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Adult-derived mesenchymal stem cells (MSCs) can be used in therapies for the treatment of various diseases. MSCs derived from aging tissues or long-term MSC cultures could have diminished therapeutic effects compared with MSCs derived from younger tissues, but the underlying mechanism has not been completely established. Dysfunction of energy metabolism is one of the main mechanisms underlying cell senescence. Although cyclic adenosine monophosphate (cAMP) is known to inhibit cell division and proliferation in vitro, its impact on MSC senescence has not been described. In this study, we used forskolin, an adenylate cyclase agonist and cAMP inducer, to disrupt metabolism in human adipose-derived MSCs and investigate the effects of metabolic dysfunction on MSC senescence. Treatment of human MSCs with forskolin resulted in senescence phenotypes, including reduced proliferation, cell-cycle arrest, and enhanced expression of the cell aging markers p16 and p21. Furthermore, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several inflammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress-induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related-inflammatory-phenotype (SASP). Our results indicate that forskolin can cause senescence of human MSCs through oxidative stress-induced mitochondrial metabolic dysfunction, and thus the results provide a basis for developing strategies for improving the quality and efficacy of cultured MSCs for clinical use.

Diabetic foot ulcer (DFU) is a multifactorial complication of diabetes, mainly manifested as infection, ulcer or destruction of deep tissue and there is currently no effective treatment. Several pre-clinical and clinical studies have proved that the transplantation of mesenchymal stem cells (MSCs) improved wound healing. Here, we evaluated the therapeutic efficacy of human umbilical cord (hUC-MSCs) in DFU rat model. One dose of hUC-MSCs (1×106 cells) were subcutaneously injected around wounds in male SD rats. Wound healing was evaluated macroscopically (wound closure) every three days. In addition, we measured growth factors and specific proteins (MMP-9 and MMP-8) on Day 14 post hUC-MSCs transplantation. Results showed significant differences in the wound healing kinetics of lesions that received hUC-MSCs compared to lesions that received vehicle (PBS) (P0.05). ELISA analyses indicated that MMP-9 protein contents were significantly upregulated in DFU animals, while MMP-8 were downregulated compared to the diabetic rats (P0.05). After MSCs treatment, the level of MMP-9 and MMP-8 decreased and increased compared to the vehicle group, respectively. These findings suggest hUC-MSCs transplantation can ameliorate the healing process of DFU rats and a potential mechanism through which MSCs enhance DFU wound healing by decreasing MMP-9 expression and increasing MMP-8 expression. This study represents a promising opportunity to gain insight into how MSCs mediate wound healing.

As an important component of the dynamic tumor microenvironment, mesenchymal stem cells (MSCs) can interact with tumor cells to promote tumor growth. Treatment with tumor cell-derived exosomes can change the biological functions of MSCs. We want to study the mechanism by which exosomes derived from gastric cancer cells affect the biological functions of MSCs. After MSCs were treated with adenocarcinoma gastric cells (AGS) cell-derived exosomes, circular RNAs differentially expressed in MSCs were verified using existing RNA microarray results combined with quantitative real-time polymerase chain reaction (qRT-PCR). Then, circular RNAs were knocked down or overexpressed by plasmids, and the functions of circular RNAs were evaluated by Migration and invasion assay. Dual luciferase reporter assay was used to evaluate the potential mechanism of circular RNAs. After treatment with exosomes secreted by AGS, the results showed that some circular RNAs expressed by human adipose-derived MSCs showed significant differences. The elevated circ_0004303 promoted the migration and invasion of human adipose-derived MSCs in vitro. Circ_0004303 upregulated the expression of activated leukocyte cell adhesion molecule (ALCAM) by acting as a miR-148a-3p sponge, thereby enhancing the migration and invasion functions of human adipose-derived MSCs. Therefore, exosomes secreted by AGS can affect the expression of circular RNAs in human adipose-derived MSCs. Hsa_circ_0004303 can regulate the migration and invasion of human adipose-derived MSCs via the miR-148a-3P/ALCAM axis. This study suggests that tumor cells can promote the migration and homing of MSCs in adjacent tissues by secreting exosomes.

