저자는 현재 영화 &#39;씨 카일 홀덴 및 슬라이드 편집 그들의 도움 씨 조나단 루카스에 대한 세포 격리 그들의 기술 도움을 양 Haiying 팬 씨와 이안 Bellayr의 크게 감사하고 있습니다. DRS : 지난 년 동안 수정된 preplate 기술의 개발에 관여했습니다 사람의 다음과 같은 목록에 많은 감사드립니다. Burhan Gharaibeh, BaoHong 카오, Deasy 브리짓, 토마스 R. 페인, Aiping 루, Hairong 펭, 히데키 오시마, 보 Zeng, Xiaodong 뮤, Kimimasa 도비타, 론 Jankowski, Makato Ikezawa. 우리는 제임스 H. 커밍, 라이언 Pruchnic, 요셉 Feduska, 레베카 C. Schugar, Haiying 팬과 제시카 Tebbets의 기술 지원에 감사하고 있습니다. 저자는 또한 근육질 영양 장애 협회 (MDA), 국립 보건원 (NIH), 국방부 (DOD), UPMC의 피츠버그의 아동 병원의학과이 작품에 대한 재정 지원을 인정하고 싶습니다 정형 외과 외과, 그리고 피츠버그 대학.

일반적으로 전체 수정 preplate 절차는 준비 1-2 일, 기술적인 절차를 수행하기 1 주, immunocytochemistry와 세포 식별로부터 2-3 일 및 줄기 세포 인구 확장의 추가 2~3주이 필요합니다. 줄기 세포 배양에 필요한 장비는 벤치 - 최고 원심 분리기, CO 2 배양기, 층류 조직 문화 후드와 형광과 디지털 카메라가 장착된 거꾸로 현미경을 포함하는 다른 세포 배양 시스템의 비슷합니다. 분리된 줄기 세포는 복제 수 있으며 현재 또는 미래의 연구 1,2을위한 액체 질소의 장기 저장할 수 있습니다. 이 절차에 사용된 모든 시약 및 재료 살균하고 무균 처리합니다. 1 단계 : 준비 <ol><li> 콜라겐 코트 조직 문화 요리와 flasks : 송아지 가죽 (일리터에 0.1 g, 시그마 - 알드리치)에서 파생된 유형 I 콜라겐. 코팅에 plasticware은 다음과 같습니다으로 1,2 앞에 설명한 T - 25 flasks, 6 또는 12 자 접시, 요리 (60 100mm, BD 팰컨). 부드럽게 콜라겐의도 코팅을 보장하기 위해 4 시간, plasticware에게 매 30 분마다 흔들어. 콜라겐 솔루션은 다음 제거됩니다. 그리고 코팅 plasticware는 다음 불임을 보장하기 위해, 자외선과 후드에 모두 조직 문화 후드의 건조 수 있으며, 마지막으로 recapped 또는 나중에 사용하기 위해 있었다. </li><li> ;, 50mL 열 Inactivated 태아 소 혈청 (FBS, Invitrogen), 50mL 호스 세럼 (HS, Invitrogen), 5 ML의 칙의 배아 추출 (시, 정확한 화학 (주) 400mL DMEM (Invitrogen DMEM, 높은 혈당) : 세포 배양 매체 준비 ) 일회용 0.22 - μm의 500 ML 살균 필터 시스템 (코닝)를 사용하여 필터링 진공 것입니다. 무균 페니실린 / 스트렙토 마이신 솔루션 (Invitrogen)가 마침내 100 단위 / ML 추가됩니다. 준비 솔루션은 최소한 한 달 동안 4 ° C에 저장할 수 있습니다. 이 솔루션은 술병에서 세포를 분리하는 데 사용되는 경우 분할 문화 또는 이식을위한 세포를 준비. </li><li> 세포 분리를위한 솔루션을 따뜻하게 다음 시약 37을 예열해야 ° C 세포 분리 위해 사용하기 전에 : 행크의 버퍼 소금 솔루션 (HBSS, Invitrogen), 0.2 % (WT / 권) collagenase 타입 XI (시그마 Dispase 2.4 units/1mL (Invitrogen), 0.5 % (WT / 권) 트립신 (Invitrogen) ML HBSS 당 3,200 콜라겐 소화 단위의 평균 - 알드리치). </li><li> 준비 수술 장비를 소독 : 서로 다른 크기의 포셉, 마이크로 가위, 가위 홍채 및 / 또는 메스 블레이드 : 수술을 포함한 살균 수술 도구가 필요합니다. 또한, 15 50 ML의 폴리 프로필렌의 원심 분리기 튜브 (BD 팔콘), 셀 스트레이너 (기공 크기 70m) (BD 팔콘), 18 -, 23 - 27 - 게이지 바늘 (BD PrecisionGlide) 10 - 또는 20 - ML 무균 주사기 (BD). </li></ol> 2 단계 : 조직 biopsies, 절연 및 기계적 해리 1-4 조직 biopsies은 살균 문화 후드 아래에 수행됩니다 갓 수확 힘줄과 골격 근육을 포함은 야생 형 생쥐의 hindlimb (여성 C57BL/6J, 4-5 세 주 이하, 잭슨의 경골 또는 soleus 근육에서 얻을 수 있습니다 연구소). 모든 보이는 결합 조직, 혈관, 그리고 지방 조직은 다음 생검 샘플에서 제거됩니다. 힘줄과 근육의 조직은 다음 조심스럽게 피부와 뼈의 잔해를 제거하는 수술 해부 현미경으로 식별 및 포함된 차가운 (4 ° C) HBSS (FBS의 5 %를 추가로 보충).