Sca-1+Lin-CD117- mesenchymal stem/stromal cells induce the generation of novel IRF8-controlled regulatory dendritic cells through Notch-RBP-J signaling.

Mesenchymal stem/stromal cells (MSCs) can influence the destiny of hematopoietic stem/progenitor cells (HSCs) and exert broadly immunomodulatory effects on immune cells. However, how MSCs regulate the differentiation of regulatory dendritic cells (regDCs) from HSCs remains incompletely understood. In this study, we show that mouse bone marrow-derived Sca-1(+)Lin(-)CD117(-) MSCs can drive HSCs to differentiate into a novel IFN regulatory factor (IRF)8-controlled regDC population (Sca(+) BM-MSC-driven DC [sBM-DCs]) when cocultured without exogenous cytokines. The Notch pathway plays a critical role in the generation of the sBM-DCs by controlling IRF8 expression in an RBP-J-dependent way. We observed a high level of H3K27me3 methylation and a low level of H3K4me3 methylation at the Irf8 promoter during sBM-DC induction. Importantly, infusion of sBM-DCs could alleviate colitis in mice with inflammatory bowel disease by inhibiting lymphocyte proliferation and increasing the numbers of CD4(+)CD25(+) regulatory T cells. Thus, these data infer a possible mechanism for the development of regDCs and further support the role of MSCs in treating immune disorders.

Mesenchymal stem/stromal cells induce the generation of novel IL-10-dependent regulatory dendritic cells by SOCS3 activation.

Suppression of immune response by mesenchymal stem/stromal cells (MSCs) is well documented. However, their regulatory effects on immune cells, especially regulatory dendritic cells, are not fully understood. We have identified a novel Sca-1(+)Lin(-)CD117(-) MSC population isolated from mouse embryonic fibroblasts (MEF) that suppressed lymphocyte proliferation in vitro. Moreover, the Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced hematopoietic stem/progenitor cells to differentiate into novel regulatory dendritic cells (DCs) (Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs) when cocultured in the absence of exogenous cytokines. Small interfering RNA silencing showed that Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced the generation of Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs via IL-10-activated SOCS3, whose expression was regulated by the JAK-STAT pathway. We observed a high degree of H3K4me3 modification mediated by MLL1 and a relatively low degree of H3K27me3 modification regulated by SUZ12 on the promoter of SOCS3 during SOCS3 activation. Importantly, infusion of Sca-1(+)CD117(-)Lin(-) MEF-MSCs suppressed the inflammatory response by increasing DCs with a regulatory phenotype. Thus, our results shed new light on the role of MSCs in modulating regulatory DC production and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.

miR-21 modulates the ERK-MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation.

The ERK-MAPK signaling pathway plays a pivotal role during mesenchymal stem cell (MSC) differentiation. Studies have demonstrated that ERK-MAPK promotes adipogenesis and osteogenesis through the phosphorylation of differentiation-associated transcription factors and that it is the only active signaling in all three lineages (adipogenic, chondrogenic, and osteogenic) during MSC differentiation. Recent studies pointed to the significant roles of microRNA-21 (miR-21) during several physiological and pathological processes, especially stem cell fate determination. The miR-21 expression pattern is also correlated with ERK-MAPK activity. Here, we found that miR-21 expression is elevated and associated with an increased differentiation potential in MSCs during adipogenesis and osteogenesis. The overexpression of miR-21 elevated the expression level of the differentiation-associated genes PPARγ and Cbfa-1 during MSC differentiation, whereas miR-21 knockdown reduced the expression level of both genes. The ERK-MAPK signaling pathway activity had an increasing tendency to respond to miR-21 upregulation and a decreasing tendency to respond to miR-21 down-regulation during the first 4 days of adipogenesis and osteogenesis. Our data indicate that miR-21 modulated ERK-MAPK signaling activity by repressing SPRY2 expression, a known regulator of the receptor tyrosine kinase (RTK) signaling pathway, to affect the duration and magnitude of ERK-MAPK activity. The ERK-MAPK signaling pathway was regulated by Sprouty2 (SPRY2) expression via a miR-21-mediated mechanism during MSC differentiation.

Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2-dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

Concise review: bone marrow-derived stem/progenitor cells in cutaneous repair and regeneration.

