; 박사 루시 센터, 닥터 Brigit Willeford 및 MSU ALAC 공인 마우스 시설에서 교육 및 동물 관리와 지원을위한 마이크 Basett, 박사 드웨인 와이즈 박사 닐 첫번째 원고 편집을위한 Kourtney 윌킨슨 : 저자의 귀중한 도움을 인정하고 싶습니다 세자르 Monroy, 한나 스우프 및 MSU에서 동영상 제작과 함께 자신의 도움 바비 허들 스턴, 현미경 및 이미지 캡처와 함께 자신의 도움을. 이 연구는 미시시피 주립 대학에서 기관 연구 및 생물 과학학과 사무실에서 후원했다.

1 부 : 임산부 마우스 개복술 <ol><li> 자궁 경부 전위를 사용하여, 10.5-11.5 DPC에서 임신 여성 C57BL6J 마우스를 안락사. </li><li> 다음 항균 비누로 복부를 청소하고, 그것을 면도. </li><li> 면도 후, 생리 식염수로 복부를 씻으십시오. </li><li> 그리고, 멸균 거즈로 복부를 건조. </li><li> 새로운 멸균 분야로 마우스를 커버. </li><li> 포셉 및 해부 가위를 사용하여 복부 절개를합니다. </li><li> 식별 및 복강에서 전체 자궁을 제거합니다. 마우스 배아는 자궁 안에 표시됩니다. </li><li> D - PBS로 가득 배양 접시에 자궁을 전송하고, 얼음에 보관하십시오. </li><li> 살균 메스와 집게를 사용하여 태반 및 각 배아의 여분의 배아 조직을 제거합니다. </li><li> D - PBS로 가득 신선한 페트리 접시에 배아를 전송합니다. </li><li> 마우스 배아의 길이를 측정합니다. 우리는 크기가 배아의 연령에 따라 다릅니다 것으로 나타났습니다. 예를 들어 8.5 DPC는 6mm의 평균 측정, 10.5 DPC는 11mm의 평균에서 측정하고, 12.5 DPC는 16 mm의 평균 측정. </li><li> 그런 다음, DNA 추출을 위해 자신의 꼬리를 제거합니다. </li></ol> 2 부 : Gonadal 리지 해부 stereomicroscope 및 광원 쇼트 Fostec 아래 <ol><li> 페트리 접시에 여과지를 놓습니다. 그런 다음, 배아를 건조하고 해부에 대한 고정하기 위해 필터 종이 위에 마우스 배아를 놓습니다. </li><li> 멸균 메스를 사용하여 탯줄의 기원 위에 마우스 배아의 가로 절단합니다. </li><li> stereomicroscope에서 살균 고급 포셉 및 살균 고급 괴롭히고 바늘을 사용하여 비뇨기 시스템이 볼 수 있도록 창자와 간장을 제거하십시오. 신장은 복강에 옆으로 위치하고 있으며, 방광은 신장에 비해 중간과 꼬리에 위치하고 있습니다. gonadal 산등성이가 방광의 양쪽에 위치하고 있기 때문에 배아는 남자로 확인할 수 있습니다. 여성에 gonadal 산등성이는 단단히 신장의 꼬리 끝에 측면에 붙어있다. </li><li> 그 뒤에 바늘을 슬라이딩하여 gonadal 리지 밖으로 껍질. gonadal 봉우리는 그것을 지원하는 조직을 떠나 절단하여 제거됩니다. </li><li> 신선한 D - PBS로 가득한 새로운 배양 접시에 gonadal 능선을 전송합니다. 미세, 남성 gonadal 능선은 크고 모양 타원형 송두리째해야합니다. 반면, 여성 gonadal 능선을 발견해야 길쭉한 모양을하고 남성 gonadal 능선에 비해 작은 수 있습니다. </li></ol> 파트 3 : Gonadal 리지 소화 아래 stereomicroscope : <ol><li> Mesonephros의 해부 : 좋은과 날카로운 바늘을 사용하여 mesonephros과 능선에서 분리 생식선. <ul><li> 그것은 PGC의 유도 과정에서 자라다하는 체세포 조직이기 때문에 gonadal 산등성이에서 mesonephros을 제거하는 것이 중요합니다. </li></ul></li><li> Gonadal 리지 소화 : 신선한 D - PBS의 0.5ul 드롭 깨끗한 gonadal 산등성이를 수집합니다. Collagenase / Dispase 솔루션 20 ML (1mg/ml 최종 농도)를 추가합니다. <ul><li> 배아는 개별적으로 처리되어야합니다. 각각의 배아 자체 별도의 표시 페트리 접시가 있어야합니다. </li></ul></li><li> 리지가 민싱 Gonadal : 살균 멸균 바늘과 좋은 포셉 제 4를 사용하여 작은 조각으로 gonadal 리지 컷. </li><li> 배양 : 배양 접시가 37 인큐베이터으로 전송됩니다 ° C 15 분. <ul><li> 배양 시간은 조직간에 다를 수 있습니다. </li></ul></li><li> Pipetting : 15 분 후, 조직 pipetting하여 dissociated 수 있습니다. 40 -100 mm 사이의 직경과 함께 뽑아 유리 모세관 pipettes를 사용하면하고 아래로 pipetting하여 작품을 브레이크. 이것은 작은 대단히 짧은 시간을 형성합니다. 최종 세척을 위해 미리 예열 D - PBS 드롭의 0.5ml에 대단히 짧은 시간을 전송합니다. </li><li> 세척 후, eppendorf 튜브에 대단히 짧은 시간을 전송하기만하면됩니다. <ul><li> 이상 collagenase로 조직을 노출시키지 마십시오. </li><li> 단일 세포 현탁액을하지 마십시오. 