Profiling of Differentially Expressed Genes in Human Stem Cells by cDNA Microarray

김철근,이종주,정대영,전진선,허현석,강호철,신준호,조윤신,차경준,김찬길,도병록,김경숙,김현수 한국분자세포생물학회 2006 Molecules and cells Vol.21 No.3

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation, although a wide variety of adult stem cells as well as embryonic stem cells have been identified and stem cell plasticity has recently been reported. To identify genes implicated in the control of the stem cell state as well as the characteristics of each stem cell line, we analyzed the expression profiles of genes in human embryonic, hematopoietic (CD34+ and CD133+), and mesenchymal stem cells using cDNA microarrays, and identified genes that were differentially expressed in specific stem cell populations. In particular we were able to identify potential hESC signature-like genes that encode transcription factors (TFAP2C and MYCN), an RNA binding protein (IMP-3), and a functionally uncharacterized protein (MAGEA4). The overlapping sets of 22 up-regulated and 141 downregulated genes identified in this study of three human stem cell types may also provide insight into the developmental mechanisms common to all human stem cells. Furthermore, our comprehensive analyses of gene expression profiles in various adult stem cells may help to identify the genetic pathways involved in self-renewal as well as in multi-lineage specific differentiation.

Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

Shin Eun-Young,Park Seah,Choi Won Yun,이동율 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.4

Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism. Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.

미국의 인간배아줄기세포 연구의 규제 동향

김민우 ( Min Woo Kim ),류화신 ( Hwa Shin Ryoo ) 홍익대학교 법학연구소 2015 홍익법학 Vol.16 No.1

As the stem cell enables the treatment of fundamental cause of disease and the customized treatment for individual, so is expected to become a driving force of future medical industry. As a stem cell-utilizing medicine can replace a damaged organ with a new one or restore it into a normal organ, so is expected to delete the fundamental cause of disease. Moreover, as the stem cell can be extracted from an individual`s body and can make appropriate medicines and organs for the individual, so each patient-customized treatment will be possible. Though most medicines vary the degrees of its therapeutic effects and side effects depending on different characteristics by race and by individual, but the stem cell-utilizing medicine has the advantages to complement such disadvantages. So the stem cell is also called as the ‘gold mine of medical industry.’ U.S administration`s initial policy on the researches dealing with the human embryonic stem cell was the conditional approval only to the private field`s research support. After then, Obama administration enabled to support the U.S. federal fund to the researches on the human embryonic stem cell, and as the results from the U.S` such active support, international competition for preoccupying the superior position in the researches human embryonic stem cell became fierce based on each nation`s tremendous researchsupporting fund. Contrary to Korean position supporting the stem cell research, the U.S.A is the nation where the intensive confrontation between the competitiveness in the stem cell research and the bioethics has long been kept. In the U.S. administration and the Congress during the period from Clinton administration to Obama administration, there have continually been raised ethical, political controversies due to the both values. This thesis investigated the U.S` regulation trend on the researches about human embryonic stem cell by the institution, the presidential term, and each state.

인간배아줄기세포 연구와 임상시험의 문제점

류화신 경북대학교 법학연구원 2013 법학논고 Vol.0 No.42

This paper examines the research and a clinical testing of human embryonic stem cells in Korea. Now the treatment of Human embryonic stem cells is in the spotlight in the regenerative medicine. However, a few problems still remain: an ethical problem of destroying human embryo and technological difficulty avoiding immune rejection of human embryonic stem cells, Though a clinical testing of human embryonic stem cells was first approved in 2011, it is not only the violation of regulations about external use of Human embryonic stem cells according to “a Law concerning Bioethics and Safety” but also has a problem in the extent of informed consent on clinical testing by an egg donor. For these discussions, this treatise reviews about the value of an human embryo as resources in the regenerative medicine and legal position of an embryo, looking into regulations on the research of an embryo and human embryonic stem cells. And I mention the problem of the absence in the regulation on the clinical testing of human embryonic stem cells and some considerations in enacting that regulation. 본 논문은 한국에서 배아줄기세포 임상연구의 문제점에 관하여 다루고 있다. 배아줄기세포치료는 재생의학 분야에서 획기적인 치료법으로 각광받고 있으나 인간 배아를 파괴한다는 윤리적 문제와 면역거부반응과 같은 안전성 문제를 안고 있다. 2011년에 배아줄기세포 임상시험이 국내에서 첫 승인되었지만 생명윤리및안전에관한법률상 배아줄기세포주의 체외이용에 관한 규정에 위배될 뿐 아니라 배아 생성시 난자제공자 등의 동의의 범위가 문제되고 있다. 이러한 논의를 위하여 이 논문은 먼저 배아의 자원으로서의 가치, 배아의 법적 지위에 관하여 살펴보고, 배아연구 및 배아줄기세포주의 연구에 관한 법규정을 검토한 다음, 배아줄기세포 임상시험에 관한 별도의 지침이 부재함에 따른 문제점과 그러한 지침을 마련함에 있어서 고려하여야 할 사항 등에 관하여 언급하고 있다.

