이 작품은 베테랑 어페어스와 NIH 보조금 5U01HL100398 및 1R01HL104040 지원했다.

1. 잘 microtiter 접시를 96 라운드 바텀를 사용하여 배아 바디 (EB) 형성 <ol><li> 난생과 보충 마우스 ES 세포 매체와 10cm의 세포 배양 접시에 마우스 ES 세포를 성장. </li><li> ES 세포는 (일반적으로 50~70%의 합류에서) 사용을위한 준비가되면, ES 미디어를 제거하고 멸균 PBS 5ml로 한 세포를 씻어. </li><li> 3 ~ 5 분 37 각 플레이트와 부화 플레이트 ° C에 2ml 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)을 추가, 3 ML ES 매체와 트립신 EDTA (에틸렌 다이아 민 테트라 초산)이 끄다. </li><li> 3 분 1,000 rpm으로 50ml 팔콘 튜브와 스핀에 세포를 전송합니다. </li><li> 뜨는 제거하고, resuspend 세포 펠릿 EB 미디어의 원하는 금액과 함께. </li><li> 휴대폰 번호를 카운트하고 EB 미디어 5 X10 3 셀 / ML로 세포를 희석. </li><li> 라운드 잘 microtiter 접시 - 96 각 우물에 세포를 포함 EB 미디어 100μl를 추가하는 멀티채널 피펫을 사용합니다. </li><li> 플레이스 96 - 인큐베이터에 둥근 잘 microtiter 접시. </li></ol> 2. cardiogenesis에 대한 주요 신호 경로 (S)의 중요한 시간 창을 확인 <ol><li> 각 96 예 일 0 일 1 일 2 일 3 일 4 우물의 등 반 등 다양한 시간 지점에서 ES 세포를 포함하는 96 - 웰 microtiter 플레이트의 각 우물에 특정 신호 경로 및 차량 제어 모듈 레이터 추가 - 잘 플레이트는 (48 웰스) 치료의 각 시작 시점에 사용됩니다. </li><li> 다른 정지 시간 지점에서 매 48 우물을위한 EB 미디어를 변경하여 모듈 레이터를 씻으십시오. 예를 들어, 하루 0부터 시작하는 ES 세포의 두 가지 일 치료를 위해 하루에 2 변조기를 씻으십시오. 더 이상 치료 48 시간보다 원하는 정지 시간이 지점까지 이틀 신선한 변조기와 보충 EB 미디어를 변경합니다. </li><li> 일 6.25 후 현미경 EB 수축을 검사합니다. 각기 다른 치료 시간 코스 EBS를 계약의 비율을 얻기 위해서는 긍정적인 사람으로 EBS를 계약으로 우물을 점수. </li><li> ES의 cardiogenesis에 대한 중요한 시간이 지점은 차량 제어에 비해 EBS는 신호 변조기에 의해 처리 계약의 높은 비율을 포함하는 시간 프레임입니다. </li></ol> 3. EBS에서 cardiogenesis에 대한 신호의 시간 창을 확인 매달려 방울로 만든 <ol><li> 증발을 방지하기 위해 PBS의 3-4 ML로 추가하여 배양 접시를 준비합니다. </li><li> 단계 1.1 ~ 1.5 2.5x10 4 셀 / ML 최종 세포 농도에서 ES 세포를 준비를 따르십시오. </li><li> 쉽게 접근할 세균 그릇에 세포 혼합물을 붓고. 멀티채널 피펫을 사용하여 거꾸로 뚜껑에 20 μl 방울 (약 500 세포를 포함) 추가합니다. 개인 방울 연락하는 것을 허용하지 않습니다. 일반적으로 하나의 뚜껑 80 방울까지 들어갈 수 있습니다. PBS를 포함하는 요리를 통해 다시 뚜껑을 플립. 인큐베이터에서 EB 형성을 허용 24 시간 또는 48 시간 동안 부화. </li><li> EB 미디어과 수영장 새로운 배양 접시에 EBS 2 뚜껑 3 ML 각 뚜껑에서 방울을 걸려에서 형성 EB를 씻으십시오. 10ml의 총 볼륨 EB 미디어를 추가합니다. </li><li> 0.2 % 일 4 젤라틴 코팅 6 - 잘 접시에 정지에 전송 EBS. 일반적으로 30 EBS는 각 잘으로 전송됩니다. 각 잘에 대한 2 ML 최종 볼륨을 얻을 EB 미디어를 추가합니다. </li><li> 96 - 웰 플레이트 실험에서 확인된 기간에 변조기 신호 품어. </li><li> RT - PCR 추가, immunostaining 등, 필요한 경우의 특징 수 일 7 cardiogenesis 후 현미경 EB의 수축을 관찰. </li></ol>