이것은 식민지 형성에 중요합니다. 그것이 하나의 세포 현탁액 경우, PGCs는 차별과 마이 그 레이션하는 경향이 있습니다. </li><li> 이 과정은 20 분 이상하지 않아야, 또는 세포 생존을 잃게됩니다. </li></ul></li><li> 실온에서 5 분간 2000 rpm으로 원심 분리기. </li><li> centrifuging 후, 뜨는을 제거하고, 미디어 밖으로 보충 노크 (37 ° C에서 미리 예열)의 0.5 ML의 펠렛을 resuspend. </li></ol> 4 부 : 원시 종자 세포 문화 &quot;당신은 격리와 유도를위한 PGCs의 생존을 유지하기 위해 신속하게 다음 단계를 수행합니다. <ol><li> 24시간이 과정을하기 전에 장 J. 외 2008 프로토콜에 따라 mitotically inactivated MEF - CF1 피더 레이어를 준비한다. <ul><li> 한 pregna 20 요리를 준비NT 여성 마우스 (6-8 배아). </li><li> MEFs은 5-6 구절 후 (성장을 도모하고 차별을 방지) 피더 레이어 수있는 능력을 잃게됩니다. </li><li> 48시간보다 오래된 MEFs를 사용하지 마십시오. </li></ul></li><li> 즉시 도금 PGCs 전에 MEF 매체는 서서히 MEF 피더 레이어에서 제거되어야합니다. </li><li> 그렇다면, 0.5 ML / 음의 MEF 피더 계층에 미리 예열 보충 녹아웃 매체를 추가합니다. <ul><li> MEF 미디어 PGCs의 차별을 유도 태아 소 혈청이 포함되어 있습니다. </li><li> 마네 매체가 정화 특정 성장 요인과 함께 보충해야하는 것은 바로 전에 성장 인자의 활동을 보장하기 위해 사용합니다. </li></ul></li><li> MEF 피더 층 이상의 대칭 유통, 판 PGCs을 사용합니다. <ul><li> 이 조각이 너무 가까이있다면, 그들은 집계하는 경향과 저조한 첨부하거나 차별 시작 밀도 식민지합니다. </li><li> 문화 요리는 배아 본원 세포주 이름, 통로 번호 및 날짜와 함께 표시해야합니다. </li></ul></li><li> 신중하게, 37 ° C와 5% CO 2 배양기에 문화 요리를 전송할 수 있습니다. </li><li> 세포마다 24 시간 모니터링합니다. </li><li> 48 시간 후, 식민지의 첨부 파일을 방해하지 않도록하기 위해 문화의 맨에서 미디어 250 ML를 제거합니다. 그런 다음, 신선한 보충 노크 아웃 미디어 250 ML (37 ° C에서 미리 예열)을 추가합니다. </li><li> 7 단계 후, 또 다른 48 시간을 기다린 후, 완전히 모든 미디어를 제거하고 신선한 노크 아웃 보충 미디어 (37 ° C에서 미리 예열)로 바꾸십시오. PGC 식민지 양식까지 80~10일이 이일 모든 마십시오. </li></ol> 제 5 부 : 본원 배아 세포 <ol><li> PGC 절연 10 일 후, 본원 배아 세포 식민지가 형성됩니다. </li><li> 태아의 새싹 세포 라인 수동 구절마다 80-10일에 의해 생명을 유지하고 있습니다. 그들은 undifferentiated 형태를 제시하고 같은 alkalyne 인산 가수 분해 효소, 10 - 4, SSEA - 1, 및 SOX - 2로 pluripotency 마커를 표현하기 위해 계속 </li></ol> 노트 <ul><li> 멸균 / 무균 조건은 문화 실에 필수적입니다. </li><li> 파생 문화, PGCs는 정수기 클래스 II 바이오 안전 내각에 처리됩니다. </li><li> Incubations가 humidified 37 아르 ° C와 5% CO 2 배양기. </li><li> 모든 미디어 및 시약 4에 저장된 음 0.2 필터 ° C에서 사용하기 전에 필터링하고, 사용하기 전에 ° C 37 미리 예열하고 있습니다. </li><li> 해부 단계 동안, 모든 배아와 조직은 얼음에 보관해야합니다. </li><li> 모든 시약의 flasks는 캐비닛에 배치하기 전에 에탄올로 소독하고 있습니다. </li><li> 장갑, 연구실 코트, 그리고 간호사 모자의 사용은 필수입니다. </li></ul> 결론 우리는 당신이 분리하는 방법을 보여줍니다 비디오, 파생 및 10.5-11.5 DPC 마우스 배아의 gonadal 산등성이에서 문화 배아 새싹 세포를 제공하고 있습니다. 배아 새싹 세포 라인의 재현성 분리 및 장기 문화가 초기 배아 발달, 유전 및 epigenetic 본원 패턴, 생식선 형성의 연구를위한 중요한 기반을 제공, 남자와 여자 태아에 gametogenesis의 발달 과정에서 주변 장기의 환경적 영향, 그리고 암, 불임로 연결 통로의 결정. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="그림 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

