Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

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.

MicroRNAs (miRNAs) function as a key regulator of diverse cellular functions. To find out novel miRNAs that promote the differentiation of mouse embryonic stem cells (mESCs), we compared the miRNAs expression profiles of mESCs under self-renewal vs. differentiation states. We noticed that miR-222 was highly expressed during the differentiation of mESCs. Quantitative RT-PCR analysis revealed that expression of miR-222 was up-regulated during the embryonic bodies formation and retinoic acid -dependent differentiation. When miR-222 was suppressed by antogomiR-222, the differentiation of mESCs was delayed compared to control. Self-renewal marker expression or cell proliferation was not affected but the expression of lineage specific marker was suppressed by the treatment of miR-222 inhibitor during the differentiation of mESCs. Taken together, these results suggest that miR-222 functions to promote the differentiation of mESCs by regulating expression of differentiation related genes.

후성유전학적 조절은 DNA 서열상의 변화 없이도 유전자의 기능을 변화시킬 수 있는 현상을 뜻한다. 염색체의 후성유전학적 상태는 히스톤 변형, DNA 변형 그리고 RNAi에 의한 유전자 침묵 등에 의해 조절된다. 본 총설에서는 배아줄기세포에서의 후성 유전학적 조절에 영향을 주는 요인으로서 히스톤(histone)의 메틸화에 초점을 맞추었다. 배아줄기세포에서 발현되는 유전자의 조절에는 두 가지 단백질 복합체가 관여한다. Polycomb repressive c

We previously reported that purified hepatocyte-like cells derived from human embryonic stem cell (hESC) promoted the liver tissue recovery not only by cell replacement, but also by delivering proteins (secretome) that enhance endogenous host liver regeneration. In this study, we investigated possible therapeutic effects of secretomes obtained from undifferentiated hESC and mesenchymal stem cell (hMSC), and explored the underlying mechanism in a mouse model of chronic liver injury. Mice pre-intoxicated with dimethylnitrosamine (DMN) were treated with single intraperitoneal injection of secretome or medium used to support the growth of hESCs or hMSCs. Both hESC- and MSC-secretomes induced robust host liver regeneration, as determined by biochemical and histological analyses. The expression of MMP2 was significantly increased in the liver that received hESC- or hMSC-secretome, compared to control groups. In contrast, expression of α-SMA, a hallmark of activated hepatic stellate cells, was profoundly decreased after administration of both secretomes. These results suggest that hESCs and MSCs may release soluble factors that support the host tissue regeneration of chronically injured liver.

Human embryonic stem cells (hESCs) have the potential for use in regenerative medicine and in the field of basic research. Therefore, effective cryopreservation and storage of hESCs are important for preservation of newly established cell line for various purposes. Despite poor survival and slow recovery after thawing, the conventional slow freezing method is most commonly used for cryopreservation of hESCs due to its simplicity and ease of use for freezing a large number of hESCs appropriate to clinical applications. Here we controlled the clump size (Group Ⅰ; 400～450 ㎛, Group Ⅱ; 800～900 ㎛, and Group Ⅲ; 1500～1700 ㎛) of hESCs at 5 days after plating using a glass pipette during cryopreservation in order to obtain a larger amount of hESCs after thawing. Attachment rates differed significantly (P<0.05) in each of the three groups and the average of attachment rate of GroupⅡ was highest in SNUhES4 and H1. In particular, the attachment rate of Group Ⅱ in SNUhES3 showed a significant improvement with ROCK inhibitor Y-27632. These results indicate that clump size and cell-cell adhesions of GroupⅡ are appropriate for cryopreservation compared to the Group Ⅰ and Group Ⅲ. This method increased cell viability and reduced the recovery time leading to various experiments, and therefore has an advantage for use with hESCs like newly established in particular. We demonstrated that use of this effective cryopreservation method with control of the clump size of hESCs can effectively improve the attachment rate and survival of post-thaw hESCs with and without Y-27632.

The development of humanized culture system of human embryonic stem cells (hESCs) hold promise for therapeutic applications. However, conventional culture system contain animal-derived components such as fetal bovine serum and mouse embryonic fibroblasts that bear a risk of transmitting non-human pathogens and incorporation of non-human immunogenic molecules to hESCs. In this study, we developed an efficient xeno-free hESCs culture system using humanized materials, the CELLstartTM, human foreskin feeder and xeno-free medium containing knockOutTM SR XenoFree (XF-medium) without animal-derived material. The hESCs were gradually adapted to the XF-medium; 25:75, 50:50, 75:25 and 100:0. Two karyotypically normal hESC lines, SNUhES4 and H1, were used for the experiments of xeno-free culture condition. The attachment rates at xeno-free culture system were 52.6±12.4%, 67.0±16.6%, 59.0±13.9%, 28.3±2.9% in SNUhES4, 79.3±5.4%, 53.8±20.9%, 69.4 ±6.4%, 59.8±12.6% in H1 and the spontaneous differentiation rates were 42.2±12.7%, 31.4±2.9%, 40.8±14.5%, 55.2±35.5% in SNUhES4, 35.6±8.5%, 36.4±13.5%, 48.4±7.8%, 80.1±6.0% in H1 in the first four passage. Although the attachment rates were low and the spontaneous differentiation rates were high compared to that of conventional system in the early passages using this humanized culture condition, hESCs in this culture condition were found to maintain hESC characterizations; morphology, expression of cell surface markers and stable karyotype. Our results indicate that simplified compositions of humanized culture system can be applicable to the further optimization for a xeno-free culture of hESCs without the loss of pluripotency and contamination from xenogenic sources.

