Name: isolation of endothelial progenitor cells from human umbilical cord blood
Uploaded: 2017-09-14T14:00:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

이 자료는 부여 번호 아래 국립 과학 재단에서 지 원하는 작업 기반 CMMI 1452943 고 아칸소 대학 명예 대학에 의해. 우리 또한 아칸소 코드 혈액 은행 코드 혈액 단위를 제공 하 고 싶습니다.

이 연구 아칸소 기관 검토 위원회 (승인 번호 16-04-722) 대학교의 승인을 실시 되었다. 탯 줄 혈액 단위 구 연산 인산 포도 당 (일일) 솔루션 아칸소 코드 혈액 은행에서 수집 그리고 저장에 대 한 요구 사항을 충족 하지 않았다 단위 연구에 대 한 기증 했다. 코드 혈액 단위 했다 주위 온도에 컬렉션의 24 시간 이내 실험실에 couriered.
 1. 절연의 내 피 조상 세포 탯 줄 혈액에서 <ol><li>시 약의 준비.
	인슐린 같은 성장 인자 (IGF)의 20 ng/mL, 1 µ g/mL 의약품, 5 ng/mL 재조합 인간 표 피 성장 인자 (rhEGF), 22.5 µ g/mL <ol><li>준비 EGM 10% 태아 둔감 한 혈 청 (FBS)에 내 피 기저 매체 (EBM)을 추가 하 여 보완 헤 파 린, 그리고 0.5 ng/mL 혈관 내 피 성장 인자 (VEGF)와 10 ng/mL의 재조합 인간 섬유 아 세포 성장 인자 B (rhFGF-B), 0.2 µ g/mL 날린, 2% 페니실린-스-글루타민. 0.2 µ m polyethersulfone (PES) 멤브레인 진공 필터를 사용 하 여 배양 소독.</li>
		<li>준비 쥐 나 콜라겐 솔루션 0.02 N 초 산을 사용 하 여 50 µ g/mL의 농도에서 꼬리. 0.2 µ m 주사기 필터를 사용 하 여이 솔루션을 소독.</li>
		<li>사전 코트 6-잘 접시 준비 쥐의 2 개 mL 나 각 잘 콜라겐 솔루션 꼬리. 뿌리기 전에 5% CO 2와 적어도 1 시간 동안 37 ° C에서 유지 하는 인큐베이터에 배치.</li>
		<li>탯 줄 혈액으로 행 크 균형 소금 솔루션 (HBSS) 1:1 농도의 약 25 mL를 희석.</li>
		<li>준비 라디오 면역 강 수 분석 결과 150 m m 염화 나트륨, 1% 트라이 톤 X-100, 0.5% 나트륨 deoxycholate, 1 m m β-glycerophosphate, 0.1 %SDS, 및 50 mM Tris (pH 8.0)를 사용 하 여 (RIPA) 버퍼.</li>
	</ol></li> <li>내 피 조상 세포의 고립.
	<ol><li>따뜻한 모든 준비 물 목욕 격리 전에 37 ° C에서 유지 시 약.</li>
		<li>밀도 그라데이션 중간 시 약 20 mL 50 mL 원심 분리기 튜브 추가.</li>
		<li>신중 하 게 20 mL 튜브의 측면 벽 쪽으로 피 펫을 목표로 하 여이 레이어를 깨지 않고 밀도 그라데이션 매체를 포함 하는 원심 분리기 튜브에 희석된 코드 혈액의 레이어.</li>
		<li>에서 원심 분리기 회전자의 브레이크와 함께 실 온에서 30 분 동안 800 x g에서 centrifuging 그들의 밀도 ( 그림 1)에 따라 혈액의 구성 요소를 구분 합니다. 다른 레이어를 방해 하지 마십시오.</li>