<ol>
	<li>Fukuda, K., Yuasa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+a+source+of+regenerative+cardiomyocytes.">Stem cells as a source of regenerative cardiomyocytes.</a> Circulation research. 98, 1002-1013 (2006).</li><li>Foley, A. C., Mercola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+induction+by+Wnt+antagonists+depends+on+the+homeodomain+transcription+factor+Hex.">Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex.</a> Genes Dev. 19, 387-396 (2005).</li><li>Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., Komuro, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stage-specific+biphasic+roles+of+Wnt/beta-catenin+signaling+in+cardiomyogenesis+and+hematopoiesis.">Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis.</a> Proc Natl Acad Sci U S A. 103, 19812-19817 (2006).</li><li>Hao, J., Daleo, M. A., Murphy, C. K., Yu, P. B., Ho, J. N., Hu, J., Peterson, R. T., Hatzopoulos, A. K., Hong, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin,+a+selective+small+molecule+inhibitor+of+BMP+signaling,+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Dorsomorphin, a selective small molecule inhibitor of BMP signaling, promotes cardiomyogenesis in embryonic stem cells.</a> PloS one. 3, e2904-e2904 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

매달려 드롭 방식 EB 형성과 체외의 차별화에 사용되는 종래의 방법을하고 있습니다. 그러나, 그것은 힘드는이며 물류 문제로 인해 실험 유연성을 제한합니다. 실험자의 기술이 달린 방울로 성공 EB 형성 및 조작하는 데있어 매우 중요합니다 같은 토큰에 의해 결과도 확인이 더 어렵습니다. 간단한 방법은 단일 단계 과정으로 둥근 바닥 96 - 웰 플레이트에 EBS를 형성하는 것입니다. 이 포맷은 표준으로 체외 차별에 수 있으며 EB 형성 및 다운 스트림 조작 (예 : 변조기 추가 또는 제거)의 용이성으로 인해 더 많은 실험 조건을 수용할 수 있습니다. 또한, 96 - 웰 플레이트 형식은 마우스 ES 세포에서 효율적인 심사 도구로 최적화할 수 있습니다.

<ul><li> 마우스 CGR8 세포는 주로 사용됩니다. </li><li> ES 매체 : 10 % 열 inactivated FBS와 보충 GMEM 미디어, 2 MM L - 글루타민, 2 - 메르 캅 토 에탄올 0.05 MM, 200 U / ML murine 평범한. </li><li> EB 매체 : 20 % 열 inactivated FBS와 IMDM 미디어를 준비, 1.6 MM L - 글루타민, 2 - 메르 캅 토 에탄올 0.08 MM, 그리고 1X 멤 비 필수 아미노산 솔루션입니다. </li></ul>

modified mouse embryonic stem cell based assay for quantifying  cardiogenic induction efficiency

pluripotent 줄기 세포의 분화는 밀접하게 여러 주요 신호 경로의 시간적 및 공간적 규정에 의해 제어됩니다. 의 이해에 장애물 중 하나는 분화의 효율을 주요 신호 이벤트의 변경 내용을 연관의 다양한 방법을하고 있습니다. 우리는 여기서 / β - catenin Wnt와 cardiogenic 유도하는 동안 BMP 신호 활성화를위한 중요한 시간 윈도우를 식별하는 마우스 배아 줄기 (ES) 세포 기반 분석의 사용을 설명합니다. 96 - 웰 플레이트 형식으로 배아 기관 (EBS)을 계약에 대한 점수로, 우리는 신속하게 cardiogenic 효율성을 계량 및 / Wnt β - catenin 및 특정 변조기 트리 트먼트 다음 시간 코스 BMP 신호 활성화를위한 중요한 시간 창을 확인할 수 있습니다. 여기에 설명된 교장 선생님은 혼자 심장 유도에 한정되지 않고 여러 셀 lineages의 연구 방향으로 적용할 수 있습니다. 또한, 96 - 웰 형식은 더 높은 처리량, 더 정교한 실험 가설의 시험 수 있도록 자동화된 분석으로 개발할 수있는 잠재력이 있습니다.

Watch this Scientific Journal Video about Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency at JoVE.com

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

우리는 / β - catenin Wnt와 cardiogenic 유도하는 동안 BMP 신호 활성화를위한 중요한 시간 윈도우를 식별하는 마우스 ES 세포 기반 분석의 사용을 설명합니다. 방법은 안정 cardiogenic 효율성을 단정 표준화된 플랫폼을 제공하고, 다른 세포 lineages의 연구에 적용됩니다.

Cardiogenic 유도 효율을 Quantifying에 대한 수정 마우스 배아의 줄기 세포 기반 어세이

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

modified-mouse-embryonic-stem-cell-based-assay-for-quantifying

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9.6K 조회수.  Vanderbilt University School of Medicine. 우리는 / β - catenin Wnt와 cardiogenic 유도하는 동안 BMP 신호 활성화를위한 중요한 시간 윈도우를 식별하는 마우스 ES 세포 기반 분석의 사용을 설명합니다. 방법은 안정 cardiogenic 효율성을 단정 표준화된 플랫폼을 제공하고, 다른 세포 lineages의 연구에 적용됩니다.

비디오: Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

연구

생물학

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

인터뷰 : 줄기 세포 연구에 대한 Bioreactors와 분수 - 수정된 3D - 공사장 공중 발판

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

모터 뉴런에 마우스 배아 줄기 세포의 효율적인 분화

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

마우스 배아 줄기 세포 유지 보수 및 자체 갱신을 조절 유전자를 공부하는 Oct4GiP 리포터 분석

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...