저자는 원래 프로토콜 쟈넷 Rossant 감사합니다. 이 연구는 WH하는 보건 부여 CA106308 국립 연구소에 의해 지원되었다.

이 프로토콜에서는, 우리는 마우스 blastocysts의 준비와 TS 세포 라인의 설립에 대해 설명합니다. 이전 자연 교배에 설정하고 슈퍼 배란의 유도를 포함 blastocysts의 수집, 일반적인 마우스 조작은 기본적으로 나지 외 의해 설명된 표준 프로토콜을 따르십시오. 2003 (pp146 - 150). TS 세포의 유도 및 유지 보수 <ol><li> 관심 마우스 사이에 설치 사육 크로스. </li><li> 피더 세포로 마우스 배아 섬유아 세포 (MEFs)를 준비합니다. 이틀 blastocysts의 수집 전에 MEFs는 (2 × 10 6 세포) 100mm 요리 도금입니다. 그 다음날, 이들 세포는 37 2 1 시간 배양을위한 ° C, 5 % CO 2 mitomycin C (20 MG / ML)이있는 TS 매체의 10 ML로 취급됩니다. 이것은 PBS로 두 번 세포를 세척옵니다. mitomycin C 처리 전지는 다음 12 잘 접시 (1 X 10 5 셀 / 음)에 씨앗을 품고 있습니다. </li><li> blastocyst 수집 (3.5 DPC, 첫날이라 함)의 날, MEF 문화 매체는 TS 중간 플러스 1X FGF4/Heparin (0.5 ML / 음)으로 대체됩니다. </li><li> 자궁 수집 준비 : 마우스를 안락사와 자궁 (그림 1A)을 찾습니다. </li><li> 자궁 경부 (방광 뒤에 위치)에서 집게로 쥐고하여 자궁을 제거하고 자궁 및 자궁 경부 (그림 1B)의 교차점을 가로질러. 자궁을 당겨, 멀리 멤브레인 지방 조직을 장식하고 수란관 (그림 1C)와 접합 아래 자궁 잘라. 60mm 판에서 TS 매체의 작은 볼륨 (0.2 ML)의 uteri (그림 1D)를 놓으십시오. </li><li> 26 게이지 바늘로 1 - ML 주사기를 사용하여 TS 매체 1 ML 각 자궁 경적을 플러시. 자궁의 기본에 바늘을 삽입하고 1 ML의 TS 매체와 새로운 60mm 판으로 천천히 플러시. 자궁이 비정상적으로 증가하고 내리는 동안 완벽하게 확장해야합니다. </li><li> 수집하고 입 제어 피펫을 사용하여 TS 매체를 포함하는 깨끗한 접시에 12 잘 신중하게 blastocysts을 전송. 12 잘 접시에 TS 매체를 통해 직렬 전송으로 blastocysts 세 번 씻으십시오. 37 mitomycin C - 대우 MEF 모이통과 문화를 포함하는 12 잘 플레이트에 잘마다 놓으 blastocyst (그림 2A) ° C, 5 % CO 2. </li><li> blastocysts은 조나 pellucida에서 부화하고 24~36시간에있는 우물에 연결합니다. 작은 가지는 쉽게 일 3 관찰됩니다. 500 μl 신선한 TS 매체 플러스 1X FGF4/Heparin과 문화를 피드. </li><li> 4 ~ 5 일에, blastocyst의 가지는 크기에 따라 disaggregation 준비되어야합니다. TS 세포 disaggregation에 대한 이상적인 크기는 그림 3B의 그림입니다. 0.5 ML PBS로 한 문화를 씻으십시오. 37 오분 ° C, 5 % CO 2 100 ML 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)과 부화를 추가합니다. P100의 pipetteman과 함께 적극적으로하고 아래로 pipetting하여 작은 대단히 짧은 시간에 가지 봐라. 잘으로 TS - 70CM - 1.5F4H (30 % TS 매체 70 % MEF - CM 플러스 1.5x FGF4/Heparin)을 추가하여 trypsinization를 중지합니다. disaggregation 후 중간 8 시간 변경하고 모든 이일 세포를 먹이로 진행합니다. </li><li> 일 6 10 (매우 변수) 사이 TS 세포 식민지가 볼 수 있습니다. 그들은 명확한 콜로니 경계 (그림 2C)와 형태와 같은 평면, 상피 시트 수 있습니다. 매 2 일 문화 먹이를 계속 </li><li> TS 세포가 50 % confluency (대개 일 15 20 사이)에 도달하면, PBS로 한번 씻어 100 ML 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)를 추가합니다. 가까운 단일 세포 현탁액을 보장하기 위해 trypsinization 동안 아래 피펫. mitomycin C - 대우 MEF 모이통 (2.5 X 10 5 cells/6-well 또는 5 X10 5 cells/60mm 플레이트) 포함하는 6 자 또는 60mm 판에서 새로 만들기를 TS - 70CM - 1.5F4H 및 전송을 추가하여 trypsinization를 중지합니다. </li><li> 통로 중간 이후 8 시간 변경 2 일 모든 문화 먹이를 계속합니다. </li><li> MEF 모이통에 하나 또는 두 개 더 구절 (구절 사이 3-5일) 후, TS 세포는 그들없이 교양 수 있습니다. MEFs 훨씬 빠른 trypsinization 후 TS 세포보다 문화 판에 연결되기 때문에, 자기편 요금 이러한 차이는 MEF 모이통에서 TS 세포를 분리하기 위해 이용하실 수 있습니다. 교양 세포를 통과 후, ° C, 5 % CO 2 30 분 37 그들을 품어. 대부분의 MEFs는 판에 연결되며 TS 인구는 현탁액에 남아 있습니다. 