Hyun-Jin Do

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.

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.

CHARACTERIZATION OF PUTATIVE CIS-REGULATORY ELEMENTS THAT CONTROL THE TRANSCRIPTIONAL ACTIVITY OF THE HUMAN OCT4 PROMOTER

Octamer‐binding transcription factor‐4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5′ upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5′ upstream region and identified cis‐regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis‐regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis‐acting element located between CR1 and CR2. In addition, the sequence −380/−1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site‐directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the −380/−1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis‐regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.

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.

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.

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.

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.

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 α (hEF1α), human cytomegalo-virus, and chicken β-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 hEF1α 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.

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. J. Cell. Biochem. 106: 1079–1089, 2009.