		<li>다른 셀 피 펫 팁 MNC에 도달할 수 있게 될 때까지 레이어 레이어 ( 그림 1 참조)을 방해 하지 않고 튜브에서 신중 하 게 pipetting으로 플라즈마 레이어를 제거 합니다. 제거 된 플라즈마 삭제. 
		참고: 피 펫 팁을 사용 하 여 플라즈마를 제거를 하는 동안 떠나 MNC 레이어 ( 그림 1) 장애를 줄이기 위해 위에 플라즈마의 작은 층.</li>
		<li>MNCs 18 게이지 바늘에 장착 한 주사기를 사용 하 여 포함 된 버 피 코트를 수집 하 고 신선한 원심 분리기 튜브 전송.</li>
		<li>EBM의 동일한 볼륨 수집된 MNCs. 원심 분리기를이 관에 추가 10 분 동안 500 x g 4에 ° C. 삭제는 상쾌한. 
		참고: 셀 펠 릿 나타납니다 붉은 적혈구/적혈구의 존재 때문에.</li>
		<li>붉은 혈액 세포를 lyse 염화 염화 물 해결책의 5 mL를 추가 합니다. 가끔 흔들어와 5-10 분 동안 얼음이 튜브를 품 어. 
		참고: 주의 고려 해야한다이 단계 동안에 지속 시간을 초과 하지 그것은 MNCs에 유해할 수 있다.</li>
		<li>원심 4 ° C에서 10 분 동안 500 x g에서 튜브와 삭제는 상쾌한. 적혈구 계속 되 면, 단계 단계를 반복 1.2.8 및 1.2.9 아니 붉은 색 펠 릿에서 관찰 됩니다.</li>
		<li>준비 EGM에 펠 릿을 resuspend를 사용 하는 hemocytometer는 MNCs 계산.</li>
		<li>Aspirate 쥐 꼬리를 나 콜라겐 코팅 6 잘 플레이트 (1.1.3 단계)에서 고 인산 염 버퍼 식 염 수 (PBS) x 1와 3 번 웰 스 세척.</li>
		<li>1 x 10 7에 각 잘 MNCs의 농도에 콜라겐 플레이트 EGM에 resuspended MNC 펠 렛 씨앗. 37 ° C, 5% CO 2 배양 기에.</li>
		<li>후 24 h 매체를 발음 하 고 한번 EGM와 웰 스를 씻어. 각 잘을 EGM 매체의 3 mL를 추가 하 고 37 ° C, 5% CO 2 배양 기에 다시 배치.</li>
		<li>신선한 EGM 매체와 접시 7 일 동안 매일 보충. 7 일 후 변경 EGM 매체 매일.</li>
		<li>식민지 내 피 세포의 진행을 추적 하기 위해 매일 6 잘 플레이트의 이미지를 받아 밝은 분야 현미경을 사용 하 여. 식민지 그들의 성장을 위해 발생 하는 플레이트 표시. 
		참고: 그것은 일반적으로 5 ~ 9 일 문화에 식민지를 위한 걸립니다.</li>
	</ol></li> <li>내 피 조상 세포의 확장.
	<ol><li>코트 쥐와 T-25 문화 플라스 크 나 콜라겐 (50 µ g/mL) 꼬리를 37 ° C, 5% CO 2 인큐베이터 적어도 60 분 Aspirate에에서 배치 하 고 셀을 뿌리기 전에 1 x PBS와 세 번 T-25 셀 문화 플라스 크를 씻고.</li>
		<li>Trypsinize 내 피 세포 식민지 각 우물에서 0.05% 트립 신-EDTA 용액의 150 µ L를 사용 하 여 크기 약 3 m m를 도달할 때. 6 잘 플레이트 37 ° C, 5% CO 2 배양 기 2-3 분에 대 한 다시 넣습니다</li>
		<li>는 부드럽게 연결 된 셀을 꺼내 려 6 잘 플레이트를 누릅니다. 즉시 EGM의 2 개 mL를 추가 하 고 15 mL 원심 분리기 튜브에 세포를 복구.</li>
		<li>는 hemocytometer를 사용 하 여 셀의 개수 및 초기 시드 밀도 계산.</li>