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and are essential for tumorigenesis and development. Regorafenib is a multikinase inhibitor that targets CAFs and suppresses tumor growth. In this study, we investigated the effects of regorafenib on gastrointestinal CAFs and the underlying molecular mechanisms. First, we established two in vivo tumor models, the cancer cell line HCT116 with and without mesenchymal stem cells (MSCs), and treated them with regorafenib. We found that application of regorafenib potently impaired tumor growth, an effect that was more pronounced in tumors with a high stromal ratio, thus demonstrating that regorafenib can inhibit CAF proliferation and induce CAF apoptosis in vivo. Moreover, we showed that regorafenib affected macrophage infiltration by reducing the proportion of CAFs in tumors. Second, we induced MSCs into CAFs with exosomes to establish an in vitro model. Then, we used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt and flow cytometry to detect the effects of regorafenib on proliferation and apoptosis of CAFs and western blot to determine the expression level of apoptosis-related proteins. We found that regorafenib inhibited proliferation of CAFs and induced apoptosis in CAFs in vitro. Furthermore, western blot results showed that regorafenib downregulated the expression of B cell lymphoma-2 (Bcl-2) and concurrently upregulated the expression of Bcl-2-associated X (Bax), and regorafenib inhibited the phosphorylation pathway of AKT in CAFs. In conclusion, our results provide a model in which regorafenib induces CAF apoptosis by inhibiting the phosphorylation of AKT and regorafenib affects macrophage infiltration by reducing the proportion of CAFs in tumor tissues.

It has been shown that the KCa3.1 channel-specific blocker, TRAM34, is a promising antiatherosclerosis (AS) agent, but its side effects restrict its clinical application. Notably, its effect on endothelial progenitor cells (EPCs) is unclear. We aim to unravel the effect of TRAM34 on EPCs and identify the underlying mechanism. Rats were injected intraperitoneally with TRAM34, and EPCs were isolated from bone marrow. The gene and protein levels of corresponding factors were detected by real-time PCR, enzyme-linked immunosorbent assay, western blotting, and fluorescence-activated cell sorting. Liquid chromatography-tandem mass spectrometry (LC-MS) was applied to detect metabolite differences. We showed that when rats were treated with TRAM34 in vivo, colony formation and proliferation of early EPCs were reduced, but their senescence and apoptosis were enhanced. Moreover, TRAM34 enhanced NOX activity, promoted an increase in intracellular ROS levels, increased PKC expression, and subsequently promoted EPC senescence, which is unfavorable for EPC angiogenesis in vivo and in vitro. Combining these results with LC-MS data, we found that TRAM34 significantly promoted pyrimidine and purine metabolism, leading to cellular senescence. Furthermore, the NOX inhibitor, Setanaxib, enhanced antioxidant metabolic pathways, especially S-adenosylmethioninamine (SAM) metabolism, to exert an antisenescence effect. Finally, we confirmed that SAM alleviates TRAM34-induced cellular senescence, suggesting an efficient approach to improve the quality of endogenous EPCs. This study reveals the mechanism of TRAM34-induced EPC senescence, providing a solution for the extended application of KCa3.1 inhibitor in AS.

Photoaging is a degenerative biological process. As a kind of pluripotent stem cells, adipose-derived stem cells (ADSCs) are widely used in the treatment of photoaging. Therefore, we aimed to find an effective way to improve the antiaging ability of ADSCs. In this study, we isolated ADSCs and assessed multilineage differentiation ability and markers. Cultured ADSCs were preconditioned with astragaloside IV (ASI) at 10-7, 10-6, and 10-5 M. Cell proliferation was assessed by CCK-8 assay and cytokine secretion by enzyme-linked immunosorbent assay (ELISA). A fibroblast photoaging model was established and cocultured with normal ADSCs or ASI-treated ADSCs. Matrix metalloproteinase-1 (MMP1) and type I procollagen (PC-I) secreted by human dermal fibroblasts were measured by ELISA. The effects of ASI-treated ADSCs on skin texture, including dermal thickness, collagen content, and microvessel density, in a photoaging animal model were analyzed using H&E staining, Masson staining, and CD31 immunohistochemistry, respectively. We found that 10-6 M ASI could significantly promote cell proliferation and stimulate robust secretion of growth factors in ADSCs. Furthermore, our data showed that ASI-treated ADSCs could markedly reverse the ultraviolet B-induced decrease of PC-I secretion and increase of MMP-1 release in fibroblasts. Moreover, in photoaged skin of nude mice, ASI-treated ADSCs significantly increased dermal thickness, collagen content, and microvessel density.

