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The reduction of adult stem cell self-renewal can be an important mechanism of aging. MicroRNAs have been reported to be involved in aging processes. Through a microarray approach, we have identified miR-486-5p, the expression of which is progressively expressed in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) with aging. Overexpression of miR-486-5p induces a premature senescence-like phenotype and inhibits proliferation of hAT-MSCs and inhibits adipogenic and osteogenic differentiation, whereas inhibition of miR-486-5p has the opposite effects. miR-486-5p regulates the expression of silent information regulator 1 (SIRT1), a major regulator of longevity and metabolic disorders. Decrease of SIRT1 deacetylase activity in hAT-MSCs is correlated with their passage number. miR-486-5p inhibits SIRT1 expression through a miR-486-5p binding site within the 3'-untranslated region of SIRT1. Overexpression of miR-486-5p inhibits SIRT1 deacetylase activity in hAT-MSCs, and transfection of miR-486-5p inhibitor shows the opposite effect. Downregulation of SIRT1 in hAT-MSCs induces senescence and inhibits cell proliferation. Exposure to high glucose increases miR-486-5p expression and inhibits SIRT1 expression in hAT-MSCs. Our data pinpoint miR-486-5p as an endogenous inhibitor of SIRT1 that promotes hAT-MSCs senescence and is potentially applicable to therapeutic manipulation of hAT-MSCs dysfunction in metabolic disorders.

beta-catenin/Tcf and NF-kappaB signaling pathways play an important role in biological functions and crosstalk between these pathways has been reported. We found that the modulation of NF-kappaB activity showed a direct correlation with beta-catein/Tcf pathway in human adipose tissue (hASCs) and bone marrow (hBMSCs)-derived mesenchymal stem cells. Expression of lzts2, which inhibits nuclear translocation of beta-catenin and its transactivation activity, was regulated by NF-kappaB activity. Downregulation of lzts2 by RNA interference increased the nuclear translocation of beta-catenin and NF-kappaB activity in hASCs. NF-kappaB activation by the downregulation of lzts2 was accompanied by the increase of beta-TrCP1 expression and the decrease of IkappaB level. Downregulation of lzts2 increased the proliferation of hASCs and hBMSC, and blocked the NF-kappaB inhibitor-induced inhibitory effect on their proliferation and Tcf promoter activation. These findings provide the first evidence that the reciprocal crosstalk between beta-catenin/Tcf pathway and NF-kappaB signaling in hMSCs is mediated through the regulation of lzts2 expression.

Human mesenchymal stem cells(hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum(FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. Previously, we have shown that hADSC can be cultured in human serum(HS) during their isolation and expansion, and that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34 cells mobilized from bone marrow in NOD/SCID mice. In this study we determined whether hADSC grown in HS maintain surface markers expression similar with cells grown in FBS during culture expansion and compared gene expression profile by Affymetrix microarray. Flow cytometry analysis showed that HLA-DR, CD117, CD29 and CD44 expression in HS-cultured hADSC during culture expansion were similar with that in FBS-cultured cells. However, the gene expression profile in HS-cultured hADSC was significantly different from that in FBS-cultured cells. Therefore, these data indicated that HS-cultured hADSC should be used in vivo animal study of hADSC transplantation for direct extrapolation of preclinical data into clinical application.

Adult mesenchymal stem cells (MSCs) are promising tools for such applications as tissue engineering and cellular therapy. It is not clear how stem cells exposed to unfavorable conditions (e.g., hypoxia or inflammation) respond to signals of danger after in vivo transplantation. Toll-like receptors (TLRs) play a major role in the immune system, participating in the initial recognition of microbial pathogens and pathogen-associated components. This study was designated to determine the role of TLRs in human MSCs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analysis demonstrated that MSCs derived from human adipose tissue and bone marrow express TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, and TLR-9. We investigated induction of the differentiation and proliferation of human adipose tissue stromal cells (hADSCs) by TLR agonists, including flagellin, peptidoglycans (PGN), lipopolysaccharide (LPS), the synthetic double-stranded RNA analog poly(I:C), and synthetic CpG oligodeoxydinucleotide (CpG-ODN). None of these agonists, except ODN, affected the proliferation of hADSCs. LPS and PGN increased osteogenic differentiation, but CpG-ODN decreased it. Poly(I:C) itself did not affect adipogenic or osteogenic differentiations, but exerted a synergistic effect on LPS- or PGN-induced osteogenic differentiation. RT-PCR analysis demonstrated that LPS and PGN induce osteogenic markers in hADSCs. TLR agonists affected the expression of chemokines and cytokines differentially. Furthermore, hADSCs affected the expression of specific TLRs in vitro under hypoxic conditions. These data provide evidence of a nonimmune role for TLR signaling on MSCs and may provide clues to the behavior of transplanted MSCs in vivo.

Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation.