		<li>스핀 15 mL 원심 분리기 튜브 5 분에 대 한 400 x g에서 EGM에 셀 펠 릿 Resuspend 고 ~ 500000 셀 T-25 플라스 크를 시드합니다. 통로 1이 술병을 표시 하 고 37 ° C, 5% CO 2 배양 기에 다시 T-25 플라스 크를 배치. 
		참고: 셀은 EBM과 DMEM EGM와 성장 비교에서 도금 될 수 있다.</li>
		<li>추가 구절에 대 한 플라스 크에 도달 하면 90% 합류, 단계 1.3.1-1.3.5 고 T-75 또는 T-175 셀 문화 플라스 크에 10 4 셀/c m 2에서 셀을 다시 시드합니다. 고립 된 세포 (2 단계와 3 단계)의 특성을.</li>
	</ol></li></ol> 2. 격리 된 셀 사용 하 여 서 부 럽의 격리 된 셀 특성 확장 단계를 따라 모든 통로에 <ol><li>추가 50-100 µ L의 세포의 용 해 버퍼. 약 500000 이상의 세포에서 단백질을 수집.</li>
	<li>세포 파열 풀어 단백질을 적극적으로 위쪽 및 아래쪽 플라스틱. 수집 하 고 전송 버퍼 microcentrifuge 튜브.</li>
	<li>500 x g에서 microcentrifuge 튜브를 회전 하 고 신중 하 게 새로운 microcentrifuge 튜브에는 상쾌한 전송. 레이블 및 장기 저장을 위한-80 ° C에서 튜브를 저장.</li>
	<li>어느 브래드 퍼 드를 사용 하 여 각 튜브에 있는 단백질의 양을 계량 &#39; s 또는 BCA 시험 27 , , 28 29.</li>
	<li>서양 blotting 표준 절차 27 , , 28 29를 사용 하 여 수행 합니다. 5 µ g CD31, CD34, VEGFR2, 및 α-SMA의 표현에 대 한 총 단백질의 분석. 데이터 정규화에 대 한 α-tubulin 사용.</li>
</ol> 3. 간접 면역 형광 <ol><li>Sonicate 50% 에탄올에서 18 m m 유리 coverslips 고 그들을 건조. 두고 깨끗 한 coverslips biosafety 두건에서 하룻밤 12-잘 접시에 UV 빛에 그들을 소독.</li>
	<li>코트 50 & # 18와 coverslips1; g/mL의 쥐 나 콜라겐 꼬리 및 접시를 37 ℃, 5% CO 2 배양 기 적어도 60 분에 대 한 전송</li>
	<li>는 콜라겐을 발음 하 고 씻고는 coverslips 12-잘 접시에 1 x PBS의 1 mL를 추가 합니다. 1 x PBS를 발음 하 고 두 번 반복.</li>
	<li>씨앗 250000 경작에 각 잘 37 ° C, 5% CO 2 배양 기 immunostaining를 수행 하기 전에 적어도 3 일 동안 다시 Epc.</li>
	<li>세척 3 번 1 x PBS 가진 접시. 각 우물에 얼음 처럼 차가운 메탄올의 1 mL을 추가 하 고 셀을 해결 하기 위해 15 분 동안 실 온에서 품 어.</li>
	<li>Immunostaining 표준 프로토콜 27 , , 28 29를 사용 하 여 수행 합니다. 그들의 적절 한 희석에 항 체를 사용 하 여 (예를 들어, CD31 (1:20), CD34 (1:50), α-SMA (1: 100), 및 VEGFR2 (1:50)).</li>
	<li>Epifluorescence 현미경을 사용 하 여 immunostained coverslips의 이미지.</li>
</ol>

