저자는 친절 하 게 제공 하는 난소 UNESP FCAV의 개 살 균 프로그램을 인정 합니다. 이 작품은 FAPESP (프로세스 번호 2013/14293-0)와 망 토에서 교부 금에 의해 지원 되었다.

이 실험은 4 개의 잡종 여성 개 개 살 균 프로그램에 선택 수술 후 기증의 난소와 함께 수행 되었다. 이 실험은 UNESP-FCAV (프로토콜 번호 026991/13)의 동물의 사용에 윤리 위원회에 의해 승인 되었다.
1. 실험 준비
<ol><li>준비 하거나 메 마른 Dulbecco 인산 염 버퍼 식 염 수 (DPBS) 칼슘 또는 마그네슘의 500 mL를 구매 합니다.</li>
	<li>준비는 콜라 나 DPBS의 1 mL에 효소의 40 µ g을 혼합 하 여 솔루션을 재고. 0.2 µ m 주사기 필터를 사용 하 여 필터링 하 고 저장 2 mL aliquots-20 ° c.에</li>
	<li>항생제의 1%와 10% 태아 혈 청 Dulbecco의 수정된이 글의 중간 (DMEM) 낮은 혈당에서 배양을 준비 합니다.</li>
	<li>살 균 자료 수집:가 위, 집게, 조직 배양 플라스 크, 요리, 튜브, 그리고 5 mL와 10 mL 펫.</li>
	<li>표준 셀 문화 실험실 장비를 사용 하 여: 생물 안전 캐비닛 (BSC), 5% CO2 와 38 ° C 원심 분리기에서 설정 셀 문화 인큐베이터.</li>
	<li>BSC를 청소: 70% 소독 장갑 낀 손으로 EtOH, UV 빛을 사용 하 여.</li>
</ol>2. 난소에서 중간 엽 줄기 세포의 분리
<ol><li>수술 후, 실험실에서 그들의 도착까지 난소 원뿔 튜브에 얼음에 PBS에 계속.</li>
	<li>PBS의 10 mL로 두 100 mm 페 트리 접시를 채우십시오.</li>
	<li>튜브에서 난소를 제거 하 고 PBS 가진 첫 번째 페 트리 접시에 그들을 전송. 움직임을 혼합 함께 그들을 씻어.</li>
	<li>부드럽게 난소를 검색 하 고 다른 접시에 놓습니다. 멸 균 핀셋과가 위, 아주 작은 조각, 약 1 m m 크기에서에 조직을 말하다.</li>
	<li>난소 조직 35 mm 페 트리 접시에 전송 하 고 콜라의 2 개 mL를 추가. 좀 더 조직을 말하다.</li>
	<li>3 h는 38 ° C에 인큐베이터에서 배양 접시를 놓고 부드럽게 원형 움직임 마다 20 분 페 트리 접시를 흔들.</li>
	<li>3 h 후 인큐베이터에서 배양 접시를 제거 합니다.</li>
	<li>15 mL 튜브에 접시의 내용을 전송 합니다. 확장 미디어의 5 mL를 추가 합니다.</li>
	<li>부드럽게 원심 분리 전에 튜브 반전. 원심에서 2100 x g 7 분 제거 튜브는 상쾌한 고 확장 미디어의 3 mL와 펠 릿을 resuspend.</li>
	<li>T25 병에 펠 릿을 전송 합니다. 5%와 인큐베이터에 병을 배치 3 h 38 ° C에서 CO2 . 참고: 프로시저의 가장 중요 한 부분은 외피의 3 h 후 미디어를 변경 하는.</li>
	<li>인큐베이터에서 병을 제거 하 고 나머지 조직으로 미디어를 제거 합니다. 병에 신선한 확장 미디어의 3 mL를 추가 합니다.</li>
	<li>미디어 모든 48 h 변경 하 고 관찰 셀룰러 합류에 대 한 병. 참고: 절차의 주요 단계는 그림 1에서 볼 수 있습니다.</li>
</ol>3. 난소에서 중간 엽 줄기 세포의 확장
