their niches in tissue homeostasis and regeneration., In the dental pulp and the periodontium, mesenchymal stem cells (MSCs) are highly similar, are mostly located around blood vessels, and express NOTCH3. These NOTCH3 + MSCs are important for the reaction of dental tissues to injury. In both tissues, endothelial cells adjacent to NOTCH3 + MSCs express NOTCH ligands, which could thus mediate the molecular crosstalk between MSCs and their perivascular niche.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high‐throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1‐[(4‐nitrophenyl)sulfonyl]‐4‐phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase‐polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1‐[(4‐Nitrophenyl)sulfonyl]‐4‐phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI‐ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen., The intestinal stem cell niche responds to radiation with apoptosis of intestinal stem cells and subsequent loss of tissue integrity. Treatment with compound #5 activates Hedgehog signaling, expands the number of intestinal stem cells and regenerating crypts, and allows for survival of the animals after a lethal dose of radiation.

Cancer stem cells play a critical role in disease initiation and insensitivity to chemotherapy in numerous hematologic malignancies and some solid tumors, and these stem cells need to be eradicated to achieve a cure. Key to successful targeting of cancer stem cells is to identify and functionally test critical target genes and to fully understand their associated molecular network in these stem cells. Human chronic myeloid leukemia (CML) is well accepted as one of the typical types of hematopoietic malignancies that are derived from leukemia stem cells (LSCs), serving as an excellent model disease for understanding the biology of LSCs and developing effective, selective, and curative strategies through targeting LSCs. Here, we discuss LSCs in CML with a focus on identification of unique biological features of these stem cells to emphasize the feasibility and significance of specific targeting of LSCs while sparing normal stem cell counterparts in leukemia therapy. Stem Cells Translational Medicine 2019;8:768–774

Patients with type 1 diabetes mellitus (T1DM) often suffer from osteopenia or osteoporosis. Although most agree that T1DM-induced hyperglycemia is a risk factor for progressive bone loss, the mechanisms for the link between T1DM and bone loss still remain elusive. In this study, we found that bone marrow-derived mesenchymal stem cells (BMSCs) isolated from T1DM donors were less inducible for osteogenesis than those from non-T1DM donors and further identified a mechanism involving bone morphogenetic protein-6 (BMP6) that was produced significantly less in BMSCs derived from T1DM donors than that in control cells. With addition of exogenous BMP6 in culture, osteogenesis of BMSCs from T1DM donors was restored whereas the treatment of BMP6 seemed not to affect non-T1DM control cells. We also demonstrated that bone mineral density (BMD) was reduced in streptozotocin-induced diabetic mice compared with that in control animals, and intraperitoneal injection of BMP6 mitigated bone loss and increased BMD in diabetic mice. Our results suggest that bone formation in T1DM patients is impaired by reduction of endogenous BMP6, and supplementation of BMP6 enhances osteogenesis of BMSCs to restore BMD in a mouse model of T1DM, which provides insight into the development of clinical treatments for T1DM-assocaited bone loss. Stem Cells Translational Medicine 2019;8:522–534

Islet transplantation has the potential to cure type 1 diabetes, but current transplantation protocols are not optimal and there is extensive loss of islet β‐cell insulin secretory function during the immediate post‐transplantation period. Studies using experimental models of diabetes have shown that the coculture of islets with mesenchymal stromal cells (MSCs) prior to transplantation improves graft function, but several variables differed among research groups (e.g., type of MSCs used and the treatment conditions). We have therefore assessed the effects of MSCs on mouse and human islets by investigating the importance of tissue source for MSCs, the coculture protocol configuration and length, the effect of activated MSCs, and different β‐cell secretory stimuli. MSCs derived from adipose tissue (aMSCs) were the most effective at supporting β‐cell insulin secretion in both mouse and human islets, in a direct contact coculture configuration. Preculture with aMSCs enhanced both phases of glucose‐induced insulin secretion and further enhanced secretory responses to the non‐nutrients carbachol and arginine. These effects required a coculture period of 48–72 hours and were not dependent on activation of the MSCs. Thus, direct contact coculture with autologous, adipose‐derived MSCs for a minimum of 48 hours before implantation is likely to be an effective addition to human islet transplantation protocols. Stem Cells Translational Medicine 2019;8:935&944

