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Background: Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical. Methods: We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro. Results: NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids. Conclusions: We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration. [ABSTRACT FROM AUTHOR]

For reconstructive surgeons, critically skeletal damage represents a major challenge. Growing evidence indicate that bone repair is dynamically regulated by the mesenchymal stem cell (MSC)‐macrophage interaction. Mechanical strain plays a fundamental role in bone repair and regeneration by influencing MSCs differentiation. Recently, a few findings indicate that macrophages may be mechanically sensitive and their phenotype can be regulated, in part, by mechanical cues. However, how macrophages subjected mechanical stretch influence the osteogenic differentiation of MSCs remain unclear. Thus, the purpose of this study is to explore the effect of macrophages stimulated with mechanical stretch on MSCs osteogenesis. By using a coculture system, we discover that macrophages efficiently induce osteogenic differentiation of MSCs under specific stretch conditions. A synergy mechanism between M2 polarization and YAP/BMP2 axis are identified through molecular and genetic analyses. Macrophages are activated by cyclic stretch and polarized to M2 phenotype that produce anti‐inflammatory cytokines such as IL‐10 and TGF‐β to regulate the local inflammatory microenvironment. Furthermore, mechanical stretch induces YAP activation and nuclear translocation, subsequently regulates downstream BMP2 expression to facilitate MSCs osteogenesis. These findings not only advance our understanding of the complex influence among the mechanical strain, macrophage inflammatory response as well as the osteogenic differentiation of MSCs, but also reveal a control system from mechanical signals to chemical response then to cell behaviors during bone repair and regeneration. [ABSTRACT FROM AUTHOR]

Osteoporosis is one of the most prevalent age-related diseases worldwide, of which vertebral fracture is by far the most common osteoporotic fracture. Reduced bone formation caused by senescence is a main cause for senile osteoporosis, however, how to improve the osteogenic differentiation of osteoporotic bone marrow mesenchymal stem cells (BMSCs) remains a challenge. This study aimed to investigate the autophagic level changes in osteoporotic BMSCs derived from human vertebral body, and then influence osteogenesis through the regulation of autophagy. We found that hBMSCs from osteoporotic patients displayed the senescence-associated phenotypes and significantly reduced autophagic level compared to those derived from healthy ones. Meanwhile, the osteogenic potential remarkably decreased in osteoporotic hBMSCs, suggesting an inherent relationship between autophagy and osteogenic differentiation. Furthermore, rapymycin (RAP) significantly improved osteogenic differentiation through autophagy activatoin. However, the osteogenesis of hBMSCs was reversed by the autophagy inhibitor 3-methyladenine (3-MA). To provide more solid evidence, the hBMSCs pretreated with osteogenesis induction medium in the presence of 3-MA or RAP were implanted into nude mice. In vivo analysis showed that RAP treatment induced larger ectopic bone mass and more osteoid tissues, however, this restored ability of osteogenic potential was significantly inhibited by 3-MA pretreatment. In conclusion, our study indicated the pivotal role of autophagy for the osteo-differentiation hBMSCs, and offered novel therapeutic target for osteoporosis treatment. [ABSTRACT FROM AUTHOR]