The novel coronavirus disease 2019 (COVID-19) has grown to be a global public-health emergency since patients were first detected in Wuhan, China, in December 2019. As of April 9, 2020, the novel coronavirus (named as SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11) has infected 83,251 and 1,484,811 patients in China and the world, respectively. However, we have neither confirmed effective antiviral medications nor vaccines available to deal with this emergency. In this commentary, we offer an alternative promising therapy for COVID-19, that is, mesenchymal stem cell transplantation.

Mesenchymal stem cells (MSCs) have a superior immunomodulatory capacity compared to other cells of the immune system, and they hold great promise for treating various immune disorders. However, their regulatory effects on the maturation of immature dendritic cells (imDCs) are not fully understood. In this study, we show that Sca-1+Lin-CD117-MSCs restrain the lipopolysaccharide-stimulated maturation transition of imDCs cocultured without exogenous cytokines. The Notch signaling pathway plays a critical role in the process by controlling interferon regulatory factor 8 (IRF8) expression in an RBP-J-dependent manner. We observed a high degree of H3K27me3 modification mediated by SUZ12 and a relatively low degree of H3K4me3 modification regulated by WDR5 at the IRF8 promoter during coculture. These data reveal a possible mechanism by which Sca-1+Lin-CD117-MSCs modulate imDC maturation and further support the role of MSCs in treating immune disorders.

Angiogenesis is a complicated and sequential process that plays an important role in different physiological processes. Mesenchymal stem cells (MSCs), which are pluripotent stem cells, are widely used for the treatment of ischemic and traumatic diseases, and exosomes derived from these cells can also promote angiogenesis. Therefore, we aimed to uncover mechanisms to improve MSC exosome-mediated angiogenesis. For this study, we isolated human adipose-derived MSCs (hAD-MSCs) and assessed differentiation ability and markers. Cells were divided into hypoxia-treated MSCs (H-MSCs) and normoxia-treated MSCs (N-MSC), and exosomes were extracted by ultrafiltration. Exosomes (100 μg/mL) from H-MSCs and N-MSCs were added to human umbilical vein endothelial cells (HUVECs). Exosome uptake and the ability of endothelial cells to form tubes were detected in real time. Protein samples were collected at different time points to detect the expression of inhibitors (Vash1) and enhancers (Angpt1 and Flk1) of angiogenesis; we also assessed their related signaling pathways. We found that exosomes from the hypoxia group were more easily taken up by HUVECs; furthermore, their angiogenesis stimulatory activity was also significantly enhanced compared to that with exosomes from the normoxia group. HUVECs exposed to exosomes from H-MSCs significantly upregulated angiogenesis-stimulating genes and deregulated angiogenesis-inhibitory genes. The expression of vascular endothelial growth factor (VEGF) and activation of the protein kinase A (PKA) signaling pathway in HUVECs were significantly increased by hypoxia-exposed exosomes. Moreover, a PKA inhibitor was shown to significantly suppress angiogenesis. Finally, we concluded that hypoxia-exposed exosomes derived from hAD-MSCs can improve angiogenesis by activating the PKA signaling pathway and promoting the expression of VEGF. These results could be used to uncover safe and effective treatments for traumatic diseases.

Mesenchymal stem cells (MSCs) have been proved to be an important element in cell-based therapy. Photobiomodulation used extremely low-level lasers (LLLs) to affect the behavior of cells. The effect mechanism of LLLs on MSCs from human remained to be discovered. In this study, cell viability was assessed using MTS assays and cell cycle was evaluated by fluorescence-activated cell sorting (FACS). The influence of LLLs on mitochondrial biogenesis (fission or fusion) and function (ATP, reactive oxygen species [ROS], nitric oxide [NO]) was evaluated by transmission electron microscope, FACS, quantitative real time polymerase chain reaction (q-PCR), and immunocytochemistry. Cell migration and cytoskeleton alteration (actin and tubulin) were evaluated using transwell assay, immunocytochemistry, enzyme-linked immunosorbent assay, and western blotting. Cell apoptosis was evaluated using FACS, immunocytochemistry, and western blotting. We investigated that certain influence of LLLs on MSCs in vitro 6 or 24 h after 1 h of LLL irradiation. The mechanism of the effects included proliferation rate increase mediated by increased S phase proportion; mitochondrial biogenesis and function alteration mediated by fusion (Mfn1, Mfn2, and Opa-1) and fission (Fis1, Drp-1, and MTP18)-related proteins, NRF1, TFAM, PGC-1a, and upregulated intracellular ROS and NO concentration; migration acceleration through the ERK1/2 and FAK pathway and upregulation of growth factors such as HGF and PDGF; and resistance to apoptosis with increased Bcl-2 and decreased Bax, or through tunneling nanotube formation between LLL-treated MSCs and 5-fluorouracil-induced apoptotic MSCs. These observations suggested that LLLs enhanced stem cell survival and therapeutic function, which could appear to be an innovative pretreatment in the application of MSCs.

