GCNF regulates OCT4 expression through its interactions with nuclear receptor binding elements in NCCIT cells.

We demonstrate that OCT4 expression is regulated by germ cell nuclear factor (GCNF) via its interactions with three nuclear receptor (NR) binding sites within OCT4 promoter conserved regions (CRs) in human embryonic carcinoma (EC) NCCIT cells. OCT4 expression is gradually reduced during the retinoic acid-induced differentiation, while GCNF temporarily increased after 2 days and then significantly decreased. In addition, OCT4 expression is significantly reduced by overexpression of exogenous GCNF, but increased by GCNF shRNA-mediated knockdown. The transcriptional activity of OCT4 is significantly inhibited by dose-dependent overexpression of GCNF. While mutants at each of the NR binding sites retain the repressive effects of GCNF on OCT4 promoter activity, the repressive effect was completely eliminated in the reporter construct with all binding sites mutated even in the presence of GCNF. Furthermore, the transcriptional activity of native minimal promoter (CR1-Luc) containing the first NR binding site was significantly reduced by GCNF overexpression, while the mutant retained basal activity to some extent. Next, an exogenous minimal ti promoter-inserted CR2 reporter construct containing the second and third NR binding sites (CR2-ti-Luc) was co-transfected with GCNF expression vector. The transcriptional activity of CR2-ti-Luc was significantly decreased by GCNF overexpression, while mutation of both binding sites retained the transcriptional activity of the reporter construct. Binding assays confirmed the direct interaction of GCNF with all three NR binding sites cooperatively. Taken together, GCNF acts as a transcriptional repressor in the regulation of OCT4 gene expression through cooperative interaction with three NR binding elements in pluripotent NCCIT cells.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells.

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (-285 to -138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (-196AGGATT-191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.

Growth and Differentiation Factor 3 Is Transcriptionally Regulated by OCT4 in Human Embryonic Carcinoma Cells.

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.

Transcriptional regulation of OCT4 by the ETS transcription factor ESE-1 in NCCIT human embryonic carcinoma cells.

The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.

Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation.

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

Identification of a putative nuclear export signal motif in human NANOG homeobox domain.

NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ((125)MQELSNILNL(134)) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-ΔNLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

Regulation of OCT4 gene expression by liver receptor homolog-1 in human embryonic carcinoma cells.

We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.

Epithelium-specific ETS transcription factor-1 regulates NANOG expression and inhibits NANOG-induced proliferation of human embryonic carcinoma cells.

The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.

Fli-1 promotes proliferation and upregulates NANOGP8 expression in T-lymphocyte leukemia cells.

Friend leukemia integration 1 (Fli-1) is a member of the E26 transformation-specific (ETS) transcription factor family. Fli-1 regulates normal hematopoiesis and vasculogenesis, and its aberrant expression underlies virus-induced leukemias and various types of human cancers. NANOGP8, a retro-pseudogene of stem cell mediator NANOG, is expressed predominantly in cancer cells and plays a role in tumorigenesis. In this study, we demonstrate that Fli-1 expression enhances human acute T-cell leukemia Jurkat cell proliferation and that Fli-1 acts as a transcriptional activator of NANOGP8 expression in these cells. NANOGP8 and Fli-1 are highly expressed in Jurkat cells, whereas NANOG was undetectable at both the RNA and protein levels. Moreover, the expression of endogenous NANOGP8 was significantly influenced by gain of function and loss of function of Fli-1. Promoter-reporter assays showed that NANOGP8 transcription was significantly upregulated by dose-dependent Fli-1 overexpression. A series of deletion mutagenesis of NANOGP8 promoter sequence revealed that NANOGP8 promoter activity was tightly regulated and found the minimal promoter region sufficient to activate NANOGP8 transcription mediated by Fli-1. Moreover, site-directed mutagenesis of the putative binding site abolished both NANOGP8 full-length and minimal promoter activities. Binding assays revealed that Fli-1 directly interacts with the potent binding site in NANOG promoter region. Taken together, our data demonstrate that Fli-1 is a novel upstream transcriptional activator of NANOGP8 and provide the molecular details of Fli-1-mediated NANOGP8 gene expression. Ultimately, these findings may contribute to understanding the expanded regulatory mechanisms of oncogenic NANOGP8 and ETS family transcription factors in leukemogenesis.

Two potent transactivation domains in the C-terminal region of human NANOG mediate transcriptional activation in human embryonic carcinoma cells.

The core embryonic stem cell transcription factors Oct4, Sox2, and Nanog are expressed in germ cell tumors (GCTs) and have been proposed to play a regulatory role in tumorigenesis. However, little is known about the mechanism of regulation of tumorigenesis by the complicated network of these proteins. Nanog is a novel homeobox-containing transcription factor that is expressed in pluripotent cells as well as GCTs. To understand the molecular and functional role of human NANOG (hNANOG) in germ cells, mutagenesis of the C-terminal domain (CD) of hNANOG and transient transfection assays in NCCIT human embryonic carcinoma cells were carried out to identify critical transactivation motifs. We divided the CD into three putative functional subdomains, CD1, tryptophan-repeat (WR) subdomain, and CD2. WR subdomain and CD2 independently contained transcriptional potential and, in combination, had a synergistic effect on transcriptional activity, while CD1 was transcriptionally inactive. The glutamine (Q) motif in WR subdomain, and multiple acidic residues in CD2 were required for maximal and synergistic transcriptional activation by the hNANOG CD. The results of the current study contribute to a better understanding of the complicated molecular machinery of stem cell transcription factors and their role in unregulated proliferation in germ cell tumorigenesis.