Glaucoma is one of the leading causes of blindness, and there is an ongoing need for new therapies. Recent studies indicate that cell transplantation using Müller glia may be beneficial, but there is a need for novel sources of cells to provide therapeutic benefit. In this study, we have isolated Müller glia from retinal organoids formed by human induced pluripotent stem cells (hiPSCs) in vitro and have shown their ability to partially restore visual function in rats depleted of retinal ganglion cells by NMDA. Based on the present results, we suggest that Müller glia derived from retinal organoids formed by hiPSC may provide an attractive source of cells for human retinal therapies, to prevent and treat vision loss caused by retinal degenerative conditions. Stem Cells Translational Medicine 2019;8:775–784

The positive effects of therapeutic human allogeneic cardiac stem/progenitor cells (hCPC) in terms of cardiac repair/regeneration are very likely mediated by paracrine effects. Our previous studies revealed the advantageous immune interactions of allogeneic hCPC and proposed them as part of the positive paracrine effects occurring upon their application postmyocardial infarction (MI). Currently, extracellular vesicles/exosomes (EV/Exs) released by stem/progenitor cells are also proposed as major mediators of paracrine effects of therapeutic cells. Along this line, we evaluated contribution of EV/Exs released by therapeutic hCPC to the benefit of their successful allogeneic clinical application. Through tailored allogeneic in vitro human assay models mimicking the clinical setting, we demonstrate that hCPC-released EV/Exs were rapidly and efficiently up-taken by chief cellular actors of cardiac repair/regeneration. This promoted MAPK/Erk1/2 activation, migration, and proliferation of human leukocyte antigens (HLA)-mismatched hCPC, mimicking endogenous progenitor cells and cardiomyocytes, and enhanced endothelial cell migration, growth, and organization into tube-like structures through activation of several signaling pathways. EV/Exs also acted as pro-survival stimuli for HLA-mismatched monocytes tuning their phenotype toward an intermediate anti-inflammatory pro-angiogenic phenotype. Thus, while positively impacting the intrinsic regenerative and angiogenic programs, EV/Exs released by therapeutic allogeneic hCPC can also actively contribute to shaping MI-inflammatory environment, which could strengthen the benefits of hCPC allogeneic interactions. Collectively, our data might forecast the application of allogeneic hCPC followed by their cell-free EV/Exs as a strategy that will not only elicit the cell-contact mediated reparative/regenerative immune response but also have the desired long-lasting effects through the EV/Exs. Stem Cells Translational Medicine 2019;8:911–924

Mesenchymal stem cells (MSCs) are proven to be beneficial in islet transplantation, suggesting a potential therapeutic role of them in total pancreatectomy with islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) patients. We investigated whether MSCs derived from CP patients are suitable for use in autologous cell therapy. MSCs from healthy donors (H-MSCs) and CP patients (CP-MSCs) were studied for phenotype, colony formation potential, multilineage differentiation ability, proliferation, senescence, secretory characters, and immunosuppressive functions. The potential protective effect of CP-MSCs was evaluated on hypoxia-induced islet cell death. Cell surface markers were similar between H-MSCs and CP-MSCs, as well as the ability of colony formation, multilineage differentiation, secretion of vascular endothelial growth factor and transforming growth factor (TGF-β), senescence, and inhibition of T cells proliferation in vitro. We found that growth differentiation factor 6 and hepatocyte growth factor (HGF) were significantly downregulated, whereas TGFβ and matrix metalloproteinase-2 were significantly upregulated in CP-MSCs compared with H-MSCs, among 84 MSC-related genes investigated in this study. MSCs from CP patients secreted less HGF, compared with the H-MSCs. A higher interferon-γ-induced indoleamine 2,3-dioxygenase expression was observed in CP-MSCs compared to H-MSCs. Moreover, CP-MSCs prevented hypoxia-induced β cell deaths to a similar extent as H-MSCs. Regardless of moderate difference in gene expression, CP-MSCs possess similar immunomodulatory and prosurvival functions to H-MSCs, and may be suitable for autologous cell therapy in CP patients undergoing TP-IAT. Stem Cells Translational Medicine 2019;8:418–429

