• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.135-141
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• 2010

Embryoid bodies (EBs) generated from human embryonic stem cells (hESCs) include spontaneously induced endodermal lineage cells (ELCs). Activin-A plays important roles in the endoderm differentiation of hESCs. Despite studies on the generation of ELCs from hESCs with treatment of Actvin-A, it was unclear for localization and pattern of ELCs by Activin-A during differentiation of hESCs. Accordingly in this study, we knew that Actvin-A increased the cystic EBs formation, including the highly enriched AFP (endoderm lineage specific marker)-expressing cells in the surface of cystic EBs. To induce the EBs formation from undifferentiated hESCs, cells were transferred onto petri-dish and cultured in suspension condition without bFGF removed hESC media (EB media) for 3 days. Next to investigate the effect of Activin-A, EBs were subsequently cultured in EB media supplement with 100 ng/ml Activin-A for 3 days. After 5~7 days of Activin-A treatment, cystic EBs began to appear which increased in numbers reaching ~60% of initially formed EBs over 5 days. Endoderm lineage marker, AFP were highly expressed and specifically localized at the surface region of cystic EBs comparison with normal EBs. We next attached the cystic EBs onto gelatin-coated plates and cultured for 5 days. In the results of real-time PCR and immunocytochemistry analysis, AFP-expressing cells migrated and localized at the outgrowth region of attached cystic EBs. To obtain the AFP-expressing cells of the outgrowth region, we manually isolated by using micro-dissection and cultured them. These cells strongly express AFP over 70% of isolated cells post re-plating. Here, we first showed an expression pattern of specifically localized ELCs by Activin-A during differentiation of hESCs. From this observation, we could highly purified ELCs from undifferentiated hESCs. Taken together, our system will provide a novel and efficient option to generate ELCs from hESCs.

• Molecules and Cells
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• v.37 no.6
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• pp.497-502
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• 2014

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.229-233
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• 2010

Pluripotency and self-renewal capacity of human embryonic stem cells (hESCs) are retained by hESCs related genes as OCT4, SOX2 and NANOG. These genes are shown high expression level in diverse cancer cells and have potential role in the carcinogenesis. On the contrary to this, several genes which are up-regulated in the differentiated hESCs are involved to suppress the carcinogenesis or proliferation of cells. We discovered several genes in immortalized lung fibroblast (WI-38 VA13) by suppression subtractive hybridization. Among them, we focused chromosome 6 open reading frame 62 (C6orf62) which is uncharacterized, mapped to 6p22.3 and generated to Hepatitis B virus X-transactivated proteins (HBVx-transactivated proteins, XTP). Aim of this study was to characterize C6orf62 through analyzing of expression pattern in various cell lines. Expression of C6orf62 was significantly upregulated in diverse normal cell lines than cancer cell lines. And C6orf62 was up-regulated in differentiated hESCs (endothelial cells, neural cells) compared to those of undifferentiated hESCs. Also, C6orf62 in WI-38 cells was highly up-regulated during G1/S transition of the cell cycle. Taken together, C6orf62 is shown expression pattern similar to differentiated hESCs-associated genes which down-regulated in cancer cells. Therefore, we assume that C6orf62 may participate to suppress the proliferation and to induce differentiation through regulating the cell cycle.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.1-6
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• 2010

Mesenchymal stem cells (MSCs) have the multipotent capacity and this potential can be applied for obtaining valuable cell types which can use for cell therapy on various regenerative diseases. However, insufficient availability of cellular source is the major problem in cell therapy field using adult stem cell sources. Recently, human embryonic stem cells (hESCs) have been highlighted to overcome a limitation of adult cellular sources because they retain unlimited proliferation capacity and pluripotency. To use of hESCs in cell therapy, above all, animal pathogen free culture system and purification of a specific target cell population to avoid teratoma formation are required. In this study, we describe the differentiation of a mesenchymal stem cell-like cells population from feeder-free cultured hESCs(hESC-MSCs) using direct induction system. hESC-MSCs revealed characteristics similar to MSCs derived from bone marrow, and undifferentiated cell markers were extremely low in hESC-MSCs in RT-PCR, immunostaining and FACS analyses. Thus, this study proffer a basis of effective generation of specialized human mesenchymal stem cell types which can use for further clinical applications, from xenofree cultured hESCs using direct induction system.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4295-4300
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• 2012