새 접시에 뜨는를 전송합니다. 순수한 TS 인구를 얻기 위해 몇 구절이 과정을 반복합니다. </li><li> TS - 70CM - 1.5F4H에서 TS 세포를 유지하고 모든 사일을 패스 (1시 10분에서 1시 20분까지 분할) 또는 때 문화의 70 % confluency에 도달합니다. 고밀도 (1시 5분 분할 이상) 또는 culturing을 지나치게 합류들이 차별의 원인이 될 수있는 세포가 나온다. </li><li> TS 세포의 신분은 세포 마커, Cdx2 (그림 3) immunostaining으로 확인하실 수 있습니다. </li></ol> 냉동 TS 세포 <ol><li> 냉동 중간 (50 % FBS, 30 % TS 매체 20 % DMSO)를 2 배 준비합니다. </li><li> 공동trypsinization로 llect의 TS 세포는, 6 분 450g에서 원심 분리하여 따라갔다. 2X 동결 매체의 동일한 볼륨 TS 매체에서 그들을 Resuspend. </li><li> 천천히 -70에서 이소 프로필 알코올 챔버를 사용하여 세포를 고정 ° C와 24-48시간 후 액체 질소로 전송할 수 있습니다. </li><li> 60mm 판 (합류 70 %) 보통 2 유리병에 보관됩니다. </li></ol> TS 세포를 해동 <ol><li> 신속하게 37 병을 해동 ° C는 (450g, 6 분) 원심 분리 10 ML TS 미디어에서 그들을 수집, 30 % TS 미디어, 70CM - 1.5F4H에 resuspend. </li><li> 세포 중 하나 유리병은 60mm 판에 씨앗을 품고있다. </li></ol> TGC에 TS 세포 분화 trophoblast 거대 세포 (TGCs)에 TS 세포의 분화는 MEF - CM, FGF4 및 헤파린의 철수에 의해 유도된 수 있습니다. TGC 표현형은 (대형 핵) 4 일 유도 후에 감지된 수 있습니다 (다나카 외., 1998 애기 외. 2008). p450scc의 Immunostaining 것은 차별화된 문화에 TGC 마커 (그림 4A)의 표현을 감지합니다. PI의 흐름 cytometric 분석 (propidium 요오드화물의) 스테인드 세포가 더 높은 DNA 내용 (그림 4B)이있는 차별화된 세포의 존재를 보여줍니다. 이러한 결과는 TS 세포가 polyploid TGCs 될 endoreduplication를 받아야하는 것이 좋습니다. 대표 결과 <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="그림 1" /> 그림 1. 마우스 uteri의 해부. (A) uteri의 위치는 화살표로 표시됩니다. 그림과 같이 (B) 자궁 및 자궁 경부의 교차점을 가로질러. (C) 그냥 난소 (OV) 아래 수란관 (OD) 아래 자궁을 잘라요. uteri의 (D) 이미지. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="그림 2" /> 그림 2. 대표 사진 마우스 blastocyst, blastocyst의 파생물 및 trophoblast들의 서식지를 보여줍니다. (A) E3.5에서 수집된 마우스 blastocyst가 표시됩니다. ICM, 내부 세포 덩어리, MT, 뮤럴 trophectoderm, PT, 북극 trophectoderm. (B) MEF 모이통에 trophoblast의 파생물이 표시됩니다. TS, trophoblast의 줄기 세포. (C) trophoblast의 줄기 세포 콜로니는 MEF 모이통에 명확한 가장자리를 보여줍니다. 화살표는 셀 경계를 나타냅니다. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="그림 3" /> 그림 3. 분석을 immunostaining하여 TS 세포의 식별. TS (AD)과 ES (제어, EH) 세포 마커의 식별이 적용됩니다. 전지는 Oct4 (A, E) 또는 Cdx2 (B, F)을 인식하는 항체와 immunostained하고, 통합 이미지를 제공 DAPI (C, G) (D, H)에 의해 counterstained 있습니다. 스케일 바, 50 μm의. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="그림 4" /> 그림 4. 체외에서 TS 세포의 분화. (A) 문화에 Undifferentiated (Undiff) 또는 차별 (비교) 세포가 TGC 마커의 표현에 대해 검사했다, p450scc (녹색), DAPI (빨간색)와 counterstained. (B) PI 물들일 차별화된 세포의 흐름 cytometric 분석은 DNA의 내용을 (; M2, 개 이상 사본 M1, 2-4 부) 측정합니다. M2 인구 polyploid trophoblast 세포를 나타냅니다.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 비디오에서는, 우리는 프로세스가 TS 세포 라인을 확립 uteri와 실험 절차에서 E3.5 blastocysts를 수집 보여줍니다. 또한 TS 세포의 stemness를 유지하고 차별화된 세포에 자신의 차별을 유발하는 조건을 설명합니다. 순수 TS 세포 라인을 얻기 위해 두 가지 중요한 단계는 가지의 disaggregation에 대한 타이밍 (9 단계)이며, 첫 번째 통로 (11 단계)를 처리. 그것은 광범위한 blastocyst 단계 11 (Kunath 외., 2005)에 TS ?...

