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We previously demonstrated that overexpression of tropomyosin receptor kinase A (TrkA) promotes the survival and Schwann cell-like differentiation of bone marrow stromal stem cells in nerve grafts, thereby enhancing the regeneration and functional recovery of the peripheral nerve. In the present study, we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts. Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA, TrkA-shRNA or the respective control. The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect. Then, 8 weeks after surgery, hematoxylin and eosin staining showed that compared with the control groups, the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged, whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group. Western blot assay showed that compared with the control groups, the TrkA overexpressing group had higher expression of the myelin marker, myelin basic protein and the axonal marker neurofilament 200. The TrkA overexpressing group also had higher levels of various signaling molecules, including TrkA, pTrkA (Tyr490), extracellular signal-regulated kinases 1/2 (Erk1/2), pErk1/2 (Thr202/Tyr204), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. In contrast, these proteins were downregulated, while the pro-apoptotic factors Bax and Bad were upregulated, in the TrkA-shRNA group. The levels of the TrkA effectors Akt and pAkt (Ser473) were not different among the groups. These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway. All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University, China in December 2014 (approval No. AEWC-2014-001219).

Adipose tissue‐derived multipotent stromal cells (AT‐MSCs) are studied as an alternative to bone marrow‐derived multipotent stromal cells (BM‐MSCs) for immunomodulatory treatment. In this study, we systematically compared the immunomodulatory capacities of BM‐MSCs and AT‐MSCs derived from age‐matched donors. We found that BM‐MSCs and AT‐MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM‐MSCs and AT‐MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte‐derived immature dendritic cells. However, at equal cell numbers, the AT‐MSCs showed more potent immunomodulatory effects in both assays as compared with BM‐MSCs. Moreover, AT‐MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin‐6 and transforming growth factor‐β1. This is correlated with higher metabolic activity of AT‐MSCs compared with BM‐MSCs. We conclude that the immunomodulatory capacities of BM‐MSCs and AT‐MSCs are similar, but that differences in cytokine secretion cause AT‐MSCs to have more potent immunomodulatory effects than BM‐MSCs. Therefore, lower numbers of AT‐MSCs evoke the same level of immunomodulation. These data indicate that AT‐MSCs can be considered as a good alternative to BM‐MSCs for immunomodulatory therapy. This study systematically compared the immunomodulatory capacities of adipose tissue‐derived multipotent stromal cells (AT‐MSCs) and bone marrow‐derived multipotent stromal cells (BM‐MSCs) derived from age‐matched donors. It was found that BM‐MSCs and AT‐MSCs show functionally similar immunomodulatory effects, but with a different dose‐response curve, in favor of AT‐MSCs. AT‐MSCs can be considered as a good alternative to BM‐MSCs for immunomodulatory therapy.

Regenerative therapies in the musculoskeletal system are based on the suitable application of cells, biomaterials, and/or factors. For an effective approach, numerous aspects have to be taken into consideration, including age, disease, target tissue, and several environmental factors. Significant research efforts have been undertaken in the last decade to develop specific cell‐based therapies, and in particular adult multipotent mesenchymal stem cells hold great promise for such regenerative strategies. Clinical translation of such therapies, however, remains a work in progress. In the clinical arena, autologous cells have been harvested, processed, and readministered according to protocols distinct for the target application. As outlined in this review, such applications range from simple single‐step approaches, such as direct injection of unprocessed or concentrated blood or bone marrow aspirates, to fabrication of engineered constructs by seeding of natural or synthetic scaffolds with cells, which were released from autologous tissues and propagated under good manufacturing practice conditions (for example, autologous chondrocyte implantation). However, only relatively few of these cell‐based approaches have entered the clinic, and none of these treatments has become a “standard of care” treatment for an orthopaedic disease to date. The multifaceted reasons for the current status from the medical, research, and regulatory perspectives are discussed here. In summary, this review presents the scientific background, current state, and implications of clinical mesenchymal stem cell application in the musculoskeletal system and provides perspectives for future developments.