Objective: To investigate the therapeutic potential of human embryonic stem cells (hESCs) as a vaccine to induce an immune response and provide antitumor protection in a rat model. Methods: Cross-reactivity of antigens between hESCs and tumour cells was screened by immunohistochemistry. Fischer 344 rats were divided into 7 groups, with 6 rats in each, immunized with: Group 1, hESC; Group 2, pre-inactivated mitotic NuTu-19; Group 3 PBS; Group 4, hESC; Group 5, pre-inactivated mitotic NuTu-19; Group 6, PBS; Group 7, hESC only. At 1 (Groups 1-3) or 4 weeks (Groups 4-6) after the last vaccination, each rat was challenged intraperitoneally with NuTu-19. Tumor growth and animal survival were closely monitored. Rats immunized with H9 and NuTu-19 were tested by Western blot analysis of rat orbital venous blood for cytokines produced by Th1 and Th2 cells. Results: hESCs presented tumour antigens, markers, and genes related to tumour growth, metastasis, and signal pathway interactions. The vaccine administered to rats in Group 1 led to significant antitumor responses and enhanced tumor rejection in rats with intraperitoneal inoculation of NuTu-19 cells compared to control groups. In contrast, rats in Group 4 did not display any elevation of antitumour responses. Western blot analysis found cross-reactivity among antibodies generated between H9 and NuTu-19. However, the cytokines did not show significant differences, and no side effects were detected. Conclusion: hESC-based vaccination is a promising modality for immunotherapy of ovarian cancer.

• Molecules and Cells
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• v.23 no.1
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• pp.49-56
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• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.9-15
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• 2011

The functional cardiovascular system is comprised of distinct mesoderm-derived lineages including endothelial cells, vascular smooth muscle cells and other mesenchymal cells. Recent studies in the human embryonic stem cell differentiation model have provided evidence indicating that these cell lineages are developed from the common progenitors such as hemangioblasts and cardiovascular progenitor cells. Also, the studies have suggested that these progenitors have a common primordial progenitor, which expresses KDR (human Flk-1, also known as VEGFR2, CD309). We demonstrate here that sustained activation of BMP4 (bone morphogenetic protein 4) in hESC line, CHA15 hESC results in $KDR^+$ mesoderm specific differentiation. To determine whether the $KDR^+$ population derived from hESCs enhances potential to differentiate along multipotential mesodermal lineages than undifferentiated hESCs, we analyzed the development of the mesodermal cell types in human embryonic stem cell differentiation cultures. In embryoid body (EB) differentiation culture conditions, we identified an increased expression of $KDR^+$ population from BMP4-stimulated hESC-derived EBs. After induction with additional growth factors, the $KDR^+$ population sorted from hESCs-derived EBs displays mesenchymal, endothelial and vascular smooth muscle potential in matrix-coated monolayer culture systems. The populations plated in monolayer cultures expressed increased levels of related markers and exhibit a stable/homologous phenotype in culture terms. In conclusion, we demonstrate that the $KDR^+$ population is stably isolated from CHA15 hESC-derived EBs using BMP4 and growth factors, and sorted $KDR^+$ population can be utilized to generate multipotential mesodermal progenitors in vitro, which can be further differentiated into cardiovascular specific cells.

• Microbiology and Biotechnology Letters
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• v.39 no.4
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• pp.364-373
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• 2011

Human embryonic stem cells have been highlighted as a valuable cellular source in the regenerative medicine field, due to their pluripotency. However, there is the challenge of the establishment of specific functional cell type forms of undifferentiated human embryonic stem cells (hESC). To establish and purify functional cell types from hESCs, we differentiated undifferentiated hESCs into vascular lineage cells and sorted the specific cell population from the whole cell population, depending on their cell volume, and compared them with the non-sorted cell population. We observed that about 10% of the PECAM positive population existed in the VEGF induced differentiating human embryoid body (hEB), and differentiated hEBs were made into single cells for cell transplantation. After making single cells, we performed cell sorting using a fluorescence-activated cell sorter (FACs), according to their cell volume on the basis of FSC region gating, and compared their therapeutic capacity with the non-sorted cell population through cell transplantation into hindlimb ischemic disease model mice. 4 Weeks after cell transplantation, the recovery rate of blood perfusion reached 54% and 17% in the FSC regions of sorted cells- and non-sorted cells, respectively. This result suggests that derivation of a functional cell population from hESCs can be performed through cell sorting on the basis of cell volume after preliminary differentiation induction. This approach may then greatly contribute to overcoming the limitations of marker sorting.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.283-292
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• 2009

Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.