Recently, it has been suggested that cellular senescence is associated with stem cell exhaustion, which reduces the regenerative potential of tissues and contributes to aging and age-related diseases. Mesenchymal stem cells (MSCs) attract a large amount of attention in stem cell research and regeneration medicine because they possess multiple advantages and senescent MSCs could be one of the most useful stem cell models in aging studies. It is important to quantitatively evaluate senescence markers to both identify and study the mechanisms involved in MSC senescence. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the most widely used tool to quantify the mRNA levels of markers. However, no report has demonstrated the optimal reference genes that should be used to normalize RT-qPCR in senescence studies of MSCs. In this study, we compared 16 commonly used reference genes (GAPDH, ACTB, RPL13A, TBP, B2M, GUSB, RPLPO, YWHAZ, RPS18, EEF1A1, ATP5F1, HPRT1, PGK1, TFRC, UBC, and PPIA) in proliferating or replicative-senescent human adipose-derived MSCs (hAD-MSCs) that were isolated from seven healthy donors aged 29-59 years old. Three algorithms (geNorm, NormFinder, and BestKeeper) were used to determine the most optimal reference gene. The results showed that PPIA exhibited the most stable expression during senescence, while the widely used ACTB exhibited the lowest stability. We also confirmed that different reference genes lead to different evaluations of senescence markers. Our work ensures that results obtained from senescence studies of hAD-MSCs will be appropriately evaluated in both basic research and clinical trials.

Podocyte depletion is a key event in the progression of end-stage kidney disease (ESKD) resulting in nephrotic proteinuria and renal failure, but the treatment options are limited to dialysis and renal transplantation. So there is an urgent need for renal regenerative therapies. Generation of podocytes from human stem cells is regarded as a promising therapeutic strategy to repair or regenerate the damaged kidneys; however, the reliable induction system remains a challenge. In this study, we established a two-stage induction protocol for podocyte generation from human adipose-derived mesenchymal stem cells (hAD-MSCs). We initially established a condition that induces hAD-MSCs toward intermediate mesoderm cells with activin A and high concentration of retinoic acid (RA). Subsequently, by using the combination of activin A and low concentration of RA and BMP7, we generated podocyte-like cells expressing multiple podocyte-specific markers and able to integrate into a developing nephron of embryonic kidney explant culture and ameliorate proteinuria and kidney injure in adriamycin-treated mice. Furthermore, we identified that miRNA-498 inhibitor has potential to improve the differentiation of hAD-MSCs into podocyte-like cells and established a robust induction protocol. Thereby, our study advocated an efficient method for the induction of kidney podocyte-like (iPod) cells from hAD-MSCs and provided an ideal candidate for regenerative therapies of the kidney.

Osteoporosis is characterized by deterioration of bone microarchitecture and low bone mass. One of the primary causes of osteoporosis is the decrease in the osteogenic differentiation of mesenchymal stem cells (MSCs). Tissue engineering therapy with genetically modified MSCs has attracted much attention in the study of bone regeneration. In this study, we found that the expression level of miR-450b was upregulated during osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs). To explore the effect of miR-450b on the osteogenesis of hADSCs, we performed a series of gain- and loss-of-function analyses and demonstrated that miR-450b not only promoted the process of hADSC differentiation to osteoblasts in vitro but also enhanced ectopic bone formation in vivo. Bone morphogenetic protein 3 (BMP3), the most abundant BMP member in bone, was identified as a direct target of miR-450b. Downregulation of the endogenous expression of BMP3 could mimic the effect of miR-450b upregulation on the osteogenic differentiation of hADSCs. Overall, our study first demonstrated that a novel microRNA miR-450b was essential for hADSC differentiation, which could promote osteogenic differentiation in vitro and enhance bone formation in vivo by directly suppressing BMP3.

