Heung-Mo Yang

Angiotensin I-converting enzyme inhibitor from Grifola frondosa

To investigate the hypotensive compounds from edible mushroom, extracts from 10 fruit bodies of mushrooms were screened for the inhibitory activity against angiotensin I converting enzyme (ACE). The most potent ACE inhibitory activity (58.7%) was detected in a cold water extract of Grifola frondosa, with an IC50 of 0.95 mg. The ACE inhibitory activities of the cold and hot water extracts increased as the extraction time increased, but decreased slightly 15 h and 90 min, respectively, after extraction. After the purification of ACE inhibitory peptides with acetone fractionation and column chromatography, we obtained an active fraction with an IC50 of 0.13 mg and a yield of 0.7%. The purified inhibitor showed competitive inhibition on ACE and this peptide maintained inhibitory activity even after digestion by intestinal proteases.

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.

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.

A therapeutic strategy for metastatic malignant fibrous histiocytoma through mesenchymal stromal cell-mediated TRAIL production.

Objective:To overcome the therapeutic limitations of malignant fibrous histiocytoma (MFH), we evaluated human adipose tissue-derived mesenchymal stromal cells (MSCs) that secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on metastatic MFH. Background:MFH is a highly malignant and metastatic type of sarcoma but surgical removal is the only effective method for treating MFH. MSCs are easily transduced to express a high level of transgene and can migrate toward cancer. For this reason, MSCs are a promising candidate for metastatic MFH therapies. Methods:In vitro sustainability of MSC-TRAIL against MFH-ino was analyzed by apoptosis assay. For preclinical study, anti-MFH effects of MSC-TRAILs were validated in murine models for local tumorigenesis and metastasis. Furthermore, a time-interval metastasis model of MFH was applied to confirm antimetastatic ability of MSC-TRAIL for preestablished metastatic MFH. Results:We found that MFH-ino is highly susceptible to recombinant TRAIL and MSC-TRAIL, which selectively induce apoptosis via caspase-8 activation in vitro. Moreover, not only MFH-ino but xenograft explants were also significantly inhibited by MSC-TRAIL in local tumorigenesis. In particular, the metastatic ability of MFH-ino was considerably reduced by MSC-TRAIL in metastasis murine model, particularly for preestablished metastatic MFH. Conclusions:These results suggest that MSC-TRAIL is sufficiently effective in inhibiting MFH-ino metastasis and the application using MSC-TRAIL could be extended to other sarcomas and recurrent metastatic cancers for cell-mediated cancer therapy.

Three-dimensional bio-printing of hepatic structures with direct-converted hepatocyte-like cells

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8-10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 mm) expressed Albumin, and ASGR1 and HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Therapeutic Efficacy of Human Embryonic Stem Cell–Derived Endothelial Cells in Humanized Mouse Models Harboring a Human Immune System

Objective—Allogeneic transplantation of human embryonic stem cell (hESC) derivatives has the potential to elicit the patient’s immune response and lead to graft rejection. Although hESCs and their derivatives have been shown to have advantageous immune properties in vitro, such observations could not be determined experimentally in vivo because of ethical and technical constraints. However, the generation of humanized mice (hu-mice) harboring a human immune system has provided a tool to perform in vivo immunologic studies of human cells and tissues. Using this model, we sought to examine the therapeutic potential of hESC-derived endothelial cells, human embryonic fibroblasts, and cord blood–derived endothelial progenitor cells in a human immune system environment. Approach and Results—All cell types transplanted in hu-mice showed significantly reduced cell survival during the first 14 days post-transplantation compared with that observed in immunodeficient mice. During this period, no observable therapeutic effects were detected in the hindlimb ischemic mouse models. After this point, the cells demonstrated improved survival and contributed to a long-term improvement in blood perfusion. All cell types showed reduced therapeutic efficacy in hu-mice compared with NOD scid IL2 receptor gamma chain knockout mice. Interestingly, the eventual improvement in blood flow caused by the hESC-derived endothelial cells in hu-mice was not much lower than that observed in NOD scid IL2 receptor gamma chain knockout mice. Conclusions—These findings suggest that hESC derivatives may be considered a good source for cell therapy and that hu-mice could be used as a preclinical in vivo animal model for the evaluation of therapeutic efficacy to predict the outcomes of human clinical trials.

Establishment of primary xenograft model from newly characterized patient extrauterine carcinosarcoma.

Background and Objective The aim of this study was to characterize primary cells from extrauterine carcinosarcoma (CS) and to establish a primary CS xenograft mouse model. Methods Primary cells were isolated from a patient with CS and cultured in vitro. Primary CS cells were verified for their ability to consecutively generate tumorigenesis in NOD/SCID mice. The properties of xenograft tumor and explants cells were investigated by immunohistochemistry, cytogenetic, and FACS analysis. Anticancer drug susceptibility of primary CS was analyzed using CCK-8. Results Primary CS cells greater than 27 passages in vitro showed an ability of a series of xenograft tumorigenesis in vivo having the same marker expression and cytogenetic character as that of original tumor. In addition, explants of xenograft tumors retained their original characteristics in the in vitro culture system. Finally, the analysis of the susceptibility to anticancer drug revealed that primary CS cells were susceptible to both doxorubicin and nilotinib, which are tyrosine kinase inhibitors. Conclusions The primary CS cells and the primary CS xenograft tumorigenesis introduce a new therapeutic model for targeting cancer and also explore a deeper understanding of generation of the tumor itself.