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.

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.

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.

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 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.

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.

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.

Regulation of human growth and differentiation factor 3 gene expression by NANOG in human embryonic carcinoma NCCIT cells.

We investigated transactivation by NANOG in regulating growth and differentiation factor 3 (GDF3) expression in NCCIT cells. GDF3 expression was affected by shRNA-mediated downregulation and by exogenous overexpression of NANOG specifically, as well as by retinoic acid-mediated differentiation. GDF3 transcription was activated by NANOG, and the upstream region (-183 to -1) was sufficient to induce minimal transcriptional activity. Moreover, NANOG binds to the GDF3 minimal promoter in vivo and the transcriptional activity is mediated by NANOG transactivation domain. This study provides the first evidence that NANOG is a transcriptional activator of the expression of the oncogenic growth factor GDF3 in embryonic carcinoma cells.

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.

Exogenous Nurr1 gene expression in electrically-stimulated human MSCs and the induction of neurogenesis.

In this study, synergistic effects of electrical stimulation and exogenous Nurr1 gene expression were examined to induce the differentiation of human mesenchymal stem cells (hMSCs) into nerve cells in in vitro culture system. A two-step procedure was designed to evaluate the effects of electrical stimulus and exogenous gene delivery for inducing neurogenesis. First, an electrical stimulation device was designed using gold nanoparticles adsorbed to the surface of a cover glass. Gold nanoparticles, as an electrical conductor for stem cells, are well-defined particles adsorbed to a polyethyleneimine (PEI)-coated cover glass. The nanoparticle morphology was examined by scanning electron microscope (SEM). Second, a plasmid carrying Nurr1 cDNA was complexed with biodegradable poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles to support neurogenesis. To evaluate the neuronal differentiation of stem cells mediated by the treatment with either electrical stimulation and exogenous Nurr1 gene delivery, or both, the expression of neuron-specific genes and proteins was examined by RT-PCR and Western blotting. Cells transfected with exogenous Nurr1 genes plus electrical stimulation (250 mV for 1000 s) showed the greatest level of neurite outgrowth with a mean neurite length of 150 µm. Neurite length in cells treated with only one stimulus was not significant, approximately 10-20 µm. These results indicate that electrical stimulation and exogenous Nurr1 gene expression together may be adequate to induce nerve regeneration using stem cells.

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.

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.

Chondrogenesis of mesenchymal stem cells and dedifferentiated chondrocytes by transfection with SOX Trio genes.

In this study, bone marrow-derived mesenchymal stem cells (MSCs), adipose-derived mesenchymal stem cells (ASCs) and dedifferentiated chondrocytes were transfected with SOX5, 6, and 9 genes (SOX Trio) and grown under pellet culture conditions (encapsulated in a fibrin hydrogel) to evaluate the chondrogenic potential in vitro and in vivo. RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays were performed to determine the chondrogenic potential of the stem cells and dedifferentiated chondrocytes. Chondrogenic genes and proteins were more highly expressed in SOX Trio-expressing cells than in untransfected cells. In addition, not only specific genes and proteins, but cartilage-forming tissues were observed in nude mice transplanted with fibrin hydrogel encapsulated SOX Trio-expressing MSCs, ASCs, and dedifferentiated chondrocytes. Both in vitro and in vivo analyses revealed that fibrin hydrogel encapsulated cultured or transplanted cells transfected with the SOX Trio successfully differentiated into mature chondrocytes and could be used for the reconstruction of hyaline articular cartilage.

C/EBP-α and C/EBP-ß-mediated adipogenesis of human mesenchymal stem cells (hMSCs) using PLGA nanoparticles complexed with poly(ethyleneimmine).

In this study, to drive efficient adipogenic differentiation, the adipogenic transcription factors C/EBP-α and C/EBP-ß fused to green fluorescent protein (GFP) or red fluorescent protein (RFP) were complexed with poly-ethyleneimine (PEI) coupled with biodegradable PLGA nanospheres and delivered to human mesenchymal stem cell (hMSC). FACS analysis revealed that the transfection efficiency of C/EBP-α, C/EBP-ß, or both genes complexed with PEI-coated PLGA nanospheres was 12.59%, 21.74%, and 28.96% of hMSCs. Expression and localization of C/EBP-α and C/EBP-ß were confirmed by Western blotting and confocal laser microscopy. Overexpression of exogenous C/EBP-α and C/EBP-ß significantly elevated adipogenic differentiation processes as indicated by RT-PCR, real-time PCR, Western blotting, histology, and immunofluorescence microscopy. During adipogenesis, PEI-coupled PLGA nanospheres complexed with C/EBP-α and C/EBP-ß greatly increased the adipogenic capability of in vitro cultured cells, as well of in vivo transplanted cells. The expression of genes and proteins specific to adipogenic differentiation in hMSCs was significantly elevated compared to the controls.

Chondrogenesis of human mesenchymal stem cells mediated by the combination of SOX trio SOX5, 6, and 9 genes complexed with PEI-modified PLGA nanoparticles.

Target gene transfection for desired cell differentiation has recently become a major issue in stem cell therapy. For the safe and stable delivery of genes into human mesenchymal stem cells (hMSCs), we employed a non-viral gene carrier system such as polycataionic polymer, poly(ethyleneimine) (PEI), polyplexed with a combination of SOX5, 6, and 9 fused to green fluorescence protein (GFP), yellow fluorescence protein (YFP), or red fluorescence protein (RFP) coated onto PLGA nanoparticles. The transfection efficiency of PEI-modified PLGA nanoparticle gene carriers was then evaluated to examine the potential for chondrogenic differentiation by carrying the exogenous SOX trio (SOX5, 6, and 9) in hMSCs. Additionally, use of PEI-modified PLGA nanoparticle gene carriers was evaluated to investigate the potential for transfection efficiency to increase the potential ability of chondrogenesis when the trio genes (SOX5, 6, and 9) polyplexed with PEI were delivered into hMSCs. SOX trio complexed with PEI-modified PLGA nanoparticles led to a dramatic increase in the chondrogenesis of hMSCs in in vitro culture systems. For the PEI/GFP and PEI/SOX5, 6, and 9 genes complexed with PLGA nanoparticles, the expressions of GFP as reporter genes and SOX9 genes with PLGA nanoparticles showed 80% and 83% of gene transfection ratios into hMSCs two days after transfection, respectively.

The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis.

In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 days after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence.