저자 안드레아 액스텔, 사토시 이토, MD, 제프 Velotta, MD, 그랜트 호이트, 로버트 C. 로빈스, MD, 진 유, MD, 팀 도일, 박사, 그리고 기술 지원을위한 스탠포드 작은 동물 이미징 핵심 주셔서 감사합니다. 저자는 또한, 수의 장비의 지원을 오전 비크포드 (주) 감사합니다. 이 연구는 국립 보건원에서 연구 기금 (R01 HL - 75774, R01 CA098303, R21 HL085743, 1K12 HL087746), 캘리포니아 대학 (15IT - 0257 및 1514RT - 0169)의 캘리포니아 담배 관련 질병 연구 프로그램에 의해 지원되었다 및 재생 의료 (RS1 - 00183)의 캘리포니아 연구소. NH는 미국 심장 협회에서 친교에 의해 지원됩니다. 심장 협회 수 있습니다.

1. 내피 세포에 murine ESCs의 분화 <ol><li> 내피 세포에 ESCs을 차별에 대한 프로토콜은 다른 설명이 프로토콜 2,3의 초점이 아닙니다. 간단히, 세포가 차별 허용, 그리고 CD31 또는 혈관 내피 cadherin (VE - cadherin)로 내피 표시 양성 반응 세포는 다음 형광 활성 세포 분류 (외과)에 의해 정화하고 있습니다. </li></ol> 2. 더블 퓨전 리포터 유전자와 Lentiviral 도입 구축 <ol><li> Bioluminescence 그러한 반딧불 루시페라제 (fluc)로 기자 유전자의 표현을 수정 세포를 추적하는 데 사용할 수 있습니다. 이 응용 프로그램에 대한 우리의 세포는 내부 ubiquitin 발기인의 통제하에 둘 fluc 및 향상된 녹색 형광 단백질 (eGFP) 융합 유전자가 포함되어 있습니다. 세포를 수정하기 위해, 주요 최적의 유전 요소를 포함하는 transgene lentiviral 벡터가 수행하는 바이오 안전 기준뿐만 아니라 증가 전달의 효율성, fluc - eGFP 퓨전 리포터 유전자를 가지고 우리가 실험실에서 개발된 pFU - FG 벡터는 안정적으로 사용할 수 있습니다 차별화 후 세포를 transduce. 이 융합 구조는 이식 세포의 bioluminescence과 형광 추적 모두 가능합니다. 바이러스 입자 전달 및 고속 기자의 유전자가 다른 네 설명되는 레이블 세포의 생산을 생성하기위한 절차. </li></ol> 3. 허혈성 hindlimb로의 이식 ESC - 파생 내피 세포 <ol><li> 세포 이식에 대한 hindlimb 국소 빈혈 공사를 가진 마우스를 준비하여 절차를 시작합니다. 이렇게하려면 1L/min의 흐름 속도 100 % 산소 isoflurane 1~3%을 포함하고있는 마취 유도 챔버에 마우스를 놓으십시오. </li><li> 그것이 외부 자극에 응답하지 않는 때까지 유도 챔버에 마우스를 둡니다. 그런 다음 유도 챔버에서 동물을 제거합니다. </li><li> 그런 다음, 운영 테이블에 위로 향한 위치에 동물을 배치하고 1L/min의 흐름 속도 100 % 산소 isoflurane 1~3%의 지속적인 흐름에 연결합니다. </li><li> 세 교대 betadine 및 알코올 스크럽으로 hindlimb의 피부를 닦아주십시오. </li><li> 피부가 청소되면, 인산 30 μL에 백만 ESC - 파생 내피 세포를 얻을 호수 (PBS)가 버퍼. 28 게이지 바늘로 이들 세포를로드합니다. </li><li> 세포가 준비가되면, 부드럽게 리프트와 더 나은 gastrocnemius 근육의 위치를​​ 시각화하기 위해 hindlimb을 확장. 다리가 확장되는 동안, 기본 근육으로 피부를 통해 바늘을 삽입합니다. 뼈 접근하지 몸조심. 부드럽게 천천히 gastrocnemius에 30 μL 세포 혼합물을 주입. 근육내 주사의 경우, 30 μL가 주입 수있는 볼륨의 한계 가까운 거리에 있습니다. 우리의 경험, 그들이 주사기의 바늘에있는 손실 볼륨을 제거 때문에 따라서, 28 게이지 인슐린 주사기가 선호됩니다. </li><li> 주입이 완료되면 복구 케이지 마우스를 반환하고 정신 때까지 지속적으로 그것을 모니터합니다. 동물이 몇 시간 동안 회복 후 이식 세포의 생체내 bioluminescence 이미징에서 진행하도록 허용합니다. </li></ol> 4. 생체내에서 ESC - 파생 내피 세포의 Bioluminescence 이미징 <ol><li> 이식 ESC - 파생 endothelials 전지는 내부 ubiquitin 발기인의 통제하에 fluc과 eGFP 융합 유전자를 모두 표현하기 위해 수정되었습니다. 따라서, bioluminescence은 허혈성 hindlimb 내에서 세포를 추적하는 데 사용할 수 있습니다. </li><li> 이 단계를 시작하려면, bioluminescence 이미징 시스템 및 생활 이미지 수집 소프트웨어를 켭니다. 다음 수집 시스템을 초기화하고보기의 필드의 크기를 지정합니다. </li><li> 배경 방사를 흡수하기 위해 이미징 상자에 다음 장소 검은색 무광택 용지. </li><li> 이미지 상자가 준비되면, 1L/min의 출력에 산소 isoflurane 1~3%을 포함하고있는 마취 유도 챔버에 마우스를 놓으십시오. 그것이 외부 자극에 응답하지 않는 때까지 유도 챔버에 마우스를 둡니다. 그런 다음 유도 챔버에서 동물을 제거합니다. </li><li> 필요에 따라 전기 면도기를 사용하는 두 hindlimbs에서 머리를 제거합니다. </li><li> 복막에 체중 g 당 D - luciferin 10 μL를 주입. D - luciferin 15 MG / PBS에 ML의 필터링된 재고 솔루션으로 사전에 준비가되어 있습니다. </li><li> luciferin가 주입되면 isoflurane의 지속적인 흐름에 연결된 부정사 위치에 검은 종이,을 통해 영상 상자에 동물을 놓으십시오. </li><li> 이미지 상자에 동물을 배치하고 isoflurane에 연결. </li><li> 이미지가 포화되지 않은 최적의 노출 시간을 결정하기 위해 10~60초에 대한 이미지를 얻기 시작합니다. 이미지가 포화되면, 노출 시간을 줄일 수 있습니다. bioluminescence 신호가 매우 약한 경우, 노출 시간을 향상시킬 수 있습니다. </li><li> 저기서신호가 최대 후 페이드에 도달할 때까지 g 최적의 노출 시간, 사진마다 1~3분을 획득 계속합니다. 인수가 완료되면 파일을 저장합니다. </li><li> 데이터를 분석하려면, 주사 사이트를 커버 관심 지역 (로아)을 선택합니다. 대조군으로 유사한 투자 수익 (ROI)가 아닌 운영 다리를위한 선택하실 수 있습니다. 소프트웨어를 사용하여 각 timepoint에 대한 로아에 대한 광자 / 초 / cm 2 / steradian (P / S / cm 2 / SR)의 단위로 표현되는 총 빛을, 측정합니다. 최대한의 가치는 최종 데이터를 사용해야합니다. 데이터는 나중에 사용하기 위해 Excel 스프레드 시트로 내보낼 수 있습니다. </li><li> 일단 모든 데이터가 취득되어 복구 케이지에 동물을 반환하고 동물 깨우고 때까지 지속적으로 모니터링합니다. </li><li> 시간이 지남에 따라 세포를 추적하기 위해이 절차를 반복합니다. </li><li> 원하는 시점에서, 동물은 조직 기능의 평가 euthanized 수 있습니다. </li></ol> 5. 대표 결과 <img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="그림 1" width="600" height="600" /> 왼쪽 허혈성 hindlimb에 이식 세포의 대표 bioluminescence 이미지는 그림 1에 표시됩니다. bioluminescence 취득하는 동안, 강도는 시간이 증가되며, 시간 과정 중에 얻은 최대 값이 최종 값으로보고해야합니다.

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

ESCs 때문에 차별과 내피 세포를 포함한 세 세균 레이어를 구성하는 세포 lineages을 일으키다 능력 그들의 소성의 조직 국소 빈혈의 치료를 위해 유망한 전지 소스입니다. ESCs과 관련된 윤리적 문제를 극복하기 위해 유도 pluripotent 줄기 세포 (iPSCs) 윤리 문제를 극복 대안 pluripotent 줄기 세포 원천 수 있습니다. ESCs 게다가, 이러한 내피 전구 세포 (EPCs) 및 조혈 줄기 세포 (HSCs)와 같은 성인 줄기 세포도 사용할 수 있지?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
<td>&nbsp;</td>
<td>28G insulin syringe is preferred</td>
</tr>
<tr>
<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
<td>&nbsp;</td>
<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
</tr>
<tr>
<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
<td>Equipment</td>
<td>Xenogen Corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

다리와 발에 산소를 혈액과 영양소를 공급 주변 동맥이 좁아에서 주변 동맥 질환 (PAD) 결과,이 병리 같은 간헐 claudication (도보로 통증), 고통스러운 허혈성 ulcerations, 심지어 사지 위협 괴저와 같은 증상을 초래합니다. 이것은 일반적으로 혈관 내피 모든 혈액과 림프 혈관의 luminal 표면을 투자 내피 세포 monolayer이 혈관 항상성 및 혈관 재생에 지배적인 역할을 것으로 생각됩니다. 그 결과, 내피의 세포 기반의 재생을 막기위한 것이 PAD 치료를위한 유망한 접근법 수 있습니다. 이 비디오에서는, 우리는 배아 줄기 세포 (ESC)의 이식을 보여주 파생 bioluminescence 이미징에 의해 유도 세포 및 생존의 비침습 추적 다음 PAD의 모델로 일방적 hindimb 국소 빈혈의 치료에 대한 내피 세포를. 세포 전달 및 이미징에 대한 구체적인 자료와 절차가 설명됩니다. 이 프로토콜은 니이야마 외 의해 hindlimb 국소 빈혈의 유도를 설명하는 다른 간행물을 준수 1.

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

인도에 대한 수술 배아 줄기 세포 - 파생 허혈성 hindlimb에 내피 세포가 bioluminescence 이미징에 의해 비침습 추적, 증명합니다.

Hindlimb 국소 빈혈의 치료를위한 배아 줄기 세포 유래 내피 세포

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K 조회수.  Stanford University. 인도에 대한 수술 배아 줄기 세포 - 파생 허혈성 hindlimb에 내피 세포가 bioluminescence 이미징에 의해 비침습 추적, 증명합니다.

비디오: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

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 ...

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

인간 배아 줄기 세포에서 Mitochondrial 복잡한 활동에 대한 탐색

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Hindlimb 국소 빈혈의 Murine 모델

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Karyotyping를위한 인간 배아 줄기 세포의 염색체 확산 준비

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

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 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 ...