Qin Han

Effects of Mesenchymal Stem Cells on Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells

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.

Upregulation of miR-22 Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells by Repressing HDAC6 Protein Expression

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.

MicroRNA‐100 regulates osteogenic differentiation of human adipose‐derived mesenchymal stem cells by targeting BMPR2

Elucidation of the molecular mechanisms governing human adipose‐derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone‐related diseases. In this study, we examined the role of microRNA (miR)‐100 on the osteogenesis of hASCs. Overexpression of miR‐100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR‐100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR‐100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR‐100. Taken together, our findings imply that miR‐100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.

miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.

Transplantation of human mesenchymal stem cells loaded on collagen scaffolds for the treatment of traumatic brain injury in rats

Studies have suggested that mesenchymal stem cells (MSCs) have therapeutic effects following traumatic brain injury (TBI). However, cell distribution and survival rate are two major barriers to their success as therapeutic treatment. The improvement of cell therapy using collagen delivery matrices had been reported. However, we know very little about the mechanisms. We labeled human bone marrow-derived mesenchymal stem cells (hMSCs) with a positron emission tomography (PET) tracer, 18F-fluoro-2-deoxy-D-glucose (FDG). hMSCs were transplanted with or without collagen scaffolds into rats with experimental TBI and the whole-body nuclear images were compared. Collagen scaffolds increased the retention of hBMSC in the lesion site and limited its distribution at the transplanted region. Significantly more hMSCs were detected in the brain when transplanted with collagen scaffolds. The results showed collagen scaffolds also efficiently improved cell survival and neurite outgrowth in vivo, resulting in better neural functional recovery. In addition, brain metabolism also improved in the collagen scaffold implanted group, as evaluated by PET. We speculated that collagen scaffolds would improve early engraftment and support the survival of grafted cells post-transplantation.

Not a process of simple vicariousness, the differentiation of human adipose-derived mesenchymal stem cells to renal tubular epithelial cells plays an important role in acute kidney injury repairing.

The recent findings indicate that under conditions of severe tubular injuries, transplantation of mesenchymal stem cells (MSCs) may be a promising treatment in acute kidney diseases; nevertheless, the underling mechanism is still under debate. To investigate the differentiation characteristics and the role of MSCs in renal tubular injury, human adipose-derived MSCs (hAD-MSCs) were transplanted into ischemia-reperfusion (I/R) kidneys in C57BL/6 mouse model. Results showed that hAD-MSCs were able to differentiate toward renal tubular epithelium at an early stage of injuries. The differentiated donor cells replaced the vacant space left over by the dead cells, contributed to maintenance of structural integrity and proceeded to a subsequent tissue repair process. Furthermore, MSCs as supportive cells may promote repair via secreting cytokines. The differentiation and replacement of MSCs at an extremely early stage play important roles for the subsequent self-repair and -renewal of functional cells. Direct differentiation of MSCs, as an important mechanism of injured kidney repair, warrants further investigation.

Flk‐1+ mesenchymal stem cells aggravate collagen‐induced arthritis by up‐regulating interleukin‐6

The immunomodulatory ability of mesenchymal stem cells (MSCs) may be used to develop therapies for autoimmune diseases. Flk‐1+ MSCs are a population of MSCs with defined phenotype and their safety has been evaluated in Phase 1 clinical trials. We designed this study to evaluate whether Flk‐1+ MSCs conferred a therapeutic effect on collagen‐induced arthritis (CIA), an animal model of rheumatic arthritis, and to explore the underlying mechanisms. Flk‐1+ MSCs, 1–2 × 106, were injected into CIA mice on either day 0 or day 21. The clinical course of arthritis was monitored. Serum cytokine profile was determined by cytometric bead array kit or enzyme‐linked immunosorbent assay. Flk‐1+ MSCs and splenocytes co‐culture was conducted to explore the underlying mechanisms. Flk‐1+ MSCs did not confer therapeutic benefits. Clinical symptom scores and histological evaluation suggested aggravation of arthritis in mice treated with MSCs at day 21. Serum cytokine profile analysis showed marked interleukin (IL)‐6 secretion immediately after MSC administration. Results of in vitro culture of splenocytes confirmed that the addition of Flk‐1+ MSCs promoted splenocyte proliferation and increased IL‐6 and IL‐17 secretion. Moreover, splenocyte proliferation was also enhanced in mice treated with MSCs at day 21. Accordingly, MSCs at low concentrations were found to promote lipopolysaccharide‐primed splenocytes proliferation in an in vitro co‐culture system. We propose that Flk‐1+ MSCs aggravate arthritis in CIA model by at least up‐regulating secretion of IL‐6, which favours Th17 differentiation. When Flk‐1+ MSCs are used for patients, we should be cautious about subjects with rheumatoid arthritis.

Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excitotoxicity-induced apoptosis by upregulation of XIAP through PI3-K/Akt activation

Glutamate excitotoxicity has been implicated as one of the factors contributing to neuronal apoptosis and is involved in many neurodegenerative diseases. Previous studies suggest that mesenchymal stem cells have the ability to protect cultured neurons from excitotoxicity-induced apoptosis, although the underlying mechanisms are not clear. In this study, we evaluated whether adipose mesenchymal stem cells (AMSCs) could protect against glutamate-induced injury in PC12 cells by secreting neurotrophic factors. We found that AMSCs secreted neurotrophic factors including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) under both normoxic and hypoxic conditions. AMSC - conditioned medium (AMSC-CM) had a protective effect on excitotoxicity-injured PC12 cells, as indicated by increased cell viability, decreased number of TUNEL-staining positive nuclei and lowered caspase-3 activity. By using neutralizing monoclonal antibodies and specific inhibitors, VEGF, HGF and BDNF were identified as the mediators of AMSC effects and PI3-K/Akt and MAPK pathways were involved. Western blot analysis showed that AMSC-CM can increase the level of p-Akt, up-regulate XIAP and reduce the level of cleaved-caspase-3 in PC12 cells. These results suggest that AMSCs can effectively protect PC12 cells from glutamate excitotoxicity-induced apoptosis and support the hypothesis that AMSCs may be a useful treatment for stroke or neurodegenerative diseases which often involve excitotoxicity.

MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma

Metastasis is one of the hallmarks of cancer malignancy that usually causes more detrimental effects than a primary tumor. Many microRNAs were reported to be involved in the process of tumor metastasis. Hep11 and Hep12 cells were derived from primary and recurrence (intrahepatic metastatic) sites of hepatocellular carcinoma (HCC), respectively. Hep12 exhibited a higher invasive and migratory potential than Hep11. There was also a significantly higher expression of miR‐9 in Hep12 cells than in Hep11 cells. Further studies in HCC cell lines demonstrated that miR‐9 could promote tumor cell migration and invasion. In addition, miR‐9 downregulated KLF17 protein expression by targeting the 3′UTR region of the KLF17 gene directly. As a transcription factor, KLF17 directly acted on the promoters of EMT‐related genes (ZO‐1, Vimentin and Fibronectin (FN)) in HCC cell lines. Therefore, we conclude that miR‐9 may possibly promote HCC migration and invasion through regulation of KLF17.

Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a

ABSTRACT Angiogenesis plays crucial roles in various physiological processes including wound healing and tissue repair. It requires a tight interaction between endothelial cells and their surrounding environment. Mesenchymal stem cells (MSCs), one of the non-endothelial cell types present in the perivascular environment, have been shown to secret exosomes to modulate intercellular communications between MSCs and their target cells. In this study, we initially isolated exosomes secreted by human adipose-derived MSCs (adMSC-Exo) and examined their roles in angiogenesis. We found that adMSC-Exo could be taken up by endothelial cells and significantly promote angiogenesis in vitro and in vivo. Further study showed that miR-125a was enriched in adMSC-Exo, and repressed the expression of the angiogenic inhibitor delta-like 4 (DLL4) by targeting its 3′ untranslated region. Additionally, adMSC-Exo and its exosomal transferred miR-125a could repress DLL4 expression and modulate endothelial cell angiogenesis through promoting formation of endothelial tip cells. In conclusion, our study indicates that adMSC-Exo can transfer miR-125a to endothelial cells and promote angiogenesis by repressing DLL4. adMSC-Exo, as a pro-angiogenic factor, might be a promising candidate for therapeutical tissue repair. Summary: The miR-125a transferred from exosomes secreted by human-adipose-derived mesenchymal stem cells can repress DLL4 expression and modulate angiogenesis through promoting endothelial tip cell formation.