<ol>
	<li>Asahara, T., 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=Isolation+of+putative+progenitor+endothelial+cells+for+angiogenesis.">Isolation of putative progenitor endothelial cells for angiogenesis.</a> Science. 275 (5302), 964-967 (1997).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J Clin Invest. 105 (1), 71-77 (2000).</li><li>Shi, Q., 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=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92 (2), 362-367 (1998).</li><li>Hirschi, K. K., Ingram, D. A., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+identity,+phenotype,+and+fate+of+endothelial+progenitor+cells.">Assessing identity, phenotype, and fate of endothelial progenitor cells.</a> Arterioscler Thromb Vasc Biol. 28 (9), 1584-1595 (2008).</li><li>Ingram, D. 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=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104 (9), 2752-2760 (2004).</li><li>Yoder, M. C., 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=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109 (5), 1801-1809 (2007).</li><li>Sales, V. L., 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=Transforming+growth+factor-beta1+modulates+extracellular+matrix+production,+proliferation,+and+apoptosis+of+endothelial+progenitor+cells+in+tissue-engineering+scaffolds.">Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds.</a> Circulation. 114, 193-199 (2006).</li><li>Sales, V. L., 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=Endothelial+Progenitor+Cells+as+a+Sole+Source+for+Ex+Vivo+Seeding+of+Tissue-Engineered+Heart+Valves.">Endothelial Progenitor Cells as a Sole Source for Ex Vivo Seeding of Tissue-Engineered Heart Valves.</a> Tissue Eng Pt A. 16 (1), 257-267 (2010).</li><li>Liew, A., Barry, F., O'Brien, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+diagnostic+and+therapeutic+considerations.">Endothelial progenitor cells: diagnostic and therapeutic considerations.</a> Bioessays. 28 (3), 261-270 (2006).</li><li>Hur, J., 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=Characterization+of+two+types+of+endothelial+progenitor+cells+and+their+different+contributions+to+neovasculogenesis.">Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis.</a> Arterioscler Thromb Vasc Biol. 24 (2), 288-293 (2004).</li><li>Ingram, D. A., Caplice, N. M., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unresolved+questions,+changing+definitions,+and+novel+paradigms+for+defining+endothelial+progenitor+cells.">Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells.</a> Blood. 106 (5), 1525-1531 (2005).</li><li>Melero-Martin, J. M., 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=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109 (11), 4761-4768 (2007).</li><li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods Enzymol. 445, 303-329 (2008).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+progenitor+cells.">Human endothelial progenitor cells.</a> Cold Spring Harb Perspect Med. 2 (7), 006692 (2012).</li><li>Siddique, A., Shantsila, E., Lip, G. Y. H., Varma, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+what+use+for+the+cardiologist.">Endothelial progenitor cells: what use for the cardiologist.</a> J Angiogenes Res. 2 (6), (2010).</li><li>Camci-Unal, G., 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=Surface-modified+hyaluronic+acid+hydrogels+to+capture+endothelial+progenitor+cells.">Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells.</a> Soft Matter. 6 (20), 5120-5126 (2010).</li><li>Hill, J. M., 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=Circulating+endothelial+progenitor+cells,+vascular+function,+and+cardiovascular+risk.">Circulating endothelial progenitor cells, vascular function, and cardiovascular risk.</a> N Engl J Med. 348 (7), 593-600 (2003).