Derivation of Neural Precursor Cells from Human Embryonic Stem Cells

Kim Sehee,Hong Ji Young,Joo So Yeon,Kim Jae Hwan,Moon Shin Yong,Yoon Hyun Soo,Kim Doo Han,Chung Hyung Min,Choi Seong-Jun 한국동물생명공학회(구 한국동물번식학회) 2004 Reproductive & developmental biology Vol.28 No.4

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo. Human ES cells have the capacity to differentiate into various types of cells in the body. Human ES cells are indefinite source of cells for cell therapy in various degenerative disorders including neuronal disorders. Directed differentiation of human ES cells is a prerequisite for their clinical application. The objective of this study is to develop the culture condition for the derivation of neural precursor cells from human ES cells. Neural precursor cells were derived from human ES cells in a stepwise culture condition. Neural precursor cells in the form of neural rosette structures developed into neurospheres when cultured in suspension. Suspension culture of neurospheres has been maintained over 4 months. Expressions of nestin, soxl, sox2, pax3 and pax6 transcripts were upregulated during differentiation into neural precursor cells by RT-PCR analysis. In contrast, expression of oct4 was dramatically downregulated in neural precursor cells. Immunocytochemical analyses of neural precursor cells demonstrated expression of nestin and SOX1. When induced to differentiate on an adhesive substrate, neuro-spheres were able to differentiate into three lineages of neural systems, including neurons, astrocytes and oligo-dendrocytes. Transcripts of sox1 and pax6 were downregulated during differentiation of neural precursor cells into neurons. In contrast, expression of map2ab was elevated in the differentiated cells, relative to those in neural precursor cells. Neurons derived from neural precursor cells expressed NCAM, Tuj1, MAP2ab, NeuN and NF200 in immunocytochemical analyses. Presence of astrocytes was confirmed by expression of GFAP immuno-cytochemically. Oligodendrocytes were also observed by positive immuno-reactivities against oligodendrocyte marker O1. Results of this study demonstrate that a stepwise culture condition is developed for the derivation of neural precursor cells from human ES cells.

배아줄기세포의 임상적용

정형민 대한의사협회 2011 대한의사협회지 Vol.54 No.5

Recent advances in stem cell biology, including the development of optimized cell typespecific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have brought cell-based therapy closer to practical application. As of now, at least 250 adult stem cell therapies are being used or tested in clinical situations. Stem cells have two important properties that distinguish them from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kinds of cells depending on their culture conditions. Thus, stem cell therapy could be most effective for treating the diseases that are marked by the loss of cells. The typical examples are Parkinson s disease, Alzheimer’s disease, diabetes, heart failure, blindness, spinal cord injury, and stroke. Additionally, stem cell derivatives can be used in drug discovery as well. In the last decade, various types of stem cells have been identified from preimplantation stage embryos, fetuses, placentas, and adult tissues. Moreover, it is now almost a common practice to produce induced pluripotent stem (iPS) cells from various adult somatic cells using only a few defined factors. Thus, it is feasible that patient-specific stem cells will be generated with less controversy in the near future. However, human embryonic stem (ES) cells firmly remain the gold standard because of their greatest potential to become any type of cell in the body. The vast knowledge obtained from human ES cell research in the past decade has made cell-based therapy more promising than ever. Even the recent establishment of iPS cell technology is the culmination of human ES cells research. In our laboratory, interesting human cardiovascular cells including endothelial precursor cells and beating myocardiac cells, artificial blood cells, and retinal pigment epithelial cells were successfully differentiated and their therapeutic potential was confirmed after cell transplantation into animal models. Thus, here, the current research status of human embryonic stem cell-based therapy will be introduced and the future directions of stem cell applications in clinical trials will be discussed.