시약 TS 매체 : RPMI - 1640, 20 % FBS, 1 ㎜의 나트륨 pyruvate, 100 MM 베타 - 메르 캅 토 에탄올 100 MG / ML 페니실린 - 스트렙토 마이신. Mitomycin C : 2 ML PBS 2 MG mitomycin C (시그마 M - 0503)을 디졸브 200 ML TS 매체 (10 NG / ML)로이 2 ML 혼합물을 추가합니다. 사용하기 전까지 -20 ° C 20 aliquots (10 ML)과 저장을합니다. 100mm 플레이트 당 한 나누어지는를 추가합니다. 마우스 배아 fibroblast - 조건 매체 (MEF - CM) : 100mm 접시에 플레이트 MEFs (1 × 10 6 / 플레이트). 다음날, 요리 당 10 ML TS 미디어를 추가하고 48 시간 동안 문화를 계속합니다. 그들을 필터 (0.45 ㎜) 35 ML의 aliquots에서 -20 ° C에서 저장, 문화 매체를 수집합니다. 4 저장할 수 있습니다 필요한 각 나누어지는 ° C.를 녹여 MEFs 그들이 합류되기 전에 CM 두 이상 일괄적으로 준비하는 데 사용할 수 있습니다. PBS / BSA : 0.45 mm 주사기 필터를 통해 필터 PBS (10 ML)에 BSA, 분수 V (시그마 A3311, 0.1 % (W / V)를) 디졸브와 1.5 ML 튜브 1 ML aliquots합니다. -70에서 보관 ° C와 FGF4을 준비하는 데 필요한 때 하나 튜브를 녹여. FGF4 (1000x) PBS / BSA 용액 1 ML과 Resuspend 동결 건조된 FGF4 (시그마 F8424, 25 MG). -70에서 잘 섞어 1.5 ML 튜브 및 저장소로 10 aliquots (100 ML)를 만들 ° C. 필요할 경우 각각의 튜브를 해동하고 4 나머지를 저장할 ° C는 다시 냉동하지 않습니다. 1X FGF4을 (25 NG / ML)을 구하는 TS 매체에서 1,000 회 희석. 헤파린 (1000x) 1 최종 농도로 PBS에 Resuspend 헤파린 (시그마 H3149, 10,000 단위) MG / ML (1000x). -70 1.5 ML 튜브와 가게에 100 ML aliquots 만들기 ° C. 해동의 aliquots 필요와 4 나머지를 저장할 ° C는 다시 냉동하지 않습니다. 1X 헤파린 (1 NG / ML)을 구하는 TS 매체 1000 시간을 희석.