Considerable effort has been directed toward deriving endothelial cells (ECs) from adipose-derived mesenchymal stem cells (ASCs) since 2004, when it was first suggested that ECs and adipocytes share a common progenitor. While the capacity of ASCs to express endothelial markers has been repeatedly demonstrated, none constitute conclusive evidence of an endothelial phenotype as all reported markers have been detected in other, non-endothelial cell types. In this study, quantitative phenotypic comparisons to representative EC controls were used to determine the extent of endothelial differentiation being achieved with ASCs. ASCs were harvested from human subcutaneous abdominal white adipose tissue, and their endothelial differentiation was induced using well-established biochemical stimuli. Reverse transcription quantitative real-time polymerase chain reaction and parallel reaction monitoring mass spectrometry were used to quantify their expression of endothelial genes and corresponding proteins, respectively. Flow cytometry was used to quantitatively assess their uptake of acetylated low-density lipoprotein (AcLDL). Human umbilical vein, coronary artery, and dermal microvascular ECs were used as positive controls to reflect the phenotypic heterogeneity between ECs derived from different vascular beds. Biochemically conditioned ASCs were found to upregulate their expression of endothelial genes and proteins, as well as AcLDL uptake, but their abundance remained orders of magnitude lower than that observed in the EC controls despite their global proteomic heterogeneity. The findings of this investigation demonstrate the strikingly limited extent of endothelial differentiation being achieved with ASCs using well-established biochemical stimuli, and underscore the importance of quantitative phenotypic comparisons to representative primary cell controls in studies of differentiation. Stem Cells Translational Medicine 2019;8:35–45

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl−/− mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl−/− mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl−/− mice that carried scaffolds with either WT or LVhsRL-transduced Rankl−/− MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl−/− mice. Stem Cells Translational Medicine 2019;8:22–34

Fibrous dysplasia (FD) is a disease of postnatal skeletal stem cells caused by activating mutations of guanine nucleotide-binding protein alpha-stimulating activity polypeptide (GNAS). FD is characterized by high proliferation and osteogenesis disorder of bone marrow stromal cells (BMSCs), resulting in bone pain, deformities, and fractures. The cAMP-CREB pathway, which is activated by GNAS mutations, is known to be closely associated with the occurrence of FD. However, so far there is no available targeted therapeutic strategy for FD, as a critical issue that remains largely unknown is how this pathway is involved in FD. Our previous study revealed that histone deacetylase 8 (HDAC8) inhibited the osteogenic differentiation of BMSCs via epigenetic regulation. Here, compared with normal BMSCs, FD BMSCs exhibited significantly high proliferation and weak osteogenic capacity in response to HDAC8 upregulation and tumor protein 53 (TP53) downregulation. Moreover, inhibition of cAMP reduced HDAC8 expression, increased TP53 expression and resulted in the improvement of FD phenotype. Importantly, HDAC8 inhibition prevented cAMP-induced cell phenotype and promoted osteogenesis in nude mice that were implanted with FD BMSCs. Mechanistically, HDAC8 was identified as a transcriptional target gene of CREB1 and its transcription was directly activated by CREB1 in FD BMSCs. In summary, our study reveals that HDAC8 associates with FD phenotype and demonstrates the mechanisms regulated by cAMP-CREB1-HDAC8 pathway. These results provide insights into the molecular regulation of FD pathogenesis, and offer novel clues that small molecule inhibitors targeting HDAC8 are promising clinical treatment for FD. Stem Cells Translational Medicine 2019;8:148&14