Implication of human OCT4 transactivation domains for self-regulatory transcription.

OCT4 plays a crucial role in pluripotency and self-renewal of embryonic stem cells. OCT4 is also expressed in testicular germ cell tumors (GCTs), suggesting the important function of OCT4 as an oncogenic factor in GCTs. To understand the molecular mechanism of human OCT4 (hOCT4) in tumorigenesis as well as stemness, we identified hOCT4 transactivation domains in human embryonic carcinoma cells. Context analyses of heterologous GAL4 and natural hOCT4 revealed that each N-terminal domain or C-terminal domain independently stimulated transcriptional activity, and that both domains are required for synergistic transactivation by deletion mapping analysis. Dose-dependent overexpression of exogenous hOCT4 significantly decreased the transcriptional activity of the hOCT4 promoter. This inhibition was reversed by the removal of one or both domains. These results suggest that the inhibitory effect of hOCT4 is mediated by transactivation domains, and that the self-regulation of hOCT4 may be mediated via a negative feedback loop in pluripotent cells.

The expression of the embryonic gene Cripto-1 is regulated by OCT4 in human embryonal carcinoma NCCIT cells.

Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.

Fermented Moringa oleifera Decreases Hepatic Adiposity and Ameliorates Glucose Intolerance in High-Fat Diet-Induced Obese Mice.

Metabolic diseases, such as glucose intolerance and nonalcoholic fatty-liver disease (NAFLD), are primary risk factors for life-threatening conditions such as diabetes, heart attack, stroke, and hepatic cancer. Extracts from the tropical tree Moringa oleifera show antidiabetic, antioxidant, anti-inflammatory, and anticancer effects. Fermentation can further improve the safety and nutritional value of certain foods. We investigated the efficacy of fermented M. oleifera extract (FM) against high-fat diet (HFD)-induced glucose intolerance and hepatic lipid accumulation and investigated the underlying mechanisms by analyzing expression of proteins and genes involved in glucose and lipid regulation. C57BL/6 mice were fed with normal chow diet (ND) or HFD supplemented with distilled water (DW, control), nonfermented M. oleifera extract (NFM), or FM for 10 weeks. Although body weights were similar among HFD-fed treatment groups, liver weight was decreased, and glucose tolerance test (GTT) results improved in the FM group compared with DW and NFM groups. Hepatic lipid accumulation was also lower in the FM group, and expressions of genes involved in liver lipid metabolism were upregulated. In addition, HFD-induced endoplasmic reticulum (ER) stress, oxidative stress, and lipotoxicity in quadriceps muscles were decreased by FM. Finally, proinflammatory cytokine mRNA expression was decreased by FM in the liver, epididymal adipose tissue, and quadriceps of HFD-fed mice. FMs may decrease glucose intolerance and NAFLD under HFD-induced obesity by decreasing ER stress, oxidative stress, and inflammation.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor.

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment.

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

Identification of a putative transactivation domain in human Nanog.

Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.

Efficient gene delivery in differentiated human embryonic stem cells.

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Transcriptional activation of OCT4 by the ETS transcription factor PEA3 in NCCIT human embryonic carcinoma cells.

We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.

Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector.

Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.

Increase of chondrogenic potentials in adipose-derived stromal cells by co-delivery of type I and type II TGFß receptors encoding bicistronic vector system.

Stem cell therapy has been developing rapidly as a potential cure for repairing or regenerating the functions of diseased organs and tissues. Adipose-derived stromal cells (ASCs) are an attractive cell source for stem cell therapy because they can be isolated easily from fat tissue in significant numbers and exhibit multiple differentiation potential under appropriate in vitro culture conditions. However, ASCs derived from individual donors can show wide variations in differentiation potential. In addition, the regulatory mechanisms underlying stem cell differentiation remain unclear. Transforming growth factor ß (TGFß) is a well-known ASC chondrogenic differentiation factor that stimulates ASC signaling pathways by activating transmembrane type I and type II receptors. We hypothesized that the chondrogenic differentiation potential of ASCs is dependent upon the expression of TGFß receptors and could be improved by the co-delivery of type I (TGFßRI) and type II (TGFßRII) TGFß receptors. To prove this, heterogeneity within the chondrogenic potential of ASCs isolated from 10 donors was examined and their susceptibility to TGFß during the process of chondrogenic differentiation investigated. In addition, the results showed that co-delivery of the TGFßRI and TGFßRII genes increased the expression of TGFß receptor signaling in ASCs with low chondrogenic potential, resulting in increased chondrogenic differentiation. Monitoring and delivering TGFßRI and TGFßRII may, therefore, be a powerful tool for predicting the differentiation potential of stem cells and for enhancing their differentiation capacity prior to stem cell transplantation.