미시시피 주립 대학에서이 연구 검토 및 기관 애니멀 케어 및 마우스 프로토콜 # 08030 (프로토콜을 개최하고 사육 반응 산소 세균 라인 개발 및 종양 형성에 종 - ROS의 역할)의 이용위원회 (IACUC) 승인 후 실시했다.

포셉 제 14, 15, 메스 35mm, 해부 가위, 외과 분야, 항균 비누, 생리 식염수 0.9 %, 거즈, 얼음 폼 컨테이너, 면도날, 메스 35mm, 괜찮아요 포셉 제 4, 5, 괜찮아요 올린 바늘로 핸들, 종이 시트를 필터링하고, 유리 pipettes를 추출.<br /<br /&gt; 장비 : 해부 stereomicroscope, 광원 쇼트 Fostec, 거꾸로 현미경, micropipette 10 200ul, 원심 분리기, 바이오 안전 내각.

isolation and derivation of mouse embryonic germinal cells

배아 새싹 세포의 기능 (예) 암, 불임에 관한 우리의 가설을 해결하기 위해 완벽한 모델​​입니다 초기 발달 단계 동안 원시 종자 세포 (PGCs)로 나중에 gametes로 구별합니다. 이 프로토콜은 10.5-11.5 일 게시물 coitum (DPC) 마우스 배아의 개발 생식선에서 원시 종자 세포를 분리하는 방법을 보여줍니다. 마우스 배아에서 gonadal 능선을 개발 (C57BL6J) 다음 이전 mitotically 녹아웃 미디어의 존재에있는 mitomycin C와 inactivated과 백혈병 억제제로 보충되었다 마우스 배아 fibroblast 공급기 층 (MEF - CF1)에 도금 collagenase와 기계 파괴에 의해 dissociated되었습니다 팩터 (난생), 기본 Fibroblast의 성장 인자 (bFGF), 그리고 줄기 세포 계수 (SCF). 문화 조건 PCG 식별, 격리 및 설립을위한 이러한 최적화 방법을 사용하면 40 개 이상의 일간 EG 세포의 장기적인 문화를 수 있습니다. 태아의 새싹 세포 라인은 pluripotent 국가의 일반적인 사용 마커의 배아 표현형과 표현을 보여주었다. 분리 및 문화에 새싹 세포의 유도는 체외에서 개발을 이해하고 산화 스트레스에 노출 후 유전 및 epigenetic 수준에서 누적 손상을 모니터링하는 기회를 제공하는 도구를 제공합니다.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

초기 개발 단계 동안 원시 종자 세포로 차별화하는 배아 새싹 세포의 능력은 암, 불임에 관한 우리의 가설을 해결하기 위해 완벽한 모델​​입니다. 이 프로토콜은 10.5-11.5 일 게시 coitum 마우스 배아의 개발 생식선에서 원시 종자 세포를 분리하는 방법을 보여줍니다.

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 ...

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16.0K 조회수.  Mississippi State University. 초기 개발 단계 동안 원시 종자 세포로 차별화하는 배아 새싹 세포의 능력은 암, 불임에 관한 우리의 가설을 해결하기 위해 완벽한 모델​​입니다. 이 프로토콜은 10.5-11.5 일 게시 coitum 마우스 배아의 개발 생식선에서 원시 종자 세포를 분리하는 방법을 보여줍니다.

비디오: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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 ...

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 ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

배아 심실 내피 세포의 분리

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

개발 배아 마우스 심장 밸브 지역에서 단백질 분리

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...