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246

Chronic inflammation and severe dry eye are two important adverse factors for the successful transplant of cultured limbal stem cells. The aim of this study was to investigate the effects of inflammation and hyperosmotic stress (a key pathological factor in dry eye) on corneal epithelial stem cells (CESCs) and corneal epithelial wound healing. We observed that the CESCs exhibited significant morphological changes when treated with interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), or hyperosmotic stress. Colony-forming efficiency or colony-forming size was decreased with the increasing concentrations of IL-1β, TNF-α, or hyperosmotic stress, which was exacerbated when treated simultaneously with pro-inflammatory factors and hyperosmotic stress. However, the colony-forming capacity of CESCs recovered more easily from pro-inflammatory factor treatment than from hyperosmotic stress treatment. Moreover, when compared with pro-inflammatory factors treatment, hyperosmotic stress treatment caused a more significant increase of apoptotic and necrotic cell numbers and cell cycle arrest in the G2/M phase. Furthermore, the normal ability of corneal epithelial wound healing in the mice model was suppressed by both pro-inflammatory factors and hyperosmotic stress treatment, and especially severely by hyperosmotic stress treatment. In addition, inflammation combined with hyperosmotic stress treatment induced more serious epithelial repair delays and apoptosis in corneal epithelium. Elevated levels of inflammatory factors were found in hyperosmotic stress-treated cells and mice corneas, which persisted even during the recovery period. The results suggested that pro-inflammatory factors cause transient inhibition, while hyperosmotic stress causes severe apoptosis and necrosis, persistent cell cycle arrest of CESCs, and severe corneal wound healing delay. Stem Cells Translational Medicine 2019;8:46–57

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology., Germinal zone neural stem cells (Gz‐NSC) are isolated from the hemorrhagic cerebrospinal fluid of preterm infants with severe intraventricular hemorrhage. These cells express ventral and posterior forebrain markers, can be differentiated and do not cause tumors. Gz‐NSC represent a valuable tool for the development of new cell therapies and the study of human Gz biology.

Human lungs bear their own reservoir of endogenous mesenchymal stem cells (MSCs). Although described as located perivascular, the cellular identity of primary lung MSCs remains elusive. Here we investigated the vascular nature of lung‐resident MSCs (LR‐MSCs) using healthy human lung tissue. LR‐MSCs predominately reside within the vascular stem cell niche, the so‐called vasculogenic zone of adult lung arteries. Primary LR‐MSCs isolated from normal human lung tissue showed typical MSC characteristics in vitro and were phenotypically and functionally indistinguishable from MSCs derived from the vascular wall of adult human blood vessels (VW‐MSCs). Moreover, LR‐MSCs expressed the VW‐MSC‐specific HOX code a characteristic to discriminate VW‐MSCs from phenotypical similar cells. Thus, LR‐MSC should be considered as VW‐MSCs. Immunofluorescent analyses of non‐small lung cancer (NSCLC) specimen further confirmed the vascular adventitia as stem cell niche for LR‐MSCs, and revealed their mobilization and activation in NSCLC progression. These findings have implications for understanding the role of MSC in normal lung physiology and pulmonary diseases, as well as for the rational design of additional therapeutic approaches., Although the precise stem cell niche(s) of lung‐resident mesenchymal stem cells (LR‐MSCs) remain(s) elusive, lung injury following a pathological trigger might at least be partially mediated by endogenous MSCs. Here we show that LR‐MSCs predominately reside within the vascular stem cell niche, the so‐called vasculogenic zone of adult lung arteries, and reveal their mobilization and activation in lung cancer progression.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs‐based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose‐derived MSCs (ASCs) via its regulation of transforming growth factor‐β (TGF‐β) activation. Silencing of GARP in human ASCs increased their activation of TGF‐β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF‐β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP−/lowASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide‐induced DNA damage and apoptosis, in a TGF‐β‐dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF‐β responses with diametrically opposing effects on ASC proliferation and survival., Silencing of glycoprotein A repetitions predominant (GARP) in adipose‐derived mesenchymal stromal cells (ASCs) increases the activation of transforming growth factor‐β (TGF‐β), resulting in mitochondrial reactive oxygen species‐dependent DNA damage, inhibition of proliferation, and apoptosis. In contrast, overexpression of GARP induces a TGF‐β response characterized by an increased resistance to DNA damage, increased proliferation, and a reduction in apoptosis. We show that GARP is critical in controlling TGF‐β activation/signaling in ASCs during in vitro expansion.

Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells that play fundamental roles in coordinating loading‐induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro‐osteogenic potential of osteocyte‐derived mechanically activated extracellular vesicles, which have great potential as a cell‐free therapy to enhance bone regeneration and repair in diseases such as osteoporosis., Bone formation/regeneration requires the recruitment and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. In his study, we demonstrate that osteocytes coordinate stem cell behavior in response to loading via modulation of their secretome that is delivered via extracellular vesicles (EVs). This therefore highlights mechanically activated EVs as potential therapies for bone regeneration.