을 접시에 배치됩니다 분리된 조직은 다음 별도 추운 HBSS를 포함하는 콜라겐 코팅 요리에 위치하고 있으며 마이크로 가위 및 / 또는 메스 블레이드를 사용하여 굵은 슬러리에 (&lt;1x1 mm 3) 다진 것입니다. 3 단계 : 조직의 효소 소화 : 다진 조직 단계 2,4,5 일련의를 통해 다음 효소 dissociated입니다 <ol><li> 별도의 15 ML 튜브와 4 ~ 3,500 rpm으로 원심 분리기 ° C 5 분에 다진 조직 slurries을 전송합니다. </li><li> 뜨는 제거 HBSS로 세척하고 다시 원심 분리를 반복합니다. </li><li> 뜨는을 제거하고, prewarmed 0.2 % collagenase 타입 XI (시그마)의 10 ML을 추가하여 이러한 slurries을 소화. 37 60 분에 대해 품어 ° C 연속적으로 부드럽게 튜브 락을하면서. 또는 수동으로 매 10 분 흔들. </li><li> 원심 분리기 10 ML dispase (2.4 단위 / ML, Gibco) 솔루션에서 이러한 slurries을 resuspend. 손을 떨고 또는 부드럽게 매 10 분 로킹, 37 ° C에서 45 분에 대해 품어. </li><li> 0.2 % 트립신의 HBSS 용액 10 ML 이러한 slurries을 원심 분리기 다시 일시 중지합니다. 부화는 하나의 세포가 현미경으로 정학을 준수하여 수행할 수있는 조직에서 발표되는 수있는 정도에 따라 달라집니다. 그것은 반전 튜브 ° 37 30 분을 초과하는 C를 권장하거나 부드럽게 매 10 분 락을하지 않습니다. </li><li> 결과 세포를 원심 분리기S 다시 중단 확산 중간 (PM) 10 ML에있는 세포 펠릿합니다. </li><li> 바늘 일련의를 통해 추출을 통과하여 세포 현탁액을 떼어 놓다. 세포가 다음 18G 통해 23G를 2 번 통과하고, 마지막으로 27G 바늘입니다. </li><li> 70 μm의 셀 스트레이너를 통해 세포 추출물 패스. </li></ol> 4 단계 : 세포 접착 률 1-3에 따라 서로 다른 세포 집단을 분리 Preplating <ol><li> 원심 분리기 및 확산 매체 셀 알약을 resuspend. </li><li> 플레이트 셀룰라 콜라겐 - 코팅 T - 25 플라스크 (또는 60mm 요리)에 혼합하고 PP1로 표시합니다. 세포 현탁액은 현미경으로 굴절 핵 및 다른 파편의 큰 숫자를 가진으로 관찰해야합니다. </li><li> 37 품어 ° 2 시간 동안 humidified 5 % CO 2 배양기에서 C. </li><li> T - 25 플라스크 (또는 60mm 요리) 마크가 PP2 그대로 새로운 콜라겐 - 코팅에 nonadherent 세포를 전송합니다. PP1 표시 접시에 PM 5 ML을 추가합니다. 플라스크에 연결 초기 준수 세포가 대부분 myofibroblasts 삼아르. </li><li> (또는 접시) T - 25 플라스크 새로운 콜라겐 - 코팅에 nonadherent 세포를 전송하고 PP3로 표시, 24 시간 동안 인큐베이터에 flasks를 반환합니다. PP2 표시된 판으로 확산 중 5 ML을 추가합니다. 이 플라스크에 연결이 준수 전지는 대부분 섬유아 세포 2,3입니다. </li><li> 인큐베이터에 flasks를 반환하고 인구 PP6가 만들어 때까지 또 다른 24 시간의 절차를 반복하십시오. 정지 세포의 각 그룹을 유지하고 세포 분열 따위에 의해 번식하고 거꾸로 현미경을 사용하여 정기적으로 그들을 검토하도록 허용합니다. 근육 위성 세포가 자주 PP5 2 주로 볼 수있는 동안 PP3 - PP4은 대부분 myoblasts 있습니다. </li><li> 천천히 준수 세포의 대부분은 일반적으로 PP6로 연결합니다. 이 단계에서는 세포가 작고, 둥근, 빛의 굴절을 표시하고 번호 매우 희박한 있으며 줄기 세포의 인구 1,2로 간주됩니다. </li></ol> 5 단계 : 식별 및 격리 줄기 세포의 확장 <ol><li> 고립된 PP6 세포 수가 거의하고 매일 변경됩니다 FBS 풍부한 확산 중간 (20 % DMEM에 FBS)의 유지 관리해야합니다. 세포의 대부분은 culturing의 다음 1~2주 동안 PP6 문화 다이에 존재하지만, 대형, 건강 PP6 인구는 일반적으로 확장 추가 1-2주 후에 생성됩니다. 생쥐에서 분리된 줄기 세포는 고도로 줄기 세포 항원 1 (Sca1), CD34, 태아 간 키나제 1 (Flk1) 및 immunocytochemical 얼룩을 통해 시각 수있는 다른 줄기 마커 1,2,6,7을 표현. 이러한 줄기 세포는 여러 차별화 용량에게 3,6,8를 나타냅니다. </li><li> 고립된 세포 유지와 차별화 1,2를 피하기 위해 (물론이나 술병이나 접시에 &lt;20-30%의 합류 12 잘 50-100 세포에서 요리 /) 문화의 낮은 세포 밀도로 확장됩니다. 매우 세포 몇 확장 중에 클론을 형성하고, 이러한 복제 줄기 세포가 다른 능력 연구에 대한 오랜 시간이 확산 (LTP)를 받아야 수 있습니다. 복제된 줄기 세포는 10 % DMSO로 오후 중간 예를 들어, 일반적인 세​​포 저장 프로 시저를 사용하여 액체 질소에 저장할 수 있습니다. 다른 뒤로 최대 연구를위한 줄기 세포의 낮은 통과 주식의 번호를 확인합니다. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, 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=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