<ol><li>
셀 통로
	<ol>
<li>셀 합류에 도달, 병에서 미디어를 제거 합니다.</li>
		<li>PBS의 3 mL 병을 씻어. PBS를 제거 합니다.</li>
		<li>트립 신 고 3 분에 38 ° C에 인큐베이터에서 병 부드럽게 탭 분리를 셀 수 있도록 병의 1.5 mL를 추가 합니다. 참고: 셀 합류 약 5-7 d 초기 도금 후에 도달 한다.</li>
		<li>확장 미디어의 3 mL를 추가 하 고 내용을 원뿔 튜브에 전송.</li>
		<li>원심에서 2100 x g 7 분 제거 튜브는 상쾌한 고 확장 미디어의 1 mL를 추가 합니다.</li>
		<li>부드럽게 튜브의 내용을 균질.</li>
		<li>세 고 인큐베이터에 다시 세포와 함께 튜브를 넣어 셀을 수행 하기 위해 샘플의 10 µ L의 약 수를 가져가 라.</li>
		<li>hemocytometer로 믹스의 장소 10 µ L.</li>
	</ol>
</li>
	<li>
셀 계산
	<ol>
<li>현미경의 10 X 목표를 사용 하 고는 hemocytometer의 눈금선에 초점.</li>
		<li>5 개의 작은 사각형 (그림 2)에 있는 셀을 계산 합니다.</li>
		<li>50, 000 밀리 리터 당 셀의 수를 추정 하 여 계산된 수를 곱하면.</li>
	</ol>
</li>
</ol>4. 난소에서 중간 엽 줄기 세포의 분화
참고: 차별화 분석 힐 외 에 설치 하는 지침에 따라 수행 했다 9.
<ol><li>잘, 3 중, 4 잘 문화 접시를 사용 하 여 당 씨 1.0 x 104 셀.</li>
	<li>확장 미디어의 1 mL를 추가 합니다.</li>
	<li>부드럽게 원형 움직임으로 접시를 교 반 하십시오.</li>
	<li>24 시간 후 문화 미디어를 바람직한 차별화 미디어 바꿉니다.</li>
	<li>대 한 osteogenic, adipogenic, 및 chondrogenic 분화, 품 어 30 d 유도 미디어 셀, 미디어 교체와 함께 모든 3 디 특정 차별화 키트가 분석이 실험의 각을 위해 사용 되었다.</li>
	<li>Neurogenic 혈통 차별화 유도 미디어 미디어 교체 매 3 d와 함께 10 일에 대 한 셀을 품 어. 여기에서 사용 하는 미디어 DMEM 낮은 포도 당, 2 mM valproic 산, 1 µ M 날린, 10 µ M 산림, 염화 칼륨 5 m m, 5 µ g/mL 인슐린, 그리고 200 µ M 자 hydroxyanisole 포함 되어 있습니다.</li>
	<li>Endoderm 선구자, 5 d 시 약 제조업체의 지침에 따라 endoderm 미디어에서 품 어.</li>
	<li>원시 생식 세포 계보 차별화 유도 미디어 미디어 교체 매 3 d와 14 d 셀을 품 어. 여기에서 사용 하는 미디어 포함 된 DMEM, 10 %FBS, 1mm 나트륨 pyruvate, 10 ng/mL LIF, 1mm 불필요 한 아미노산, 2 m L-글루타민 m, 5 µ g/mL 인슐린, 0.1 m m β-mercaptoethanol, 60 µ M putrescine, 20 µ g/mL 올려진다, 10 ng/mL 마우스 표 피 성장 인자, 인간의 1 ng/mL 기본적인 섬유 아 세포 성장 인자, 40 ng/mL 인 glial 세포 선 파생 된 neurotrophic 요인, 및 15 mg/L 페니실린.</li>
</ol>