The critical role of neuroinflammation in favoring and accelerating the pathogenic process in Alzheimer's disease (AD) increased the need to target the cerebral innate immune cells as a potential therapeutic strategy to slow down the disease progression. In this scenario, mesenchymal stem cells (MSCs) have risen considerable interest thanks to their immunomodulatory properties, which have been largely ascribed to the release of extracellular vesicles (EVs), namely exosomes and microvesicles. Indeed, the beneficial effects of MSC‐EVs in regulating the inflammatory response have been reported in different AD mouse models, upon chronic intravenous or intracerebroventricular administration. In this study, we use the triple‐transgenic 3xTg mice showing for the first time that the intranasal route of administration of EVs, derived from cytokine‐preconditioned MSCs, was able to induce immunomodulatory and neuroprotective effects in AD. MSC‐EVs reached the brain, where they dampened the activation of microglia cells and increased dendritic spine density. MSC‐EVs polarized in vitro murine primary microglia toward an anti‐inflammatory phenotype suggesting that the neuroprotective effects observed in transgenic mice could result from a positive modulation of the inflammatory status. The possibility to administer MSC‐EVs through a noninvasive route and the demonstration of their anti‐inflammatory efficacy might accelerate the chance of a translational exploitation of MSC‐EVs in AD., In a preclinical model of Alzheimer's disease, characterized by neuronal damage and a high rate of inflammation (left), the intranasal (IN) administration of extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) operates in dampening inflammation (by reducing microglia activation) and in inducing neuroprotective effects (by decreasing spine loss) (right). These data suggest the possibility that the IN route administration of MSC‐EVs might accelerate the chance of a translational exploitation of MSC‐EVs toward therapy.

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion.

Cell transplantation therapy utilizing neural precursor cells (NPCs) is a conceptually attractive strategy for traumatic spinal cord injury (SCI) to replace lost cells, remyelinate denuded host axons and promote tissue sparing. However, the number of mature oligodendrocytes that differentiate from typical NPCs remains limited. Herein, we describe a novel approach to bias the differentiation of directly reprogrammed human NPCs (drNPCs) toward a more oligodendrogenic fate (oNPCs) while preserving their tripotency. The oNPCs derived from different lines of human NPCs showed similar characteristics in vitro. To assess the in vivo efficacy of this approach, we used oNPCs derived from drNPCs and transplanted them into a SCI model in immunodeficient Rowett Nude (RNU) rats. The transplanted cells showed significant migration along the rostrocaudal axis and proportionally greater differentiation into oligodendrocytes. These cells promoted perilesional tissue sparing and axonal remyelination, which resulted in recovery of motor function. Moreover, after transplantation of the oNPCs into intact spinal cords of immunodeficient NOD/SCID mice, we detected no evidence of tumor formation even after 5 months of observation. Thus, biasing drNPC differentiation along an oligodendroglial lineage represents a promising approach to promote tissue sparing, axonal remyelination, and neural repair after traumatic SCI.

Chronic and nonhealing wounds are constant health issues facing patients with type 2 diabetes. As the incidence of type 2 diabetes mellitus (T2DM) increases, the incidence of chronic wounds and amputations will rise. T2DM is associated with peripheral arterial occlusive disease, which leads to the development of nonhealing skin ulcers after minor trauma. Patients develop severe pain limiting their mobility and ability to work and take care of themselves, thus putting a significant burden on the family and society. CD34+ cells from umbilical cord blood (UCB) grown in fibroblast growth factor-4 (FGF-4), stem cell factor, and Flt3-ligand produced a population of cells that have the ability to proliferate and develop properties enabling them to enhance tissue regeneration. The goal of this study was to assess in vitro cultured CD34+ cells in a setting where they would eventually be rejected so we could isolate paracrine signaling mediated therapeutic effect from the therapeutic effect due to engraftment and differentiation. To achieve this, we used db/db mice as a model for diabetic skin ulcers. Here, we report that in vitro cultured UCB CD34+ cells from frozen units can accelerate wound healing and resulted in the regeneration of full thickness skin. This study demonstrates a new indication for banked UCB units in the area of tissue regeneration.

