Hyun Hwa Cho

Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia.

Background/Aim: 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. Methods&Results: 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. Conclusion: 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.

Role of Toll-Like Receptors on Human Adipose-Derived Stromal Cells

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.

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue‐derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT‐PCR and Real‐time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP‐2, and Runx2 expression. The addition of noggin inhibited VPA‐induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.

NF-κB activation stimulates osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue by increasing TAZ expression†

Tumor necrosis factor‐alpha (TNF‐α) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF‐α inhibits the mineralization of osteoblasts, the effect of TNF‐α on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF‐α on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF‐κB activation on TNF‐α activity. TNF‐α treatment dose‐dependently increased osteogenic differentiation over the first 3 days of treatment. TNF‐α activated ERK and increased NF‐κB promoter activity. PDTC, an NF‐κB inhibitor, blocked the osteogenic differentiation induced by TNF‐α 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‐α‐ and TLR ligand‐induced osteogenic differentiation. TNF‐α 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‐α also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF‐α enhances osteogenic differentiation of hADSC via the activation of NF‐κB and a subsequent increase of TAZ expression. J. Cell. Physiol. 223: 168–177, 2010.

MicroRNA 21 regulates the proliferation of human adipose tissue-derived mesenchymal stem cells and high-fat diet-induced obesity alters microRNA 21 expression in white adipose tissues†

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. J. Cell. Physiol. 227: 183–193, 2012.

Expression of Telomerase Extends Longevity and Enhances Differentiation in Human Adipose Tissue-derived Stromal Cells

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.

Role of CD9 in proliferation and proangiogenic action of human adipose-derived mesenchymal stem cells

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-κB 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.

Role of Thioredoxin 1 and Thioredoxin 2 on Proliferation of Human Adipose Tissue-Derived Mesenchymal Stem Cells

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.

The Role of Chemokines in Proangiogenic Action Induced by Human Adipose Tissue-Derived Mesenchymal Stem Cells in the Murine Model of Hindlimb Ischemia

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.

Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells.

Toll-like receptors (TLRs) sense microorganism components and are critical host mediators of inflammation during infection. Recently, TLRs have been reported to be involved in cell proliferation and differentiation. We previously reported that TLR agonists might affect proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs). In this study, we sought to determine whether TLR signaling is dependent on MyD88 in hASCs. The hASCs were downregulated using LV-GFP-miR-MyD88, a lentiviral construct inserted siRNA against human MyD88 that significantly inhibited cell proliferation. MyD88 downregulation reduced NF-κB activation and enhancement of osteogenic differentiation induced by peptidoglycan (PGN) more significantly than that induced by lipopolysaccharide (LPS). Although LPS- and PGN-induced cytokine secretions were decreased greatly by MyD88 downregulation, IFN-gamma-induced protein-10 (IP10) and IFNβ expression were enhanced by LPS irrespective of the downregulation of MyD88. These results suggest that TLR signaling is mediated via MyD88-independent pathways as well as MyD88-dependent pathways in hASCs and that MyD88 contributes to the regulation of cell proliferation and differentiation in hASCs.