<ol>
	<li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D., Atala, A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li><li>Ahmed, N., Thompson, E. W., Quinn, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li><li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17 (2005).</li><li>Gong, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li><li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145 (2004).</li><li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li><li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391 (2017).</li><li>Dominici, M. L. B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li><li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li><li>Kuznetsov, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li></ol>

저자는 공개 없다.

여기 증거 MSCs ovariectomy 후 생물 학적 폐기물 이라고 여겨진다 개 난소 조직 으로부터 격리 될 수 있습니다 제공 합니다. 사실은 많은 세포 유형 난소에서 찾을 수 있습니다, 우리 구와 같은 형태와 단층에서 성장 하는 셀을 성공적으로 선택, 플라스틱에 그들의 급속 한 준수에 따라 MSCs를 선택 하는 프로토콜을 제안 했다.
골 수에서 MSCs의 파생의 첫 번째 보고서는 문화<sup class="...

줄기 세포에 초점 년간 과거, 강력한 재생 의학 치료 발견의 집단 목표에 의해 연료 되었습니다 연구 노력을 실질적으로 증가 했다 출판된 연구의 수입니다. 줄기 세포는 두 개의 기본 정의 마커: 자기 혁신과 차별화. 중간 엽 줄기 세포 정기 조직 회전율에 대 한 책임은 고1배아 줄기 세포 비해 분화의 보다 제한 된 용량을가지고. 최근에, 많은 연구 다양 한 MSCs의 그리고 논의 중인 주제는 배아 및 성체 줄기 세포 간의 차이점 모든2에 존재 하는 여부.
Ovarium 표면 상피는 커밋되지 않은 세포의 층, 상대적으로 덜 분화, 두 상피와 중간 엽 마커3, 유지 하는 다른 종류의 세포에 대 한 응답에서으로 분화 하는 능력을 표현 하는 환경 신호4. 줄기 세포는 난소에서의 정확한 위치는 잘 알려진; 그러나, 그것은 bipotential 창시자 tunica albuginea에서 생식 세포5일으키 제안 되었습니다. 이러한 셀 stromal 기원6 또는에 있는 또는 난소 표면7에 인접 면역학 연구 가설 있다. 이후 중간 엽 줄기 세포 세포 접착8에 중요 한 역할을 재생 하는 수많은 수용 체를 표현, 실험 가설을 급속 한 접착을 가진 세포의 인구를 선택 하면 명확 하 게 세포의 인구를 분리 하는 것을 테스트 하도록 설계 되었습니다. 실제로 엽으로 characterizable. 최근, 우리의 그룹 문화의 첫 번째 3 h 문화 접시의 플라스틱 표면에 접착의 급속 한 접착9전시 세포의 순화 된 인구를 얻기 위하여 그들의 용량에 따라 난소 조직에서 MSCs의 파생을 했다. 여기는 난소 조직 으로부터 간 엽 줄기 세포 분리를 위한 개발된 방법에 설명합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DPBS&#160;</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">14190144</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Collagenase I</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">17100017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tissue flask</td>
			<td style="width:151px;">Corning</td>
			<td style="width:123px;">CLS3056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DMEM low glucose</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">11054020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FBS</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12484-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">TrypLE express</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12604021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Adipogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Chondrogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Osteogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">STEMdiff Definitive Endoderm Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:123px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Penicillin-Streptomycin</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD45</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2035S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD34</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2411GA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD90</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1036G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD44</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Nestin</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">&#946;-Tubulin&#160;</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB1637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DDX4</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:123px;">PA5 -23378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR80F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR120F</td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation and differentiation of canine ovarian mesenchymal stem cells

중간 엽 줄기 세포 (MSCs)에 대 한 관심은 절연, 확장, 및 문화의 그들의 용이성으로 인해 지난 10 년간 증가 했다. 최근에, 연구는 이러한 세포 넓은 차별화 용량을 증명 하고있다. MSCs에서 풍부 하 고 생물학 낭비로 ovariectomy 수술 후 자주 취소 됩니다는 사실 때문 난소 세포 기반 치료를 위한 유망한 후보자를 나타냅니다. 이 문서에서는 격리, 확장에 대 한 절차를 설명 합니다 및 MSCs의 세포 분류의 필요성 없이 개 난소에서 파생 된 기술. 이 프로토콜은 임상 시험에서이 매우 구별할 세포의 광범위 한 적용으로 인해 재생 의학에 대 한 중요 한 도구 나타내고 치료 사용.

송곳 니 난소에서 중간 엽 줄기 세포 분리:
난소 MSC 격리 절차는 그림 1에 요약 됩니다. 수술, 조직 닦지, 콜라 소화, 그리고 미디어 변화는 문화의 시작 후 3 h, 후 빠른 플라스틱 접착제 속성 상 상속 MSC 인구는 성공적으로 개 난소 조직 으로부터 격리. 빠르게 수확된 셀 문화 접시?...

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

여기, 우리가 격리, 확장, 및 개 난소 조직 으로부터 간 엽 줄기 세포의 분화 하는 방법을 설명합니다.

파생 및 개 난소 중간 엽 줄기 세포의 분화

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

이 절차의 전반적인 목표는 개 난소 조직에서 파생 된 중간 엽 줄기를 분리, 확장 및 차별화하는 것입니다. 이 실험은 개 살균 프로그램에서 선택 수술 후 기증 된 4 개의 mongrel 암컷 개난소와 함께 수행되었습니다. 이 실험은 상파울루 주립 대학, FVAC의 동물의 사용에 윤리위원회에 의해 승인되었다.대표적인 결과. 토론.결론.

derivation-differentiation-canine-ovarian-mesenchymal-stem

Research

JoVE Journal

Developmental Biology

5.6K 조회수.  São Paulo State University (UNESP). 이 절차의 전반적인 목표는 개 난소 조직에서 파생 된 중간 엽 줄기를 분리, 확장 및 차별화하는 것입니다. 이 실험은 개 살균 프로그램에서 선택 수술 후 기증 된 4 개의 mongrel 암컷 개난소와 함께 수행되었습니다. 이 실험은 상파울루 주립 대학, FVAC의 동물의 사용에 윤리위원회에 의해 승인되었다.대표적인 결과. 토론.결론.

비디오: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

배아 줄기 세포에서 심장 전구 세포의 유도

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

연구

발생학

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

줄기 세포에서 망막 색소 상피 세포의 효율적인 유도

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

작은 분자 변조 차별화 및 후속 포도당 기아을 사용하여 인간 유도 만능 줄기 세포에서 고도로 정제 된 심근 세포의 유도

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

배아 줄기 세포와 조직의 심장 차별화의 성적인 규정

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

생체 외에서 기능 Cardiomyocyte-같은 세포로 인간 중간 엽 줄기 세포의 분화

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

인간의 배아 또는 유도 만능 줄기 세포에서 망막 안료 상피 세포의 급속 한, 감독 차별화

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.