Our understanding of the role of bone marrow (BM)-derived cells in cutaneous homeostasis and wound healing had long been limited to the contribution of inflammatory cells. Recent studies, however, suggest that the BM contributes a significant proportion of noninflammatory cells to the skin, which are present primarily in the dermis in fibroblast-like morphology and in the epidermis in a keratinocyte phenotype; and the number of these BM-derived cells increases markedly after wounding. More recently, several studies indicate that mesenchymal stem cells derived from the BM could significantly impact wound healing in diabetic and nondiabetic animals, through cell differentiation and the release of paracrine factors, implying a profound therapeutic potential. This review discusses the most recent understanding of the contribution of BM-derived noninflammatory cells to cutaneous homeostasis and wound healing.

Profiling microRNA expression with microarrays.

The discovery of several types of small RNAs (sRNAs) has led to a steady increase in available RNA databases. Many of these sRNAs remain to be validated and functionally characterized. Recent advances in microRNA (miRNA)-expression profiling of different tissues, stages of development and physiological or pathological states are beginning to be explored using several technological approaches. In this review, these recent advances in miRNA microarray technology and their applications, particularly in basic research and clinical diagnosis, will be summarized and discussed. The methods for miRNA enrichment and probe design and labeling will also be discussed with an emphasis on evaluation of predicted miRNA sequences, analysis of miRNA expression and exploration of the potential roles of miRNA sequences in the regulation of stem cell differentiation and tissue- and time-specific profiling patterns of their target genes.

Exosomes Derived from Hypoxia-Treated Human Adipose Mesenchymal Stem Cells Enhance Angiogenesis Through the PKA Signaling Pathway.

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.

Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue.

Stem cell plasticity has created great interest because of its potential therapeutic application in degenerative or inherited diseases. Transplantation of bone marrow-derived stem cells was shown to give rise to cells of muscle, liver, nerve, endothelium, epithelium, and so on. But there are still disputes about stem cell plasticity, especially concerning the contribution of bone marrow-derived cells to skin cells. In this study, CM-DiI fluorescence-labeled Flk-(1+) bone marrow mesenchymal stem cells (bMSCs) of BALB/c mice (H-2Kd, white) were transplanted into lethally irradiated C57BL/6 mice (H-2Kb, black). By fluorescence tracing, we found that donor cells could migrate and take residency at the skin, which was confirmed by Y chromosome-specific PCR and Southern blot. The recipient mice grew white hairs about 40 days later and white hairs could spread over the body. Immunochemistry staining and RT-PCR demonstrated that skin tissue within the white hair regions was largely composed of donor-derived H-2Kd cells, including stem cells and committed cells. Furthermore, most skin cells cultured from white hair skin originated from the donor. Thus, our findings provide direct evidence that bone marrow-derived cells can give rise to functional skin cells and regenerate skin tissue. These may have important scientific implications in stem cell biology and transplantation therapy for skin tissue injury.

Effects of allogeneic bone marrow-derived mesenchymal stem cells on T and B lymphocytes from BXSB mice.

Bone marrow-derived mesenchymal stem cells (bMSCs) can differentiate into a number of different cell/tissue types, and also possess immunoregulatory functions. The present study was undertaken to elucidate the exact immunoregulatory effects of allogeneic bMSCs on T- and B-lymphocyte proliferation, activation, and function maturation of BXSB mice, which has been considered as a experimental model for human systemic lupus erythematosus (SLE). We determined that bMSCs from BALB/c mice had inhibitory effects on BXSB mice T-lymphocyte proliferation, but no inhibitory effect on their activation. In addition, they had a significant inhibitory and stimulatory effect on IL-4- and IFN-gamma-producing T cells, respectively. Also, bMSCs had inhibitory effects on the proliferation, activation, and IgG secretion of B lymphocytes. In addition, BALB/c bMSCs had an enhancing effect on CD40 expression and inhibitory effects on CD40 ligand (CD40L) ectopic hyperexpression on B cells from BXSB mice.

Allogeneic bone marrow-derived flk-1+Sca-1- mesenchymal stem cells leads to stable mixed chimerism and donor-specific tolerance.