</li><li>Young, P. P., Vaughan, D. E., Hatzopoulos, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+properties+of+endothelial+progenitor+cells+and+their+potential+for+cell+therapy.">Biologic properties of endothelial progenitor cells and their potential for cell therapy.</a> Prog Cardiovasc Dis. 49 (6), 421-429 (2007).</li><li>Mehta, J. L., Szwedo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+progenitor+cells,+microparticles+and+vascular+disease.">Circulating endothelial progenitor cells, microparticles and vascular disease.</a> J Hypertens. 28 (8), 1611-1613 (2010).</li><li>Nevskaya, T., 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=Circulating+endothelial+progenitor+cells+in+systemic+sclerosis+are+related+to+impaired+angiogenesis+and+vascular+disease+manifestations.">Circulating endothelial progenitor cells in systemic sclerosis are related to impaired angiogenesis and vascular disease manifestations.</a> Ann Rheum Dis. 66, 67-67 (2007).</li><li>Cebotari, S., 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=Clinical+application+of+tissue+engineered+human+heart+valves+using+autologous+progenitor+cells.">Clinical application of tissue engineered human heart valves using autologous progenitor cells.</a> Circulation. 114, 132-137 (2006).</li><li>Ito, H., 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=Endothelial+progenitor+cells+as+putative+targets+for+angiostatin.">Endothelial progenitor cells as putative targets for angiostatin.</a> Cancer Res. 59 (23), 5875-5877 (1999).</li><li>Vasa, M., 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=Increase+in+circulating+endothelial+progenitor+cells+by+statin+therapy+in+patients+with+stable+coronary+artery+disease.">Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease.</a> Circulation. 103 (24), 2885-2890 (2001).</li><li>Wu, X., 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=Tissue-engineered+microvessels+on+three-dimensional+biodegradable+scaffolds+using+human+endothelial+progenitor+cells.">Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells.</a> Am J Physiol Heart Circ Physiol. 287 (2), 480-487 (2004).</li><li>Boyer, M., 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=Isolation+of+endothelial+cells+and+their+progenitor+cells+from+human+peripheral+blood.">Isolation of endothelial cells and their progenitor cells from human peripheral blood.</a> J Vasc Surg. 31 (1), 181-189 (2000).</li><li>Huber, B., Czaja, A. M., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+epidermal+growth+factor+(EGF)+and+hydrocortisone+on+the+co-culture+of+mature+adipocytes+and+endothelial+cells+for+vascularized+adipose+tissue+engineering.">Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.</a> Cell Biol Int. 40 (5), 569-578 (2016).</li><li>Sturdivant, N. M., Smith, S. G., Ali, S. F., Wolchok, J. C., Balachandran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetazolamide+Mitigates+Astrocyte+Cellular+Edema+Following+Mild+Traumatic+Brain+Injury.">Acetazolamide Mitigates Astrocyte Cellular Edema Following Mild Traumatic Brain Injury.</a> Sci Rep. 6, 33330 (2016).</li><li>Lam, N. T., Muldoon, T. J., Quinn, K. P., Rajaram, N., Balachandran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valve+interstitial+cell+contractile+strength+and+metabolic+state+are+dependent+on+its+shape.">Valve interstitial cell contractile strength and metabolic state are dependent on its shape.</a> Integr Biol (Camb). 8 (10), 1079-1089 (2016).</li><li>Tandon, I., 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=Valve+interstitial+cell+shape+modulates+cell+contractility+independent+of+cell+phenotype.">Valve interstitial cell shape modulates cell contractility independent of cell phenotype.</a> J Biomech. 49 (14), 3289-3297 (2016).</li><li>Cockshell, M. P., Bonder, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Human+CD133++Non-adherent+Endothelial+Forming+Cells.">Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells.</a> Bio-Protocol. 6 (7), (2016).</li></ol>