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-, GFR-, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/ BMP4 or 0.01 RA. In the BMP4-treated group, GFR- and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR- and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-, GFR-, CD90/Thy1 and immunocytochemistry using GFR- antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

DNA 메틸화 (DNA methylation)는 유전자의 발현을 조절하는 대표적인 후생학적 조절기작 (epigenetic regulation) 중에 하나이다. DNA 메틸화 양상은 생식세포 형성과정 및 배 발생단계에서 탈메틸화 (demethylation)와 de novo 메틸화의 드라마틱한 변화가 일어난다. 또한 이러한 DNA 메틸화는 배아줄기세포 (embryonic stem cells, ESCs)에서 특징적인 양상을 보이는 것으로 알려져 있다. 본 연구에서는 생쥐 수정란 유래 배아줄기세포와 체세포핵이식 배아줄기세포 (nuclear transplanted ESCs)를 이용해서 대표적 각인유전자 (imprinting genes)로 알려진 Snrpn, Igf2r, H19 유전자들에 대한 메틸화 양상을 알아보고자 하였다. 연구 결과 H19 유전자에 대해서는 DNA 메틸화 양상은 수정란 유래 배아줄기세포와 체세포핵이식 배아줄기세포에서 비슷한 경향을 보였으나, Snrpn과 Igf2r의 경우에는 체세포핵이식 배아줄기세포에서 과메틸화 (hypermethylation) 경향을 보였다.

It has been reported that ganglioside GT1b is expressed during neuronal cell differentiation from undifferentiated mouse embryonic stem cells (mESCs), which suggests that ganglioside GT1b has a direct effect on neuronal cell differentiation. Therefore, this study was conducted to evaluate the effect of exogenous addition of ganglioside GT1b to an in vitro model of neuronal cell differentiation from undifferentiated mESCs. The results revealed that a significant increase in the expression of ganglioside GT1b occurred during neuronal differentiation of undifferentiated mESCs. Next, we evaluated the effect of retinoic acid (RA) on GT1b-treated undifferentiated mESCs, which was found to lead to increased neuronal differentiation. Taken together, the results of this study suggest that ganglioside GT1b plays a crucial role in neuronal differentiation of mESCs.

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

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

배아줄기세포를 이용한 치료법 개발을 위해서는 배아줄기세포의 자가재생산 및 분화과정을 조절하는 분자적 기전을 이해하는 것이 매우 중요하다. 지질합성경로(Mevalonate pathway)에 작용하는 HMG-CoA 환원효소(Hydroxymethylglutaryl-coenzyme A reductase)의 억제제인 스타틴은 콜레스테롤 저하제로 잘 알려져 있으며, 콜레스테롤 이외에 단백질 isoprenylation의 기질로 작용하는 아이소프레노이드(Isopren

배아 줄기세포는 세포 치료 목적을 위한 재료로써 매우 큰 잠재력을 가지고 있으며, 이러한 잠재력의 실현을 위해서 세포의 운명에 결정적인 역할을 하는 요소들을 확인하고 특정 세포의 대량 생산을 위한 방법을 개발하여야 한다. 조혈과정은 폭넓게 연구되어 왔으며, 배아 줄기세포로부터 조혈세포의 분화는 lineage commitment에 관한 연구에 좋은 모델이 된다. 본 연구에서는, 두 종류의 마우스 배아 줄기세포주 TC-1과 B6-1를 이용하여 그 특성과 조

배반포 단계의 난자에서 차이 나게 발현하는 유전자의 발굴을 통해 초기 동물 발생과 분화에 관한 기전을 알 수 있다. 본 연구에서는 새로운 차별발현 역전사효소중합법, 이름하여 에닐링 콘트롤 프라이머(ACP) 방법에 의해 생쥐 배반포에서 차이 나게 발현하는 유전자를 줄기세포와 비교하여 발굴하였다. 총 100개의 ACP를 사용하여 26개의 유전자 단편을 확인하였고, BLAST 탐색에 의해 유전자 정보 은행(GeneBank/EMBL)에 저장된 유전자와 동일하다