그것은 일부 성인 조직이 여러 개의 줄기 세포가 포함되어있다는 사실을 인정했습니다. 연구의 지난 몇 년 동안, 줄기 세포는 골격근과 힘줄을 포함한 부드러운 musculoskeletal 조직에서 발견되었습니다. 성공적으로 힘줄과 골격 근육에서 성인 줄기 세포를 분리하기 위해 실험실에서 사용되었습니다 수정된 preplate 기술로 알려진이 현재의 프로토콜은 세부 절차를. 세포 위에서 설명한 수정된 preplate ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
<tr>
<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
<tr>
<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
<tr>
<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type XI 0.2%(wt/vol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C7657</td>
<td>&nbsp;</td>
<tr>
<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>#D-2650</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolating stem cells from soft musculoskeletal tissues

성인 줄기 세포는 긴 재생 의료에서​​ 응용 프로그램에 관해서 논의했습니다. 성인 줄기 세포는 여러 혈통이 세포 분화 및 자기 갱신 능력을 받아야하기 때문에 그들의 인상적인 기능으로 부상하고 병에 걸린 조직을 치료에 대한 흥분의 큰 거래를 생성합니다. 가장 중요한 것은, 이러한 자질은 autologous 세포 이식 요법에 사용하기 위해 그들이 유리 만들었습니다. 현재 프로토콜은 musculoskeletal 시스템, 예를 들어, 힘줄 및 근육의 부드러운 조직 1의 수정된 preplate 기술에 독자를 소개합니다<sup&gt; 일</sup&gt; 매체와 콜라겐 코팅 flasks에 배치 효소 dissociated 후. 중간 나머지 부동 세포로 구성되어 있습니다 뜨는은, 순차적으로 새로운 flasks 매일 전송됩니다. 줄기 세포는 일반적으로 (시리얼 전송 또는 preplates) 5-7일 소요되는 flasks을 준수하기 위해 느린 있습니다. 이 방법을 사용하여, 성인의 줄기 세포가이 조직에있는 쉽게 상당히 비침습 절차를 통해 수확 수 있습니다.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

플라스크에 세포의 준수 속도에 따라 musculoskeletal 부드러운 조직에서 분리 성인 줄기 세포.

소프트 Musculoskeletal 조직에서 줄기 세포를 분리

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Research

JoVE Journal

Biology

13.3K 조회수.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. 플라스크에 세포의 준수 속도에 따라 musculoskeletal 부드러운 조직에서 분리 성인 줄기 세포.

비디오: Isolating Stem Cells from Soft Musculoskeletal Tissues

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

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

연구

생물학

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 ...

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 ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

인간 췌장암의 Xenografts에서 줄기 세포의 분리

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

인간 유도 Pluripotent 줄기 세포의 선택 및 분리 식민지는 성인 섬유아 세포에서 다시 프로그램

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

마우스의 심장에서 차 멜라닌 세포와 같은 세포를 분리

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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 ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

성인에서 장내 줄기 세포를 분리<em&gt; 초파리</em&gt; FACS로 Mi​​dguts는 노화 동안 줄기 세포 동작을 연구하기 위해

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...