Mesenchymal stem cells (MSCs) have a superior immunomodulatory capacity compared to other cells of the immune system, and they hold great promise for treating various immune disorders. However, their regulatory effects on the maturation of immature dendritic cells (imDCs) are not fully understood. In this study, we show that Sca-1

Bone and fat cells share a common progenitor, stromal/mesenchymal stem cells (MSCs), that can differentiate into osteoblasts or adipocytes. Osteogenesis and adipogenesis of MSCs maintain homeostasis under physiological conditions. The disruption of this homeostasis leads to bone-related metabolic diseases. For instance, reduction in bone formation, which is usually accompanied by an increase in bone marrow adipogenesis, occurs with aging, immobility, or osteoporosis. Thus, it is crucial to gain an understanding of how osteogenic and adipogenic lineages of MSCs are regulated. Here, we present evidence that let-7 is a positive regulator of bone development. Using gain- and loss-of-function approaches, we demonstrate that let-7 markedly promotes osteogenesis and suppresses adipogenesis of MSCs in vitro. Moreover, let-7 could promote ectopic bone formation of MSCs in vivo. Subsequent studies further demonstrated that let-7's effects are mediated through the repression of high-mobility group AT-hook 2 (HMGA2) expression. RNAi depletion of HMGA2 could also enhance osteogenesis and repress adipogenesis. Overall, we found a novel role of let-7/HMGA2 axis in regulating the balance of osteogenesis and adipogenesis of MSCs. Thus, let-7 can be used as a novel therapeutic target for disorders that are associated with bone loss and adipocyte accumulation.

The therapeutic effect of mesenchymal stem cells (MSCs) in tissue repair/regeneration is substantially dampened by the loss of primitive properties and poor engraftment to target organs. In this study, the multipotency and cell sizes of human MSCs, which had been expanded in monolayer culture for several passages, were dramatically restored after an episode of three-dimensional (3D) spheroid culture. Unlike MSCs derived from monolayer, which caused embolism and blindness, MSCs derived from 3D spheroids did not cause vascular obstructions, after intra-carotid artery infusion in rats. Importantly, intra-carotid infusion of 1 million 3D spheroid MSCs in rats 24 h after middle cerebral artery occlusion and reperfusion resulted in engraftment of the cells into the lesion and significant (over 70%) reduction of infarct size along with restoration of neurologic function. Moreover, the enhanced effect of spheroid MSCs was coincided with significantly increased differentiation of the MSCs into neurons and markedly increased number of endogenous glial fibrillary acidic protein–positive neural progenitors in the peri-infarct boundary zone. However, the similarly administered monolayer MSCs resulted in a modest functional improvement. Our results suggest that 3D MSCs, in combination with intra-carotid delivery, may represent a novel therapeutic approach of MSCs for stroke.

Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5×10(6) human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.

Pancreatic progenitor (PP) cells are tissue-committed cells, which can differentiate into all kinds of pancreatic cells. They are potential candidates for regeneration of pancreatic tissue. However, it is unfeasible to acquire PP cells from pancreatic tissues and expand them in vitro. Generation of PP cells from adipose tissue-derived mesenchymal stem cells (AD-MSCs) would provide an unlimited source of PP cells. Here we developed a 2-step stepwise protocol, which induced AD-MSCs to generate FOXA2- or SOX17-positive definitive endoderm (DE) (5 days) and pancreatic and duodenal homeobox gene 1 (PDX1)-positive PP cells (4-6 days). By mimicking the developmental progress in embryonic development, we optimized the timing and combination of cytokines to activate the key signaling pathways during pancreatic development. We found that activating the Nodal/Activin signal with Activin A could induce differentiation of AD-MSCs toward DE, which could be further promoted by the Wnt signaling pathway activator Wnt3a. Besides, transient T (BRACHYURY)(+) mesendodermal cells were observed during formation of DE from AD-MSCs. Subsequently, the Wnt signaling pathway inhibitor Dkk1 along with retinoic acid/FGF2 (60 ng/mL) further induced AD-MSC-derived DE cells to differentiate into PDX1-positive PP cells. The derived PP cells were capable to form pancreatic endocrine or exocrine cells. In conclusion, we established a stepwise protocol that could derive DE and PP cells from AD-MSCs. It might provide an unlimited source of autologous PP cells for pancreatic diseases.