배아줄기세포에 의한 영양세포의 지방 분화

이순례 ( Sun Ray Lee ),박현숙 ( Hyun Sook Park ) 한국조직공학·재생의학회 2010 조직공학과 재생의학 Vol.7 No.1

Human embryonic stem(hES) cells have generally been cultured on mouse embryonic fibroblast feeder cells. Paracrine factors from feeder cells have drawn much attention in their relation to the maintenance of selfrenewal and pluripotency of hES cells. There has however been no attention on factors from hES cells and their effects on feeder cells. In the present study, we examined the effect of hES cells on feeder cells, mouse embryonic fibroblasts, human mesenchymal stem cells and human skin fibroblasts, in indirect co-culture system using porous membrane. We observed lipid droplet formation in feeder cells as early as 2 days after co-culture and lipid vacuole matured with time in the influence of hESCs. Only with hES-conditioned medium adipogeneisis of feeder cells occurred but co-culture with hES cells dramatically increased adipogenesis of feeder cells. This implied adipogenesis of feeder cells occurred via paracrine communication between hES cells and feeder cells. Further investigation on how adipogenesis of feeder cells affects the niche of human embryonic stem cells is required.

In Vitro Expansion of Homogeneous Neural Precursor Cells Derived from Human Embryonic Stem Cells

Deuk-Chae Na,Sehee Kim,Won-Ik Choi,Hyun-Jin Hwang,Inho Han,Jae-hwan Kim,Keun-Hong Park,Hyung-Min Chung,Seong-Jun Choi 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.4

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III β-tubulin and MAP2ab were observed. Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.

Cancer stem cell surface markers on normal stem cells

( Won-tae Kim ),( Chun Jeih Ryu ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.6

The cancer stem cell (CSC) hypothesis has captured the attention of many scientists. It is believed that elimination of CSCs could possibly eradicate the whole cancer. CSC surface markers provide molecular targeted therapies for various cancers, using therapeutic antibodies specific for the CSC surface markers. Various CSC surface markers have been identified and published. Interestingly, most of the markers used to identify CSCs are derived from surface markers present on human embryonic stem cells (hESCs) or adult stem cells. In this review, we classify the currently known 40 CSC surface markers into 3 different categories, in terms of their expression in hESCs, adult stem cells, and normal tissue cells. Approximately 73% of current CSC surface markers appear to be present on embryonic or adult stem cells, and they are rarely expressed on normal tissue cells. The remaining CSC surface markers are considerably expressed even in normal tissue cells, and some of them have been extensively validated as CSC surface markers by various research groups. We discuss the significance of the categorized CSC surface markers, and provide insight into why surface markers on hESCs are an attractive source to find novel surface markers on CSCs. [BMB Reports 2017; 50(6): 285-298]

Distinct transcriptional profiles of angioblasts derived from human embryonic stem cells

Song, S.H.,Jung, W.,Kim, K.L.,Hong, W.,Kim, H.O.,Lee, K.A.,Lee, K.Y.,Suh, W. Academic Press 2013 Experimental cell research Vol.319 No.8

Identification of differentially expressed genes in angioblasts derived from human embryonic stem cells (hESCs) is of great interest for elucidating the molecular mechanisms underlying human vasculogenesis. The aim of this study was to define hESC-derived angioblasts at the clonal level and to perform comparative transcriptional analysis to characterize their distinct gene expression profiles. In a clonal analysis performed in cell-specific differentiation media, hESC-derived CD34<SUP>+</SUP>CD31<SUP>+</SUP> cells were identified as angioblasts in that they exhibited a significantly higher ability to form endothelial cell (EC) and smooth muscle cell (SMC) colonies than CD34<SUP>+</SUP>CD31<SUP>-</SUP> and CD34<SUP>-</SUP> cell populations did. Microarray analysis showed that many genes involved in vascular development and signaling transduction were overexpressed in hESC-derived CD34<SUP>+</SUP>CD31<SUP>+</SUP> cells, whereas those related to mitosis, the DNA damage response, and translation were substantially downregulated. In addition, comparative gene expression profiling of hESC-derived CD34<SUP>+</SUP>CD31<SUP>+</SUP> cells and human somatic primary vascular cells demonstrated that hESC-derived CD34<SUP>+</SUP>CD31<SUP>+</SUP> cells expressed key genes involved in the EC and SMC differentiation processes, which supports the result that hESC-derived CD34<SUP>+</SUP>CD31<SUP>+</SUP> cells are bipotent angioblasts. Our results may provide insights into the identity and function of hESC-derived angioblasts and may also facilitate further investigation of the molecular mechanisms regulating human embryonic vasculogenesis.