Angiitis-induced critical limb ischemia (AICLI) patients constitute a remarkable proportion of no-option critical limb ischemia (CLI) patients. Stem cell therapy has become an innovative and promising option for no-option CLI patients. As one of these promising stem cell therapies, purified CD34+ cell transplantation (PuCeT) has shown favorable short-term results. However, the long-term efficacy of PuCeT has yet to be reported. This study evaluates the long-term efficacy of PuCeT in AICLI patients. Twenty-seven AICLI patients were enrolled from May 2009 to December 2011. Granulocyte colony-stimulating factor (G-CSF) and enoxaparin sodium were administered for 5 days. On day 5, CD34+ cell isolation was performed, and cells were transplanted by intramuscular injection. The primary endpoint, major-amputation-free survival rate (MAFS), as well as secondary endpoints, such as peak pain-free walking time (PPFWT) and the Wong-Baker FACES pain rating scale score (WFPRSS), were routinely evaluated during the 5-year follow-up period. The endpoints were as follows: the MAFS was 88.89%; PPFWT increased from 3 ± 3 to 17 ± 6 minutes; WFPRSS decreased from 7 ± 2 to 0.3 ± 1.7; the ulcer healing rate was 85.71%; the recurrence rate was 11.11%; and SF-36v2 scores were significantly improved at 5 years after PuCeT. The rate of labor recovery 5 years after PuCeT was 65.38%, and no severe adverse effect was observed during the treatment. PuCeT demonstrated long-term efficacy and durability as a treatment of AICLI not only in achieving limb salvage but also in recovering the labor competence and improving the quality of life of patients.

The capacity of stem and progenitor cells to stimulate cardiac regeneration has been studied for almost 20 years, with very promising preclinical data and mixed clinical results. Several cell types have been studied, identified by their cell surface markers, differentiation capacity and their secreted growth factors. Bone marrow derived mesenchymal stem cells (MSCs) have been found to have potent regenerative capacity, through multiple mechanisms, including mesoderm lineage differentiation, immunomodulation, and paracrine stimulation. MSCs also secrete exosomes and microvesicles, which themselves contain potent angiogenic cytokines or mRNA molecules with effects on their local milieu. This concise review summarizes the mechanisms of MSC-based cardiac regeneration and highlighting results from molecular and preclinical studies. We also discuss clinical trial results to date, and ongoing studies. Furthermore, we discuss novel approaches for the enhancement of MSC based cardiac regeneration, such as genetic modification.

Renal repair after injury is dependent on clonal expansion of proliferation-competent cells. In the human kidney, the expression of CD133 characterizes a population of resident scattered cells with resistance to damage and ability to proliferate. However, the biological function of the CD133 molecule is unknown. By RNA sequencing, we found that cells undergoing cisplatin damage lost the CD133 signature and acquired metanephric mesenchymal and regenerative genes such as SNAIL1, KLF4, SOX9, and WNT3. CD133 was reacquired in the recovery phase. In CD133-Kd cells, lack of CD133 limited cell proliferation after injury and was specifically correlated with deregulation of Wnt signaling and E-cadherin pathway. By immunoprecipitation, CD133 appeared to form a complex with E-cadherin and β-catenin. In parallel, CD133-Kd cells showed lower β-catenin levels in basal condition and after Wnt pathway activation and reduced TCF/LEF promoter activation in respect to CD133+ cells. Finally, the lack of CD133 impaired generation of nephrospheres while favoring senescence. These data indicate that CD133 may act as a permissive factor for β-catenin signaling, preventing its degradation in the cytoplasm. Therefore, CD133 itself appears to play a functional role in renal tubular repair through maintenance of proliferative response and control of senescence.

Stem cell therapy is a promising strategy for tissue regeneration. The therapeutic benefits of cell therapy are mediated by both direct and indirect mechanisms. However, the application of stem cell therapy in the clinic is hampered by several limitations. This concise review provides a brief introduction into stem cell therapies for ischemic heart disease. It summarizes cell-based and cell-free paradigms, their limitations, and the benefits of using them to target disease.