Human bone marrow-derived mesenchymal stem cells (MSCs) are currently widely used in cell therapy and tissue engineering. In vitro experiments have demonstrated that apoptosis of MSCs can be induced by hypoxia, serum deprivation, and chemotherapeutic agents, and the process is p53 independent. In this study, we investigated the role of p63 (a member of p53 family) in the regulation of apoptosis of MSCs. TAp63α, a subtype of p63, is highly similar to p53 and plays a crucial role in apoptosis. In vitro exposure of MSCs to either cisplatin or etoposide resulted in an increased TAp63α expression, which was time and dose dependent. Interference of TAp63α led to drug resistance and decreased apoptosis, accompanied by reduced expression of Bax, poly(ADP-ribose) polymerase, and caspase-3. However, downregulation of TAp63α did not influence the phenotype, proliferation capacity, and differentiation potential of MSCs. These results indicate that downregulation of TAp63α in MSCs is an attractive strategy to protect against apoptosis when MSCs are used to support hematopoiesis during bone marrow transplantation.

Adult human stem cells are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue. Data suggest that adult stem cells (ASCs) generate differentiated cells beyond their own tissue boundaries, a process termed "developmental plasticity." Since early reports of developmental plasticity in animal models, researchers have made enormous advances in moving toward clinically applicable treatment options with stem cells from both bone marrow and peripheral blood. It seems China is entering this new therapeutic era more rapidly than other countries around the world. In this review, we focus on therapeutic applications of ASCs derived from bone marrow and peripheral blood. Furthermore, we also discuss concerns related to regulations monitoring stem cell-based clinical trials.

Mesenchymal stem cells (MSCs) have profound immunomodulatory functions both in vitro and in vivo. However, their effects on the differentiation of dendritic cells (DCs) are unknown. In this study, we employed an in vitro model to investigate the effects of human MSCs on the development of DCs. CD34(+) cells isolated from cord blood were cultured under conventional DC(cDC) or plasmacytoid DC (pDC) differentiation conditions, in the presence or absence of MSCs or their conditioned medium. Here we show that both MSCs and their conditioned medium dramatically increased the numbers of cells generated under either condition. The percentage of cells with the cDC phenotype is significantly reduced in the presence of MSCs or their conditioned medium, whereas the percentage of pDC increased. The capacity of cDCs from MSCs or their conditioned medium-treated CD34(+) cells to stimulate allogeneic T cells was weakened. Furthermore, MSCs can skew the DC function from cDC to pDC, thus biasing the immune system toward Th2 and away from Th1 responses. Blocking the prostaglandin E(2) (PGE(2)) synthesis of MSCs can reverse most of these influences of MSCs on DCs differentiation and function. Therefore, MSCs can significantly influence DC development through PGE(2) production.

Mesenchymal stem cells (MSCs) reportedly inhibit the mixed lymphocyte reaction. Whether this effect is mediated by dendritic cells (DCs) is still unknown. In this study, we used an in vitro model to observe the effects of MSCs and their supernatants on the development of monocyte-derived DCs. Phenotypes and the endocytosic ability of harvested DCs were determined by flow cytometry; interleukin 12 (IL-12) secreted by DCs was evaluated by enzyme-linked immunosorbent assay (ELISA); and the antigen-presenting function of DCs was evaluated by MLR. Our results show that MSCs inhibit the up-regulation of CD1a, CD40, CD80, CD86, and HLA-DR during DC differentiation and prevent an increase of CD40, CD86, and CD83 expression during DC maturation. MSCs supernatants had no effect on DCs differentiation, but they inhibited the up-regulation of CD83 during maturation. Both MSCs and their supernatants interfered with endocytosis of DCs, decreased their capacity to secret IL-12 and activate alloreactive T cells. Thus, effects of MSCs on DCs contribute to immunoregulation and development.

Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.