OBJECTIVE: To investigate the possibility of flk-1+Sca-1- bone marrow-derived mesenchymal stem cells (bMSCs) to induce stable mixed chimerism and donor-specific graft tolerance. METHODS: Allogeneic flk-1+Sca-1- bMSCs and syngeneic bone marrow (BM) cells were cotransplanted into lethally irradiated (8.5 Gy) recipient mice. FACS was used to analyze the chimerism 150 days later. Donor-type skin transplantation was performed to observe donor-specific immunotolerance in recipient mice. Mixed lymphocyte reaction (MLR) and mitogen proliferative assays were performed to evaluate proliferative response of splenocytes from recipient mice. RESULTS: More than 5% donor-derived CD3+ cells were detected in splenocytes of recipient mice. Long-term survival of donor-type skin grafts was observed. MLR and mitogen proliferative assays showed that recipient mice had low immunoresponse to donor cells but retained normal ConA-induced proliferative response compared with normal mice. CONCLUSION: Our results show for the first time that induction of stable mixed hematopoietic chimerism can be achieved with allogeneic flk-1+Sca-1- bMSC transplantation, which leads to permanent donor-specific immunotolerance in allogeneic host and results in long-term allogeneic skin graft acceptance.

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.

Effect of O-4-ethoxyl-butyl-berbamine in combination with pegylated liposomal doxorubicin on advanced hepatoma in mice.

AIM: To study the synergistic effects of calmodulin (CaM) antagonist O-4-ethoxyl-butyl-berbamine (EBB) and pegylated liposomal doxorubicin (PLD) on hepatoma-22 (H(22)) in vivo. METHODS: Hepatoma model was established in 50 Balb/c mice by inoculating H(22) cells (2.5 x 10(6)) subcutaneously into the right backs of the mice. These mice were divided into 5 groups, and treated with saline only, PLD only, doxorubicin (Dox) only, PLD plus EBB and Dox plus EBB, respectively. In the treatment groups, mice were given 5 intravenous of PLD or Dox on days 0, 3, 6, 9 and 12. The first dosage of PLD or Dox was 4.5 mg/kg, the other 4 injections was 1 mg/kg. EBB (5 mg/kg) was coadministered with PLD or Dox in the corresponding groups. The effect of drugs on the life spans of hepatoma-bearing mice and tumor response to the drugs were recorded. Dox levels in the hepatoma cells were measured by a fluorescence assay. Light microscopy was performed to determine the histopathological changes in the major organs of these tumor-bearing mice. The MTT method was used to analyze the effect of Dox or PLD alone, Dox in combination with EBB, or PLD in combination with EBB on the growth of H(22) cells in an in vitro experiment. RESULTS: EBB (5 mg/kg) significantly augmented the antitumor activity of Dox or PLD, remarkably prolonged the median survival time. The median survival time was 18.2 d for control group, but 89.2 d for PLD+EBB group and 70.1 d for Dox+EBB group, respectively. However, Dox alone did not show any remarkable antitumor activity, and the median survival time was just 29.7 d. Addition of EBB to Dox or PLD significantly increased the level of Dox in H(22) cells in vivo. Moreover, EBB diminished liver toxicity of Dox and PLD. In vitro, EBB reduced the IC50 value of Dox or PLD on H(22) cells from 0.050+/-0.006 mg/L and 0.054+/-0.004 mg/L to 0.012+/-0.002 mg/L and 0.013+/-0.002 mg/L, respectively (P<0.01). CONCLUSION: EBB and liposomization could improve the therapeutic efficacy of Dox in liver cancer, while decreasing its liver toxicity.

Three-dimensional spheroid-cultured mesenchymal stem cells devoid of embolism attenuate brain stroke injury after intra-arterial injection.

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.

The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke.

BACKGROUND AND PURPOSE: Intravascular injection of mesenchymal stem cells (MSCs) has been found to cause considerable vascular obstructions which may lead to serious outcomes, particularly after intra-arterial injection. However, the underlying mechanisms have been poorly understood. METHODS: In this study, we fractionated MSCs that had been cultured in monolayer for six passages into small (average diameter = 17.9 µm) and large (average diameter 30.4 µm) populations according to their sizes, and examined their vascular obstructions after intra-internal carotid artery injection in rats and mice in comparison with MSCs derived from 3D spheroids which were uniformly smaller in size (average diameter 12.6 µm). RESULTS: We found that 3D MSCs did not cause detectable infarct in the brain as evidenced by MRI scan and TTC stain, 2D MSCs in small size caused a microinfarct in one of five animals, which was co-localized to the area of entrapped MSCs (labeled with DiI), while 2D MSCs in large size caused much larger infarcts in all five animals, and substantial amounts of DiI-positive MSCs were found in the infarct. Meanwhile, corresponding neurological defects were observed in the animals with stroke. In consistence, injection of 2D MSCs (average diameter 26.5) caused a marked loss of cortical neurons and their axons in Thy1-GFP transgenic mice and the activation of microglia in CX3CR1-GFP transgenic mice in the area with MSC entrapment. CONCLUSIONS: Our results suggest that the size of MSCs is a significant cause of MSC caused vascular obstructions and stroke.

