인간의 불임을 극복하고 치료하기 위한 체외수정 및 배아이식술(in vitro fertilization and embryo transfer: IVF-ET)의 성공적인 임신과 출산은 1978년 영국에서 세계 최초로 성공 사례를 보고하였으며, 국내에서는 1986년에 처음으로 보고되었다. 최근에 발표된 보건복지부 통계자료에 의하면 2010년에는 130여 개의 배아생성의료기관에서 연간 42,000 건 이상의 IVF-ET가 시행되었다고 한다. 이러한 시술에 사용되

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

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.

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

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.

Recent genomic evidences from unfractionated embryonic stem cell (ESC) cultures have demonstrated high levels of concomitant activating (H3K4me3) and repressive (H3K27me3) histone methylations, termed “bivalent marks”, at lineage specific gene loci, demonstrating that all cells residing within the cultures are developmentally equipotent. However, this dogma has been challenged, indicating that ESC cultures are heterogeneous, with individual cells displaying dynamic metastability and failed to make a connection with the variations between cell lines, a broad spectrum of differentiation, continuous phenotypic oscillation, and the expression of lineage specific genes in undifferentiated state. Recently, functional in vitro assays via fractionation of ESC cultures based on comparable expression of some phenotypes (c‐KIT, A2B5, SSEA3, Nanog, Rex‐1, IGFR1, and Stella) revealed a plastic gradient of clonogenicity and lineage specification within ESC cultures reflected by the presence of bivalent marks, which are resolved down to activating “monovalent marks”. More interestingly, dynamic heterogeneity represents a conserved feature on both mouse ESCs and human ESCs as being essentially required for self‐renewal and, more importantly, differentiation. However, it is the most substantive obstacle to control and specify ESCs into desirable cell types. Mostly, differentiation from ESCs has been evaluated by measuring the responses of whole EB populations under the specific inducible conditions, making it difficult to identify, which cell populations are dominantly contributing to differentiated progeny from ESCs. Therefore, further identification of novel transcriptional and phenotypic markers may allow for the isolation and enrichment of more promising target cells for stem cell‐based clinical therapy.

The remarkable regenerative capacity of the adult liver provides a setting to test the functional consequences of grafting human cells generated from pluripotent stem cells. This presentation describes a procedure to differentiate hepatocytes from human embryonic stem and induced pluripotent stem cells using only defined factors. Two cell populations generated in vitro were grafted into the spleen of mice treated with the hepato-toxin carbon tetra-chloride. The population containing few hepatocytes generated few surviving cells that produced low levels of albumin and did not support regeneration of the host liver. The cells enriched in donor hepatocytes efficiently engrafted around the branches of the portal vein, expressed hepatic features for at least 5 weeks. These cells also contributed to the endogenous tissue regeneration and function of the host liver. These results show that the controlled differentiation of hepatocytes from human pluripotent cells provides new approaches to define the mechanisms of tissue regeneration and restore liver function.

Glycogen synthase kinase 3 beta (GSK3β)의 선택적 저해제인 Kenpaullone가 B16 멜라노마 및 사람의 멜라노 사이트에 미치는 멜라닌 합성능을 조사하였다. Kepaullone은 B16 멜라노마 및 사람의 멜라노사이트에 대하여 세포증식에는 영향이 없는 범위 내에서 농도 의존적으로 멜라닌 합성을 촉진시켰다. B16 멜라노마 세포에 Kenpaullone을 첨가 48 h 후 tyrosinase 활성이 증가하였으며, 농도별 처리에 대하여 tyrosinase 단백질의 발현 및 tyrosinase mRNA양이 증가함을 관찰하였다. 결론적으로 Kenpaullone는 B16 멜라노마 세포에서 tyrosinase 효소의 발현을 증가시켜 멜라닌 합성을 촉진하는 것으로 판단되어진다. 따라서 GSK3β 저해제가 멜라닌 합성을 촉진시키는 결과는 백반증과 같은 저색소관련 질병의 치료제 개발의 가능성을 갖고 있는 소재로서 응용가능하리라고 판단되어진다.

참나물은 광범위하게 이용되는 식용산채임에도 불구하고 그 연구는 전 세계적으로 미미한 상태이다. 본 연구에서는 최근 고급 식용 산채로서 각광받고 있는 참나물의 메탄올 추출물을 조제하고, 이로부터 n-hexane, methylene chloride, ethylacetate 및 butanol을 이용하여 순차적 유기 용매 분획물과 물 잔류물을 조제하여 각각의 항산화, 항균 및 대장암세포 생육억제 활성을 평가하였다. 참나물 메탄올 추출물의 71.51%는

Xanthine oxidase(XO)/√hypoxanthine(HX)에 대한 저먼캐모마일(Matricaria chamomile L., German chamomile)추출물에 대한 영향을 인체피부멜라닌세포(SK-MEL-3)를 배양한 후 세포부착율을 비롯한 DPPH-자유기 소거능(DPPH-radical scav-enging activity), 티로시나제의 활성, 총멜라닌량의 정량 및 광학현미경적 관찰에 의하여 조사하였다. 본 연구에서 XO/HX는 배양 SK-MEL-3세포에 처리한 농도에 비례하여 유의한 세포부착율의 감소를 나타낸 반면, 저먼캐모마일 추출물은 XO/HX에 의하여 감소된 세포부착율의 유의한 증가와 자유기 소거능을 나타냄으로서 XO/HX의 산화적 손상에 대한 방어효과를 나타냈다. 한편, 배양 SK-MEL-3세포에서 XO/HX에 대한 저먼캐모마일 추출의 멜라닌합성능을 조사하기 위하여 티로시나제의 활성 및 총멜라닌량을 측정하였다. 그 결과 80μg·mL-1, 또는 160μg·mL-1의 저먼캐모마일 추출물의 전 처리에서 XO/HX의 처리군에 비하여 유의한 티로시나제활성 감소와 총멜라닌량의 감소를 나타냈다. 한편, 광학현미경적 관찰에 있어서 저먼캐모마일 추출물을 처리한 실험군은 XO/HX만을 처리한 실험군에 비하여 세포수와 세포돌기가 더 많이 증가한 것으로 관찰되었다. 이상의 결과로 부터 XO/HX는 배양 SK-MEL-3세포에 독성효과를 나타냈으며, 저먼캐모마일 추출물은 XO/HX의 세포독성에 대한 방어효과 및 항멜라닌화를 나타냈다.