The objective of this study was to evaluate the safety and efficacy of allogeneic bone marrow mesenchymal stromal/stem cell transplantation (BM-MSCT) for patients with ursodeoxycholic acid (UDCA)-resistant primary biliary cirrhosis (PBC). Ten patients were enrolled in this trial of BM-MSCT. All patients were permitted to concurrently continue their previous UDCA treatment. The efficacy of BM-MSCT in UDCA-resistant PBC was assessed at various time points throughout the 12-month follow up. No transplantation-related side effects were observed. The life quality of the patients was improved after BM-MSCT as demonstrated by responses to the PBC-40 questionnaire. Serum levels of ALT, AST, γ-GT, and IgM significantly decreased from baseline after BM-MSCT. In addition, the percentage of CD8+ T cells was reduced, while that of CD4+CD25+Foxp3+ T cells was increased in peripheral lymphocytic subsets. Serum levels of IL-10 were also elevated. Notably, the optimal therapeutic outcome was acquired in 3 to 6 months and could be maintained for 12 months after BM-MSCT. In conclusion, allogeneic BM-MSCT in UDCA-resistant PBC is safe and appears to be effective.

Neural stem cells (NSCs) are ideal candidates in stem cell-based therapy for neurodegenerative diseases. However, it is unfeasible to get enough quantity of NSCs for clinical application. Generation of NSCs from human adipose-derived mesenchymal stem cells (hAD-MSCs) will provide a solution to this problem. Currently, the differentiation of hAD-MSCs into highly purified NSCs with biological functions is rarely reported. In our study, we established a three-step NSC-inducing protocol, in which hAD-MSCs were induced to generate NSCs with high purity after sequentially cultured in the pre-inducing medium (Step1), the N2B27 medium (Step2), and the N2B27 medium supplement with basic fibroblast growth factor and epidermal growth factor (Step3). These hAD-MSC-derived NSCs (adNSCs) can form neurospheres and highly express Sox1, Pax6, Nestin, and Vimentin; the proportion was 96.1% ± 1.3%, 96.8% ± 1.7%, 96.2% ± 1.3%, and 97.2% ± 2.5%, respectively, as detected by flow cytometry. These adNSCs can further differentiate into astrocytes, oligodendrocytes, and functional neurons, which were able to generate tetrodotoxin-sensitive sodium current. Additionally, we found that the neural differentiation of hAD-MSCs were significantly suppressed by Sox1 interference, and what's more, Step1 was a key step for the following induction, probably because it was associated with the initiation and nuclear translocation of Sox1, an important transcriptional factor for neural development. Finally, we observed that bone morphogenetic protein signal was inhibited, and Wnt/β-catenin signal was activated during inducing process, and both signals were related with Sox1 expression. In conclusion, we successfully established a three-step inducing protocol to derive NSCs from hAD-MSCs with high purity by Sox1 activation. These findings might enable to acquire enough autologous transplantable NSCs for the therapy of neurodegenerative diseases in clinic.

Mesenchmal stem cells (MSCs) can be differentiated into either adipocytes or osteoblasts, and a reciprocal relationship exists between adipogenesis and osteogenesis. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation, respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. MicroRNAs are important regulators in diverse biological processes by repressing protein expression of their targets. Here, miR-22 was found to regulate adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs) in opposite directions. Our data showed that miR-22 decreased during the process of adipogenic differentiation but increased during osteogenic differentiation. On one hand, overexpression of miR-22 in hADMSCs could inhibit lipid droplets accumulation and repress the expression of adipogenic transcription factors and adipogenic-specific genes. On the other hand, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes, indicated a positive role of miR-22 in regulating osteogenic differentiation. Target databases prediction and validation by Dual Luciferase Reporter Assay, western blot, and real-time polymerase chain reaction identified histone deacetylase 6 (HDAC6) as a direct downstream target of miR-22 in hADMSCs. Inhibition of endogenous HDAC6 by small-interfering RNAs suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22 overexpression in hADMSCs. Together, our results suggested that miR-22 acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6.