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft‐versus‐host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum‐free media that contain serum albumin, insulin, and transferrin. The ill‐defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno‐free culture media to meet the regulatory and long‐term safety requirements for cell‐based therapies. We developed and tested a serum‐free and xeno‐free culture medium (SFM‐XF) using human bone marrow‐ and adipose‐derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM‐XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum‐containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow‐derived MSCs expanded in SFM‐XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM‐XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM‐XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum‐containing media for the production of large scale, functionally competent MSCs for future clinical applications. This study developed and tested a serum‐free and xeno‐free culture medium (SFM‐XF) using human bone marrow‐ and adipose‐derived mesenchymal stem cells (MSCs). Human MSCs expanded in SFM‐XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of traditional serum‐containing culture medium (SCM). The SFM‐XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to SCM for the production of large scale, functionally competent MSCs for future clinical applications.

Background Transplantation of human bone marrow stromal cells (hBMSCs) is a promising therapy for bone regeneration due to their ability to differentiate into bone forming osteoblastic cells. However, transplanted hBMSCs exhibit variable capacity for bone formation resulting in inconsistent clinical outcome. The aim of the study was to identify a set of donor- and cell-related characteristics that detect hBMSCs with optimal osteoblastic differentiation capacity. Methods We collected hBMSCs from 58 patients undergoing surgery for bone fracture. Clinical profile of the donors and in vitro characteristics of cultured hBMSCs were included in uni- and multivariable analysis to determine their predictive value for osteoblastic versus adipocytic differentiation capacity assessed by quantification of mineralized matrix and mature adipocyte formation, respectively. Results We identified a signature that explained > 50% of variation in osteoblastic differentiation outcome which included the following positive predictors: donor sex (male), absence of osteoporosis diagnosis, intake of vitamin D supplements, higher fraction of CD146+, and alkaline phosphate (ALP+) cells. With the exception of vitamin D and ALP+ cells, these variables were also negative predictors of adipocytic differentiation. Conclusions Using a combination of clinical and cellular criteria, it is possible to predict differentiation outcome of hBMSCs. This signature may be helpful in selecting donor cells in clinical trials of bone regeneration.

Summary The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. We found that microRNA-34a-5p was a critical player in dexamethasone (Dex)-inhibited BMSC proliferation and osteogenic differentiation. MicroRNA-34a-5p might be used as a therapeutic target for GC-impaired bone formation. Introduction The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. The mechanisms of this process are still not completely understood. Recent studies implicated an important role of microRNAs in GC-mediated responses in various cellular processes, including cell proliferation and differentiation. Therefore, we hypothesized that these regulatory molecules might be implicated in the process of GC-decreased BMSC proliferation and osteoblastic differentiation. Methods Western blot, quantitative real-time PCR, and cell proliferation and osteoblastic differentiation assays were employed to investigate the role of microRNAs in GC-inhibited BMSC proliferation and osteoblastic differentiation. Results We found that microRNA-34a-5p was reciprocally regulated by Dex during the process of BMSC proliferation and osteoblastic differentiation. Furthermore, we confirmed that microRNA-34a-5p was a critical player in Dex-inhibited BMSC proliferation and osteogenic differentiation. Mechanistic studies showed that Dex inhibited BMSC proliferation by microRNA-34a-5p targeting cell cycle factors, including CDK4, CDK6, and Cyclin D1. Furthermore, downregulation of microRNA-34a-5p by Dex leads to Notch signaling activation, resulting in inhibition of BMSC osteogenic differentiation. Conclusions These results showed that microRNA-34a-5p, a crucial regulator for BMSC proliferation and osteogenic differentiation, might be used as a therapeutic target for GC-impaired bone formation.