The role of chemokines in mesenchymal stem cell homing to myocardium.

A growing body of preclinical evidence suggests that mesenchymal stem cells (MSCs) are effective for the structural and functional recovery of the infracted heart. Accordingly, clinical trials are underway to determine the benefit of MSC-based therapies. While systemic administration of MSCs is an attractive strategy, and is the route currently used for the administration of MSCs in clinical studies for myocardial infarction, the majority of infused cells do not appear to localize to infracted myocardium in animal studies. Recently, important progress has been made in identifying chemokine receptors critical for the migration and homing of MSCs. Here, we review recent literature regarding mechanisms of MSC homing and recruitment to the ischemic myocardium, and discuss potential influences of low engraftment rates of systemically administered MSCs to the infracted heart tissue on the effects of MSC-based therapies on myocardial infarction.

Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion.

Recent findings indicate that mesenchymal stem cells (MSCs) may act as a regulator of Th17 cell differentiation, however, the underlying mechanism is still under debate. To investigate the underlying mechanisms of MSCs' regulatory effect, mouse bone marrow-derived MSCs were cocultured with mouse CD4(+)CD25(low)CD44(low)CD62L(high) T cells in vitro, and the proportion of induced Th17 cells, cytokines secretion, and transcription factors expression were examined by flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blotting. For the first time, our results showed that bone marrow-derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion as the Th17 cell proportion was significantly regained when IL-10 was neutralized, or expression of IL-10 by bone marrow-derived MSCs was downregulated by RNA interference technique. Furthermore, IL-10 may suppress expression of Rorγt, the key transcription factor for Th17 cells, both by activating suppressor of cytokine signaling 3 through signal transducers and activators of transcription 5 phosphorylation, and decreasing signal transducers and activators of transcription 3 binding, which is at the promoter of Rorγt. Thus, our results demonstrate the inhibitory effect of MSCs on Th17 cells differentiation, and suggest increased IL-10 secretion might be the key factor.

Enhancer of zeste homolog 2 is overexpressed and contributes to epigenetic inactivation of p21 and phosphatase and tensin homolog in B-cell acute lymphoblastic leukemia.

Enhancer of zeste homolog 2 (EZH2) is crucially involved in epigenetic silencing by acting as a histone methyltransferase. Although EZH2 is overexpressed in many solid cancers, the role of EZH2 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely unexplored. In a microarray experiment, we found that EZH2 was significantly upregulated in Nalm-6 cells and this was associated with the silencing of tumor suppressor genes p21, p53 and phosphatase and tensin homolog (PTEN). The abnormal expression of these genes was further confirmed by quantitative realtime polymerase chain reaction and Western blot analysis on Nalm-6 cells. Chromatin immunoprecipitation assay showed that EZH2 and H3K27me3 were both enriched in the promoter region of PTEN and p21 in Nalm-6 cells but not in normal B cells. Functional analysis showed that siRNA-mediated EZH2 knockdown led to decreased proliferation and increased apoptosis of Nalm-6 cells, accompanied by the reactivation of PTEN and p21 expression. Furthermore, we found that EZH2 inhibitor deazaneplanocin A promoted vincristine sulfate-induced apoptosis of Nalm-6 cells. Taken together, our data suggest that EZH2 is overexpressed in B-ALL and promotes the progression of B-ALL by directly mediating the inactivation of tumor suppressor genes p21 and PTEN, and could serve as a potential epigenetic target for B-ALL therapy.

The role of microRNAs in self-renewal and differentiation of mesenchymal stem cells.

MicroRNAs (miRNAs) are short non-coding RNAs involved in post-trascriptional regulation of gene expression and diverse biological activities. They are crucial for self-renewal and behavior of embryonic stem cells, but their role in mesenchymal stem cells has been poorly understood. Recently emerging evidence suggests that miRNAs are closely involved in controlling key steps of mesenchymal stem cell differentiation into certain cell lineages. This review focuses on miRNAs identified recently that regulate mesenchymal stem cell differentiation and other activities.