저자는 공개 없다.

앞에서 설명 했 듯이, 점착 Epc 조약돌 형태를가지고 있습니다. 우리의 절연된 MNCs 진행 스핀 들 모양의 셀 식민지 (그림 2A 2D)에서 초기 단계에 조약돌 식민지 (그림 2E-2 층) 문화에 10 일의 기간 동안. Epc는으로 분류 되었습니다 다른 연구 그룹에 의해 다르게 즉 늦은 내 피 조상 세포10, 셀5또는...

여러 조사 특성 및 인간의 Epc5,,1011,,1213의 잠재력을 공부 했다. Epc 세포 저 산소 증, 허 혈, 부상, 또는 종양 형성의 사이트에 내 피 조직에 고착 하 고 새로운 혈관 구조4,14의 형성에 기여 하는 능력을가지고 순환 이라고 할 수 있습니다. 출생 후 vasculogenesis의 형태로 neovascularization에 그들의 관찰된 관련 이상 이러한 세포의 치료 응용 프로그램4,15, 에 그들의 사용에 대 한 이해를 주도하 고 있다 16. 개인의 Epc 수 심장 혈관 병 리9,15,,1617,18,19와 상관을 보였다 ,20. 다른 연구는 Epc 밸브 구와 같은 표현 형으로 분화 또한 있고이 세포 조직 공학 심장 밸브7,21사용할 수 제안.
Epc를 분리 하는 데 필요한 특정 세포 표면 분자는 하지 명확 하 게 확인 되었습니다 조사4사이 불일치 때문에. 다양 한 문화 조건에 노출 특정 매트릭스 MNCs의 접착 여러 그룹1,17,,2223, putative Epc 수 있습니다 제안에 의해 수행 되었습니다. 다른 phenotypic 속성을 표시 합니다. 이러한 속성에는 phagocytotic, Matrigel에 관 형성 능력과 Dil acetylated 저밀도 지 단백질의 이해의 부족 포함 됩니다. 높은 clonogenic 및 증식 잠재력 어떤 Epc hierarchized5수 하는 두 개의 속성이 있습니다. Epc 또한 생체 외에서 tubules 때 인간의 태아 폐 섬유 아 세포4cocultured를 형성할 수 있습니다. 이 세포는 내 피 세포 표면 마커를 표현 하 고 조 혈 마커13,,2425의 일부를 공유 하 알려져 있습니다. 긍정적으로 표현된 마커를 형질 Epc에 대 한 널리 받아들여진다는 CD31, CD34, 혈관 내 피 성장 인자 수용 체 2 (VEGFR2), 폰 Willebrand 인자 (vWF), CD133, c-Kit 및 혈관 내 피 cadherin (VE-cadherin)4 , 18. 공동 CD90, CD45, CD14, CD115, 또는 알파-부드러운 근육 걸 (α-SMA)를 표현 하는 세포 phagocytose 박테리아와 드 노 보 인간을 형성 하는 무 능력을 그들의 한정 된 증식 잠재력, 능력 때문에 Epc 수로 간주 되지 않습니다 선박 vivo에서4,7. 이 문서는 수정 된 프로토콜 프로토콜 정렬 셀에 대 한 필요 없이 인간 탯 줄 혈액에서 내 피 조상 세포의 고립에 대 한 설명. 이 문서에 대 한 CD31, CD34, 및 VEGFR2 부정적인 지표로 α-SMA와 긍정적인 마커로 사용 우리.
이 문서에서는, 우리는 분리 하 고 셀을 사용 하 여 정렬 없이 탯 줄 혈액에서 내 피 뿌리 세포를 배양 하는 방법 전문 성장 요인 (EGM) 보충 하는 내 피 성장 매체 제안 합니다. 이 EGM이 혈관 내 피 성장 인자 (VEGF)와 섬유 아 세포 성장 인자 (FGF), 생존, 확산, 그리고 내 피 세포26의 마이그레이션 향상 포함 되어 있습니다. 그것은 또한 포함 하는 셀;의 조약돌 형태를 유지 하는 의약품 인슐린 같은 성장 인자-1 (IGF-1), 신생 및 철새 기능; 그리고 헤 파 린,26의 중간 성장 요인의 향상 된 장기 안정성을 하면. 내 피 세포 배양에 추가 다른 성장 인자 자극 세포 증식 및 분화, EGF26 하 셀 sensitizes 날린 데 도움이 표 피 성장 인자 (EGF)와 보완을 포함 . 우리는이 특정 성장 매체를 사용 하 여 내 피 기저 매체 (EBM) 또는 Dulbecco의 수정이 글 중간 (DMEM)에 비해 Epc의 높은 숫자를 생성 보여줍니다.

