Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Hepatocytes derived from human embryonic stem cells (hESCs) may be a useful source for the treatment of diseased or injured liver. However, a low survival rate of grafted hepatocytes and immune rejection are still major obstacles to be overcome. We previously showed that secreted proteins (secretome) from hESC-derived hepatocytes had a potential therapeutic power in the tissue repair of injured liver without cell transplantation. The purpose of the present study was to discover key protein(s) in the secretome of hESC-derived hepatocytes using proteomic analysis and to study the tissue repair mechanism which may be operated by the secretomes. Purified indocyanine green+ hepatocytes derived from hESCs displayed multiple hepatic features, including expression of hepatic genes, production of albumin, and glycogen accumulation. The nano-LC/ESI-QTOF-MS analysis identified 365 proteins in the secretome of hESC-derived hepatocytes and the protein functional network analysis was conducted using the MetaCore TM from GeneGO. In addition, 20 tissue regeneration-related transcription factors (TFs) were extrapolated through further proteomic analysis. After intraperitoneal injection, the secretome significantly promoted the liver regeneration in a mouse model of acute liver injury. Protein functional network analysis on the secretome-induced regenerating liver confirmed 20 transcription factors (TFs) which were identified in the ICGhigh cells. The upreguation of these tissue repair-related TFs were validated by qPCR and western blotting on the regenerating liver tissues. These results demonstrate that application of the secretome analysis in combination with the protein functional network mapping would provide a reliable tool to discover new tissue-regenerating proteins as well as to expand our knowledge of the mechanisms of tissue regeneration.

Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

Human embryonic stem cells (hESCs) are promising cell source because of their unique self-renewal and pluripotency. Although hESC-derived cardiac cells are currently generated worldwide, cryopreservation of these cells is still limited due to low rate of post-thaw survival. Cryopreservation of hESC-derived cardiac cells is critical in that their long-term storage can accelerate their use in regenerative medicine. However, to date, there are few reports on efficient cryopreservation and post-thaw survival of hESC-derived cardiac cells. In this study, we evaluated the effects of ginsenoside, which is known to improve survival of rat embryonic cardiomyocytes against myocardial ischemia injury in diabetic rats (Wu et al., 2011), on the survival of hESC-derived cardiac cells after thawing. We induced differentiation into cardiac cells using our previously reported method (Kim et al., 2011). Differentiated, pre-beating stage cardiac cells were cryopreserved using either mass cryopreservation or vitrification. To evaluate the effects of ginsenoside (Re, Rb), we compared three sets: pre- and post-thaw treatment, pre- or post-thaw treatment only. The survival of post-thaw cardiac cells were evaluated using Trypan-blue and Annexin V staining. In addition, the three groups were treated with ROCK inhibitor Y-27632, and compared with non-treatment groups. The effect of ginsenoside was significant in post-thaw treatment group, i.e, thawed cells expressed cardiac specific genes and showed specific functionality such as spontaneous beating. Taken together, we demonstrated favorable effects of ginsenoside on the survival of hESC-derived cardiac cells after cryopreservation and thawing. These results suggest a possible application of well-known cardioprotectant ginsenoside in cell-based tissue engineering using hESC-derived cardiac cells.

Estrogens are ubiquitous signaling molecules that influence nearly every cell type, and exert profound effects on embryonic development, and differentiation. Wnt pathway, which recruits β-catenin into nuclei, and activates The Wnt-dependent transcription factors, also plays an important role in embryonic development and stem cell maintenance, and differentiation. Accumulating evidences indicate that potential convergence between these two pathways in carcinoma cells. However, physiological roles of estrogens in development and differentiation of human embryonic stem cells (hESCs) are relatively unknown. Here, we demonstrated that estrogenic compounds 17α-ethinylestradiol (EE2) and genistein (GEN) significantly increased β-catenin expression in undifferentiated hESCs cultured in feeder-free media. Interestingly, GEN treatement induced an increased trend of mesendodermal gene expressions, and significantly inhibited ectodermal gene expressions (Nestin and Pax6) in embrioid body (EB). Expectantly, GEN increased epithelial-mesenchymal transition (EMT) related gene expression (Snail2, and Twist), whereas decreased E-cadherin on day 6 of EB development. Taken together, these suggest that estrogens may in part the powerful effects on normal hESC differentiation. Mechanistic studies of estrogen signaling continue to suggest novel drug targets for stem cells and will also improve screening methods of developmental toxicity.

만능성 인간 배아줄기세포로부터 확립된 신경줄기세포 또는 신경전구세포는 퇴행성 신경질환 세포치료제로 이용될 수 있는 다양한 종류의 신경세포로 분화 유도될 수 있다. 하지만, 인간 배아줄기세포로부터 신경세포를 생산하기 위한 기술은 아직 많은 장애를 가지고 있다. 인간 배아줄기세포 유래 신경전구세포에서 특징적으로 나타나는 신경관 유사로제트에 대한 이해는 인간 배아줄기세포 신경 분화의 효율을 높이는데 유용한 정보를 제공할 것으로 사료된다. 일반적으로 신경로제트

최근 제 1형 당뇨병을 치료하기 위하여 인슐린-분비성 세포를 이식하는 세포대체요법이 새로운 치료법으로서 주목받고 있다. 그럼에도 불구하고 췌장세포 이식술은 이식원의 절대적인 부족으로 인해 광범위한 시행이 이루어지지 못하고 있는 실정이다. 무한증식과 전분화능을 보유하는 배아줄기세포는 이식할 -세포의 부족을 해결할 수 있는 잠재적 세포공급원이 될 수 있을 것으로 기대된다. 본 종설에서는 인간배아줄기세포로부터 췌장 -세포로의 유도분화방법에 관한 최근 동향을