A better understanding of the molecular mechanisms underlying the differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) could provide new insights into the pathogenesis of a number of diseases, such as obesity and diabetes, and broaden the spectrum of potential hAD-MSCs-based cell therapy. In this study, we reported that a human microRNA, hsa-miR-138, could inhibit the adipogenic differentiation of hAD-MSCs. Our results showed that miR-138 was significantly down-regulated during adipogenic differentiation. Overexpression of miR-138 in hAD-MSCs could effectively reduce lipid droplets accumulation, inhibit expression of key adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma 2 as well as several other adipogenic marker genes, such as fatty acid binding protein 4 and lipoprotein lipase. Further studies showed that the expression of adenovirus early region 1-A-like inhibitor of differentiation 1 (EID-1), a nuclear receptor coregulator, was inversely correlated with that of miR-138 when hAD-MSCs were differentiated into adipocytes. Knockdown of EID-1 by RNA interference inhibited adipocyte differentiation of hAD-MSCs. In addition, luciferase reporter assays demonstrated that miR-138 directly targeted the 3' untranslated region of EID-1, implying that the negative role of miR-138 in the adipocyte differentiation of hAD-MSCs is at least partially mediated via repressing EID-1. Taken together, this study shows that miR-138 plays a negative role in adipogenic differentiation and sheds light on the role of miRNAs during differentiation of hAD-MSCs toward adipocytes.

Duchenne muscular dystrophy (DMD) is a severe hereditary disease characterized by the absence of dystrophin on the sarcolemma of muscle fiber. This absence results in widespread muscle damage and satellite cell activation. After depletion of the satellite cell pool, skeletal muscle is then invariably replaced by connective tissue, leading to progressive muscle weakness. Herein, we isolated Flk-1(+) mesenchymal stem cells (MSCs) from adult adipose tissue and induced them to differentiate into skeletal muscle cells in culture. Within mdx mice, an animal model of DMD, adipose tissue-derived Flk-1(+) MSCs (AD-MSCs) homed to and differentiated into cells that repaired injured muscle tissue. This repair correlated with reconstitution of dystrophin expression on the damaged fibers. Flk-1(+) AD-MSCs also differentiated into muscle satellite cells. This differentiation may have accounted for long-term reconstitution. These cells also differentiated into endothelial cells, thereby possibly improving fiber regeneration as a result of the induced angiogenesis. Therefore, Flk-1(+) AD-MSC transplants may repair muscular dystrophy.

One of the clinical features of multiple myeloma (MM) is the occurrence of skeletal events, which are characterized by increased bone resorption and decreased bone formation. In contrast to enhanced osteoclastogenesis, little is known about the mechanism of impaired bone formation in MM. Because TAZ, a Runx2/Cbfa1 transcriptional co-activator, has recently been shown to modulate mesenchymal stem cell (MSC) differentiation in favor of osteoblast differentiation, we investigated whether the regulation of TAZ expression played a role in the decreased bone formation of MM. We isolated and purified Flk-1(+)CD31(-)CD34(-) cells with MSC characters from bone marrow (BM) of myeloma patients and healthy donors. We found the osteogenic potential of the MSCs from myeloma patients decreased significantly, and TAZ expression of these cells was lower than that of healthy donors. Human myeloma cell lines (HMCLs) and CD138(+) myeloma cells (MCs) from myeloma patients inhibited osteogenesis of the MSCs from healthy volunteers, which were accompanied by a reduced TAZ expression and elevated TNF-alpha concentration in the supernatant of co-culture systems. The repressed osteogenesis and TAZ expression were both partially restored by neutralization of TNF-alpha. Thus, the decreased osteogenic potential of MSCs of myeloma patients was in part due to TNF-alpha suppressed TAZ expression.

Flk-1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) are multipotent cells with extensive proliferation ability and immunomodulatory function. On the basis of our previous work showing their potential application in tissue engineering and immune diseases, we designed a preclinical and Phase I trial to evaluate the feasibility and safety of intravenous infusion of these cells after ex vivo expansion. Rhesus monkey and human Flk-1(+)CD31(-)CD34(-) MSCs were isolated from bone marrow and passaged a maximum of six passages. Karyotype analysis showed Flk-1(+)CD31(-)CD34(-) MSCs remained normally diploid within six passages of expansion. Expanded cells were transplanted into rhesus monkeys and human volunteers, respectively. During the infusion of these cells, the life signs of the recipients were normal. Laboratory tests for blood, bone marrow, kidney, and liver function were conducted and no significant changes were observed before and after cell infusion. Identification of the sry sequence from the male donor in female recipients showed that allogeneic rhesus Flk- 1(+)CD31(-)CD34(-) MSCs were capable of homing to and establishing residence within the recipients' bone marrow. No significant changes were observed in lymphocyte cell subpopulation before and after infusion. These result showed that Flk-1(+)CD31(-)CD34(-) MSCs have no obvious side effects after intravenous administration, encouraging investigators to perform further studies in stem cell therapy.