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>BDH1135-4LP</td><td></td></tr></tbody></table>

isolation of endothelial progenitor cells from human umbilical cord blood

내 피 조상 세포 (Epc) 말 초 혈액에서의 존재와 vasculogenesis에의 참여 Ashara와 동료1에 의해 처음 보고 되었다. 나중에, 다른 비슷한 유형의 골2,3에서 발생 하는 Epc의 실존 문서화. 더 최근에, 요더와 Epc 탯 줄 혈액에서 파생 했다 잉 그램은 높은 증식 잠재력 성인 주변에서 고립 된 사람에 비해 혈액4,,56했다. 출생 후 vasculogenesis에 참여 하 고, 떨어져 Epc 또한 나타났습니다 약속 만드는 조직 설계 혈관 및 심장 밸브 구조7,8셀 원본으로. 존재 하는 다양 한 격리 프로토콜, 일부는 포함 하는 단 세포 (MNCs) 내 피 및 조 혈 마커의 도움으로 설명한 소스에서 파생 된 셀 정렬 또는 특수 내 피 성장 이러한 MNCs를 경작 중간, 또는 이러한 기법9의 조합. 여기, 선물이 고립에 대 한 프로토콜 및 사용 하 여 Epc의 문화 전문 내 피 중간 immunosorting, 뒤에 서쪽에 게 더 럽 히기을 사용 하 여 격리 된 세포의 특성의 사용 없이 성장 인자를 보충 하 고 immunostaining입니다.

절연 및 내 피 조상 세포의 확장: 회로도 (그림 1) 전체 프로토콜을 묘사한 제공 됩니다. 다른 혈액 구성 요소 레이어 밀도 밀도 그라데이션 매체와 인간 탯 줄 혈액의 그라데이션 원심 다음 관찰 되었다. 콜라겐 치료 접시에 MNCs를 시드 시 식민지의 파생물 일 5와 7 (그림 2A) 사이 처음 관찰 되었다. 이?...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

이 프로토콜의 목표는 내 피 뿌리 탯 줄 혈액에서 세포를 분리. 조직 설계 만드는 혈관 및 심장 밸브 구성 및 응용 프로그램의 일부는 biomarker로 심혈 관 위험, 허 혈 성 질환 치료 환자를 식별 하는 데 이러한 세포를 사용 하 여 포함 됩니다.

인간 탯 줄 혈액에서 내 피 조상 세포의 고립

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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Developmental Biology

11.1K 조회수.  University of Arkansas. 이 프로토콜의 목표는 내 피 뿌리 탯 줄 혈액에서 세포를 분리. 조직 설계 만드는 혈관 및 심장 밸브 구성 및 응용 프로그램의 일부는 biomarker로 심혈 관 위험, 허 혈 성 질환 치료 환자를 식별 하는 데 이러한 세포를 사용 하 여 포함 됩니다.

비디오: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

말초 혈액 조혈 전구 세포에서 인간 신경 줄기 세포의 직접 유도

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

연구

발생학

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

인간 제대 베인 내피 세포의 절연 (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

생물학

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

쥐 배아 Hemogenic 내피 세포의 분리

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

흐름 조건에서 인간 제대 정맥 내피 세포 및 호중구 윤회의 연구에서 이들의 사용 격리

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

면역학 및 감염병학

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

탯줄 혈액에서 전구체 B-세포 하위 집합의 절연

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

인간 코드 혈액 유래 CD34+ 조혈 줄기 및 전구 세포의 범골수성 분화

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

심장 수술 후 혈청 샘플의 영향을 건강한 자원 봉사자와 그들의 철새 잠재력에서 내피 전구 세포의 분리

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

의학

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

내피 콜로니 형성 세포의 Phenotypic 및 기능성 특성은 인간 탯줄의 혈액에서 유래

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

인간 탯 줄 혈액 파생 CD34에서 조 혈 줄기 세포의 생체 내 확장 전+ 세포 Valproic 산 사용 하 여

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

인간 탯 줄 혈액에서 내 피 조상 세포의 고립

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