Human bone marrow stromal cells (hBMSCs) are defined as pluripotent progenitor cells with the ability to differentiate into osteoblasts, chondrochytes, adipocytes, muscle cells, and neural cells. Recently, it has been shown that telomerase expression not only extends the replicative life-span and maintains their bone-forming capability of hBMSCs. We previously reported that human adipose tissue stromal cells (hATSCs) have similar characteristics with hBMSCs. In this study, hATSCs were stably tranduced by a retrovirus containing the gene for the catalytic subunit of human telomerase (hTERT) and MSCV-neo retrovirus, and 12 clones for hTERT-hATSCs and 6 clones for MSCV-hATSCs were isolated. The tranduced clones (hATSC-TERTs) had high telomerase activity, which was maintained during subsequent subcultivation. The transduced cells of two representative clones have undergone more than 100 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 36-40 PD. The cells had a normal karyotype, and increased differentiation potential, especially osteogenic lineage. Intraventricular injection of hATSC-TERTs in ischemic rat brain showed enhancement of functional recovery as like hATSC-MSCVs. The tissue engraftment of hATSCs and hTERT-hATSCs in NOD/SCID mice after intravenous administration was identical. These results further support a similarity between hBMSCs and hATSCs. hATSCs can be used as an alternative of pluripotent stromal cells for cell replacement therapy as like hBMSCs.

A better understanding of the molecular mechanisms that govern human adipose tissue-derived mesenchymal stem cells (hASCs) differentiation could provide new insights into a number of diseases including obesity. Our previous study demonstrated that microRNA-21 (miR-21) controls the adipogenic differentiation of hASCs. In this study, we determined the expression of miR-21 in white adipose tissues in a high-fat diet (HFD)-induced obesity mouse model to examine the relationship between miR-21 and obesity and the effect of miR-21 on hASCs proliferation. Our study showed biphasic changes of miR-21 expression and a correlation between miR-21 level and adipocyte number in the epididymal fat of HFD mice. Over-expression of miR-21 decreased cell proliferation, whereas inhibiting miR-21 with 2'-O-methyl-antisense RNA increased it. Over-expression of miR-21 decreased both protein and mRNA levels of STAT3, whereas inhibiting miR-21 with 2'-O-methyl-antisense RNA increased these levels. The activity of a luciferase construct containing the miR-21 target site from the STAT3 3'UTR was lower in LV-miR21-infected hASCs than in LV-miLacZ infected cells. RNA interference-mediated down-regulation of STAT3 decreased cell proliferation without affecting adipogenic differentiation. These findings provide the evidence of the correlation between miR-21 level and adipocyte number in the white adipose tissue of HFD-induced obese mice, which provides new insights into the mechanisms of obesity.

Thioredoxin (TRX) is a ubiquitous redox protein that is involved in numerous biological functions, including the first unique step in DNA synthesis. TRX provides control over a number of transcription factors affecting cell proliferation and death through a mechanism referred to as redox regulation. In mammals, there are at least 3 members of the TRX family: TRX1, TRX2, and sperm TRX. To investigate the role of TRX1 and TRX2 in human adipose tissue-derived mesenchymal stem cells (hADSC), we modulated TRX1 and TRX2 expressions in hADSC using a lentiviral gene transfer system and small interfering RNA technique. Reverse transcription-polymerase chain reaction analysis confirmed the changes in expression of TRX1 and TRX2 in lentivirus-transduced or small interfering RNA-transfected cells. Although overexpression of TRX1 and TRX2 did not affect the differentiation of hADSC into adipogenic and osteogenic lineages, it increased the proliferation of hADSC compared with control lentivirus-transduced cells, decreased reactive oxygen species production, and inhibited oxidant-induced cell death. Downregulation of TRX1 and TRX2 inhibited cell proliferation. The treatment of U0126 blocked TRX-induced increase in cell proliferation. Overexpression of TRX1 and TRX2 increased ERK1/2 phosphorylation, nuclear factor-kappaB activation, and β-catenin/Tcf promoter activities and inhibited lucine zipper tumor suppressor 2 expression. On the contrary, downregulation of TRX1 and TRX2 expression induced inhibition of ERK1/2 phosphorylation, nuclear factor-kappaB activation, and β-catenin/Tcf promoter activities and increased lucine zipper tumor suppressor 2 expression. Activation of Wnt signal increased ERK1/2 activities in hADSC. These results indicated that TRX1 and TRX2 regulate the proliferation and survival of hADSC; these processes are mediated by the activation of ERK1/2.

Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

The proangiogenic action of human adipose tissue-derived mesenchymal stem cells (hASCs) transplantation has been shown to be mediated by secretory factors. In this study, we determined if human granulocyte chemotactic protein-2(GCP2) or monocyte chemoattractant protein-1(MCP1) is involved in the proangiogenic action of hASCs transplantation in the hindlimb ischemia model. hASCs secrete GCP2 and MCP1, which leads to increased tubule formation. The downregulation of GCP2 or MCP1 decreased MCP1 and GCP2 secretion, respectively, whereas the external addition of GCP2 or MCP1 increased MCP1 and GCP2, respectively. Additionally, the treatment of GCP2 and MCP1 increased VEGF secretion, while the downregulation of GCP2 and MCP1 showed the opposite effect on VEGF secretion. Downregulation of GCP2 and MCP1 expression also inhibited hASCs-induced proangiogenic action, while the overexpression of GCP2 increased it. Finally, the downregulation of MCP1 or VEGF inhibited the GCP2 overexpression-induced increase in blood flow recovery. Taken together, these data indicate that the proangiogenic action of hASCs transplantation is mediated by the interaction between GCP2, MCP1 and VEGF, which are secreted from the transplanted cells.

Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia.One day after creating hindlimb ischemia, mice were randomized to receive hADSC transplantation (hADSC group), hBMSC transplantation (hBMSC group), or vehicle transplantation (Control group). Two weeks after transplantation, the laser Doppler perfusion index was significantly higher in the hADSC group and hBMSC group than in the control group. Comparison between hADSC and hBMSC group showed better recovery of blood flow in hADSC group than in hBMSC group. Conditioned media from hADSC (hADSC-CM) showed better in vitro tube formation of hADSC than conditioned media from hBMSC (hBMSC-CM). hADSC showed higher expression of MMP3 and MMP9 than hBMSC. A MMP inhibitor, GM6001, and the transfection of MMP3 or MMP9 siRNA oligonucleotides inhibited in vitro tube formation of hADSC. Transplantation of MMP3 or MMP9 siRNA oligonucleotieds-transfected hADSC showed lower blood flow recovery and higher tissue injury than control oligonucelotide-transfected cells.This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease in terms of efficacy, accessibility and available tissue amounts.

CD9 belongs to the tetraspanin family and is involved in cell motility, osteoclastogenesis, metastasis, neurite outgrowth, myotube formation, and sperm-egg fusion. CD9 also promotes juxtacrine signaling involved in proliferation and attachment. Varying degrees of CD9 expression have been found in human mesenchymal stem cells. In this study, we determined the functional roles of CD9 in human adipose-derived mesenchymal stem cells (hASCs). The CD9 expression in hASCs was down-regulated during culture expansion. A colony-forming unit assay revealed that the clonal expandability of hASCs was directly correlated with the CD9 expression level of the colony. The CD9(high) cells exhibited an increased ability to proliferate, increased cell adhesiveness, and better in vitro tube formation than the CD9(low) cells. The cellular proliferation and attachment of the CD9(high) cells were inhibited upon treatment with a blocking antibody against CD9 and the transduction of a CD9 miRNA lentivirus. The CD9(high) cells showed higher NF-kappaB promoter activity and higher levels of intercellular adhesion molecule 1 than the CD9(low) cells. Reverse transcription-polymerase chain reaction analysis revealed higher endothelial nitric oxide synthase expression in the CD9(high) cells than in the CD9(low) cells. The engraftment and the proangiogenic action of hASCs in a murine model of hindlimb ischemia were significantly higher in the CD9(high) cells than in the CD9(low) cells. This study indicates that CD9 plays roles in cell proliferation and attachment in vitro as well as in in vivo engraftment and that it can be considered as a useful marker to predict the in vivo efficacy of hASCs.

Stromal-derived factor-1 (SDF-1)-mediated CXCR4 signaling plays important roles in migration, engraftment, and proliferation of stem cells. We report here that CXCR4 overexpression on human adipose tissue stromal cells (hADSCs) using a lentiviral gene transfer technique helped navigate these cells to the injured tissues in response to SDF-1 signaling. Transduced hADSCs, expressing high levels of CXCR4, displayed an increased capacity for cellular growth and protection against etoposide-induced cell death. CXCR4-overexpressed cells showed higher ERK activity than that of vector-transduced cells. U0126, an ERK inhibitor, and AMD3100, a CXCR4 antagonist, inhibited the proliferation of CXCR4 overexpression-induced proliferation and ERK phosphorylation. CXCR4-overexpressing cells showed increased level of beta-catenin and luciferase activity driven by the Tcf promoter. Our results suggest CXCR4 overexpression for improved hADSC motility, retention, and proliferation could be beneficial for in vivo navigation and expansion of stem cells.

Tbx3 is a transcription factor, the mutation of which causes ulnar mammary syndrome (UMS) characterized by abnormality and hypoplasia of the mammary gland, teeth, limbs, hair and genitalia. Tbx3 has been reported to be related to apoptosis and proliferation of rat bladder carcinoma cell and to regulate proliferation and differentiation of mouse osteoblast cells. Human adipose tissue stromal cells (hADSC) have been defined as multipotential adult stem cells, capable of differentiating into a variety of cell types such as osteoblasts, chondrocytes, adipocytes, muscle cells, and neural cells. To determine the functional roles of Tbx3 expression in hADSC, we used lentivirus siRNA vector. Expression of Tbx3 was downregulated during culture expansion. Downregulation of Tbx3 in hADSC by transduction of siTbx3 lentivirus decreased proliferation and osteogenic differentiation of hADSC. Expression of Tbx3 and the ratio of Tbx3 + 2a to Tbx3 increased during osteogenic differentiation. This report shows that Tbx3 plays an important role on osteogenic differentiation and proliferation of human mesenchymal stem cell derived from adipose tissue.