Bone-related maladies are a major health burden on modern society. Loss of skeletal integrity and regeneration capacity through aging, obesity, and disease follows from a detrimental shift in bone formation and resorption dynamics. Targeting tissue-resident adult stem cells offers a potentially innovative paradigm in the development of therapeutic strategies against organ dysfunction. While the essential role of skeletal stem cells (SSCs) for development, growth, and maintenance of the skeleton has been generally established, a common consensus on the exact identity and definition of a pure SSC population remains elusive. The controversies stem from conflicting results between different approaches and criteria for isolation, detection, and functional evaluation; along with the interchangeable usage of the terms SSC and \"mesenchymal stromal/stem cell (MSC)\". A great number of prospective bone-forming stem cell populations have been reported with various characteristic markers, often describing overlapping cell populations with widely unexplored heterogeneity, species specificity, and distribution at distinct skeletal sites, bone regions, and microenvironments, thereby creating confusion that may complicate future advances in the field. In this review, we examine the state-of-the-art knowledge of SSC biology and try to establish a common ground for the definition and terminology of specific bone-resident stem cells. We also discuss recent advances in the identification of highly purified SSCs, which will allow detailed interrogation of SSC diversity and regulation at the single-cell level.

A differentiation switch of bone marrow mesenchymal stem/stromal cells (BMSCs) from osteoblasts to adipocytes contributes to age‐ and menopause‐associated bone loss and marrow adiposity. Here it is found that osteocytes, the most abundant bone cells, promote adipogenesis and inhibit osteogenesis of BMSCs by secreting neuropeptide Y (NPY), whose expression increases with aging and osteoporosis. Deletion of NPY in osteocytes generates a high bone mass phenotype, and attenuates aging‐ and ovariectomy (OVX)‐induced bone‐fat imbalance in mice. Osteocyte NPY production is under the control of autonomic nervous system (ANS) and osteocyte NPY deletion blocks the ANS‐induced regulation of BMSC fate and bone‐fat balance. γ‐Oryzanol, a clinically used ANS regulator, significantly increases bone formation and reverses aging‐ and OVX‐induced osteocyte NPY overproduction and marrow adiposity in control mice, but not in mice lacking osteocyte NPY. The study suggests a new mode of neuronal control of bone metabolism through the ANS‐induced regulation of osteocyte NPY. Normally, norepinephrine (NE) and acetylcholine (ACh) production is maintained at a balanced level in bone, so that osteocytes cannot generate excessive neuropeptide Y (NPY) to favor bone marrow mesenchymal stem/stromal cell adipogenesis rather than osteogenesis. With aging/estrogen deficiency, sympathetic overactivity, and decreased parasympathetic activity cause NE overproduction and ACh reduction, resulting in excess osteocyte NPY generation and subsequent bone‐fat imbalance.

Stromal cell-derived factor-1 (SDF-1) is a potent chemokine for bone marrow-derived stromal stem cells (BMSCs) that express CXCR4, the receptor for SDF-1. SDF-1 is considered to play an important role in the trafficking of BMSCs. We investigated the contribution of SDF-1 to the recruitment of BMSCs to the wound area and its promotion of wound repair and neovascularization. BMSCs were pretreated with or without anti-CXCR4 blocking antibody and combined with CM-DiI label, and injected via the tail vein into mice with full-thickness skin wounds on the dorsum. Simultaneously, anti-SDF-1 antibody was injected into local wounds in another group of mice. The results show that blockade of CXCR4 on either infused BMSCs or SDF-1 in the host wounds (1) dramatically impaired the number of infused BMSCs being recruited to the injured tissue, (2) reduced the expression of growth factors involved in the repair of injured tissue such as vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor beta 1, (3) decreased the resultant neovascularization, and (4) retarded wound healing. Taken together, the findings indicate that the SDF-1/CXCR4 signal pathway facilitates wound healing through augmenting BMSC recruitment to wound tissues, responsive secretion of growth factors by BMSCs and neovascularization in the wound area.

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21 cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.