derivation of mouse trophoblast stem cells from blastocysts

trophectoderm의 사양은 포유류의 발전의 초기 분화 행사 중 하나입니다. trophectoderm의 mediates 주입에서 파생된 및 trophoblast의 일족이 태반의 태아 부분을 생성합니다. 결과적으로,이 혈통의 개발은 배아 생존을 위해 필수적입니다. 마우스 blastocysts에서 trophoblast 줄기 (TS) 세포의 유도 먼저 다나카에 의해 설명되었다<em&gt; 외.</em&gt; 1998. 적절한 자극 후 무대 및 세포 유형의 특정 마커의 trophoblast 특정 재산과 자신의 표현을 보존하기 위해 TS 세포의 능력은 초기 placentation 이벤트 recapitulating 떨어지던 trophoblast의 혈통 개발을 조사하기 위해 가치있는 모델 시스템을 제공합니다. 또한, trophoblast 세포는 자연 게놈 증폭을받은 몇 체세포 유형 중 하나입니다. trophoblast의 polyploidization를 기본 분자 경로가 해명되기 시작했지만, trophoblast의 게놈 증폭의 생리적 역할과 장점은 크게 어려운 남아 있습니다. 문화 polyploid trophoblast 세포에 diploid의 줄기 세포의 개발이 만들어<em&gt; 예 생체내</em건강과 질병의 게놈 복제 및 불안정성의 규제 메커니즘을 elucidating위한&gt; 시스템은 훌륭한 도구입니다. 여기 애기 출판 수정과 이전의 보고서를 기반으로 한 프로토콜을 설명<em&gt; 외.</em&gt; 2008.

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Derivation of Mouse Trophoblast Stem Cells from Blastocysts

이 비디오에서는, 우리는 마우스 blastocysts의 고립과 blastocysts에서 trophoblast의 줄기 세포의 유도를 보여줍니다. 우리는 또한 줄기 세포 속성의 유지 보수뿐만 아니라 문화에 분화의 유도에 대한 조건을 설명합니다.

Blastocysts에서 마우스 Trophoblast의 줄기 세포의 유도

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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18.1K 조회수.  University of Rochester. 이 비디오에서는, 우리는 마우스 blastocysts의 고립과 blastocysts에서 trophoblast의 줄기 세포의 유도를 보여줍니다. 우리는 또한 줄기 세포 속성의 유지 보수뿐만 아니라 문화에 분화의 유도에 대한 조건을 설명합니다.

비디오: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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 ...

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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 ...

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

마우스 아이의 망막 줄기 세포의 분리

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

기본 마우스 Trophoblast 셀 및 Trophoblast의 침략 분석의 절연

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

장기 성인 마우스 SSC 라인의 유도를위한 문화 Spermatogonial 줄기 및 전구 세포의 일련 농축 (SSCs)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...