BCL-2 family 단백질들은 세포사멸 신호전달 체계에서 중추적인 역할을 수행하는 것으로 알려져 있으며, BCL2L10 단백질은 그 중 하나로 세포의 사멸과 생존을 조절하는 것으로 알려져 있다. 특이하게도 BCL2L10 단백질은 세포 또는 조직 특이적으로 서로 상반되는 친 세포사멸 또는 항 세포사멸 효과가 각각 보고되어 있다. 현재까지 난소세포에서의 BCL2L10의 발현 여부 및 기능은 알려져 있지 않다. 따라서 본 연구에서 인간 난소 과립세포주인

본 실험은 transforming growth factor-(TGF-)이 첨가된 chondrogenic induction medium(CIM)을 이용하여 인간지방조직에서 유래된 중간엽 줄기세포(human adipose tissue-derived mesenchymal stem cells, ATMSCs)의 연골형성능과 gelatin-chondroitin-glucosamine scaffold(GCG-scaffold)에 접종시킨 ATMSCs의 연골형성능을 알아

본 실험은 bioceramic을 첨가하여 만든 다공성 poly D,L-lactic-co-glycolic acid(PLGA)-scaffold가 인간 지방조직에서 유래된 중간엽 줄기세포(human adipose tissue derived mesenchymal stem cells, ATMSCs)의 골 형성과정에 효과적인지를 알아보고자 수행하였다. ATMSCs를 well plate에 접종하여 골형성 유도(osteogenic induction, OI) 배양액으로

본 논문에서는 동작인식 위한 정확한 배경 분할 및 특징점 추출 방법을 제안한다. 배경 분할 과정에서는 먼저, HSV 입력 이미지를 RGB 색상 공간에서 HSV 색상 공간으로 변환한 뒤, H와 S 값에 대한 두 개의 임계치를 사용하여 살색 영역을 분할, 프레임간의 차영상을 이용하여 움직임이 있는 영역을 추출한다. 차영상에서 발생하는 잔상 영역을 제거하기 위하여 헤시안 어파인 영역 검출기를 적용하고, 잡음이 제거된 차 영상과 살색 영역의 이진화 영상을 이용하여 사람의 동작이 나타나는 영역을 분할한다. 특징점 추출 과정은 전체 영상을 블록 단위로 나눠서 각 블록 안에서 분할된 영상에 포함되는 픽셀들의 중점을 구하여 특징점을 추출한다. 실험결과 복잡한 환경에서도 정확한 배경 분할과 사용자 동작을 대표하는 특징점 추출이 약 12 fps로 가능함을 알 수 있었다.

Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self renewal requires many factors such as Oct4, Sox2, FoxD3, and Nanog. NF- is a transcription factor involved in many biological activities. Expression and activity of NF- increase upon differentiation of ES cells. Reportedly, Nanog protein directly binds to NF- protein and inhibits its activity in ES cells. Here, we found a potential binding site of NF- in the human Nanog promoter and postulated that NF- protein may regulate expression of the Nanog gene. We used human embryonic carcinoma (EC) cells as a model system of ES cells and confirmed decrease of Nanog and increase of NF- upon differentiation induced by retinoic acid. Although deletion analysis on the DNA fragment including NF- binding site suggested involvement of NF- in the negative regulation of the promoter, site-directed mutation of NF- binding site had no effect on the Nanog promoter activity. Furthermore, no direct association of NF- with the Nanog promoter was detected during differentiation. Therefore, we conclude that NF- protein may not be involved in transcriptional regulation of Nanog gene expression in EC cells and possibly in ES cells.

본 연구는 우리나라 유아교육에 대한 보편적인 입장을 담고 있는 국가고시 유치원 교육과정을 분석함으로써 시대적 요구에 맞춰 개정된 국가고시 유치원 교육과정에 나타난 유아 인권 관련 교육내용은 어떠한가를 알아보는데 그 목적이 있다. 연구 대상은 제1차 유치원 교육과정부터 현행 2007년 개정 유치원 교육과정까지이며, 분석 준거는 기본적 권리, 평등의 권리, 자유의 권리, 경제적 권리, 정치적 권리, 사회적 권리의 6개 범주와 범주별 하위 내용이다. 국가고시 유치원 교육과정에 나타난 유아 인권 관련 교육내용은 모든 국가고시 유치원 교육과정에서 나타났으며 제5차 교육과정 이후에 나타난 빈도가 큰 폭으로 증가하였으며 내용에 있어서도 큰 변화가 나타났다.

The human-following is one of the significant procedure in human-friendly navigation of mobile robots. There are many approaches of human-following technology. Many approaches have adopted various multiple sensors such as vision system and Laser Range Finder (LRF). In this paper, we propose detection and tracking approaches for human legs by the use of a single LRF. We extract four simple attributes of human legs. To define the boundary of extracted attributes mathematically, we used a Support Vector Data Description (SVDD) scheme. We establish an efficient leg-tracking scheme by exploiting a human walking model to achieve robust tracking under occlusions. The proposed approaches were successfully verified through various experiments.