This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

모든 실험 절차는 버밍엄에서 알라바마 대학의 동물 가이드 라인에 따라 수행된다. 1. hiPSC-CM이에 hiPSCs 차별화 <ol><li> 코트 밤새 4 ℃에서 사전 냉각 된 성장 인자 - 감소 된 젤라틴 단백질 혼합물로 6 웰 플레이트의 웰. 사용하기 전에 젤라틴 단백질 혼합물을 기음. 5 % CO 2, 10 μM의 ROCK 저해제와 37 ° C mTeSR1 중간 보충에있는 세포 (물론 당 1 × 10 5 세포를) 미리 코팅 된 플레이트 상에 hiPSCs 종자, 문화. </li><li> 세포가 90 % 합류에 도달 할 때까지 매일 배지를 새로 고침; 다음, 5 % CO 2, 37 ° C 두 개 더 일 동안 매체 (0.5 mg을 젤라틴 단백질 6 웰 플레이트 당 혼합물, 잘 당 2 ㎖의 중간), 문화에 세포 성장 인자 감소 젤라틴 단백질 혼합물을 추가합니다. <ol><li> 매체를 교체하려면, 부드럽게 진공 통해 페트리 접시에서 매체를 빨아아웃 셀을 터치하고, 전송 피펫을 사용하여 새로운 매체를 추가합니다. </li></ol></li><li> 보충 된 RPMI1640 배지 배지로 대체하여 분화를 시작하지 않고 인슐린 젤라틴 단백질 혼합물 B27를 성장 인자 - 감소, 100 NG / ㎖ 티빈 a 및 문화 5 % CO 2, 24 시간 동안 37 ℃에서 세포. </li><li> ; RPMI1640 인슐린없이 B27로 보충 된 배지 10 NG / ㎖ 뼈 형태 형성 단백질 4 (BMP-4), 10 ng의 / ㎖ 염기성 섬유 아세포 성장 인자 (bFGF)와 매체를 대체 문화 5 % CO 2, 96 시간 동안 37 ℃에서 세포. </li><li> B27 보충 된 RPMI1640 배지로 배지를 교체하고, 5 % CO 2 및 37 ° C에서 배양 세포를 계속; 3 일마다 배지를 새로 고칩니다. </li><li> 분화를 시작 후 3 일 ~ 현미경으로 세포를 수축의 클러스터를 관찰한다. ~ 칠일 최초의 수축을 관찰 한 후 클러스터를 수집하고 행크스 균형 소금 솔루션에 씻어. <ol><li> 부드러운 진동 37 ºC에서 10 분 동안 100 U는 / ㎖ 콜라게나 제 IV 포함 행크스 버퍼를 배양하여 접시에 클러스터 된 세포를 떼어 놓다; 이어서, 5 분 동안 에틸렌 디아민 테트라 아세트산 (EDTA) 0.25 % 트립신을 추가한다. </li><li> RPMI / B27 배지에서 10 % 소 태아 혈청 효소 용액을 중화하고 RPMI / B27 배지에서 세포를 재현 탁. </li><li> 혈구에 의해 세포 수를 계산. 문화 비 심근 세포가 배양 용기의 표면에 부착 할 수 있도록 5 % CO 2, 적어도 3 시간 동안 37 ℃에서 10cm 접시에 세포. </li></ol></li><li> hiPSC-CMS에 포함 된 세포 현탁액을 수집하고, 단백질 혼합물을 젤라틴 코팅 된 표면을 배양함으로써 (10cm 접시 당 1 × 106 세포 주위) 세포를 유지한다. </li></ol> 2. hiPSC-되는 EC에 hiPSCs 차별화 <ol><li> 일을 배양하여 단일 세포로 hiPSC 인구 해리5 % CO 2, 5 분 동안 37 ° C에서 킬레이트 제와 엠. mTeSR1 신선한 배지를 함유하는 15 mL의 원심 분리 튜브에 세포를 옮긴다. </li><li> 5 분 동안 200 XG에서 세포를 스핀 다운. 10 μM의 ROCK 저해제로 보충 mTeSR1 매체에 세포를 재현 탁. 혈구와 셀 밀도를 결정합니다. </li><li> 24 웰 플레이트 잘 하나에 20 NIH 단위 / ml의 트롬빈 용액 250 μl를 추가합니다. </li><li> 12.5 ㎎ / ㎖ 피브리노겐 용액 250 ㎕를 1 × 10 6 hiPSCs를 추가합니다. 그리고 트롬빈로드 웰에 세포 함유 피브리노겐 용액 250 μl를 추가합니다. 혼합물 분 내에 hiPSC 함유 섬유소 발판을 형성하기 위해 응고 관찰한다. </li><li> 커버 유리 집게로 6 웰 플레이트에 고화 셀 - 함유 지지체를 전송하고, 인슐린없이 2 % B27 보충 2ml의 EBM2 매체 골격을 배양하여 분화를 개시, 티빈-A (50 NG / ㎖) BMP-4 24 시간 동안 (25 NG / ㎖), 5 % CO에서 <sUB&gt;이 37 ° C를. <ol><li> 부드럽게 세포를 건드리지 않고 진공을 통해 기존의 매체를 흡입하여 매체를 교체합니다. 인슐린없이 B27 보충 된 새로운 EBM2 매체, 혈관 내피 성장 인자 (VEGF, 50 NG / ㎖) 에리트로 포이 에틴 (EPO 100 NG / ㎖) 및 변형 성장 인자 β1 (TGFβ1 10 NG / ㎖)과 문화 추가 5 %에서 비계 CO 2, 48 시간 동안 37 ° C. </li><li> 매체와 문화 5 % CO 2와 다른 48 시간 동안 37 ℃에서 공사장 공중 발판을 새로 고침; 다음에, 배지에 200 U의 콜라게나 제 IV (100 U / ㎖)을 첨가하여 지지체로부터 세포를 분리. </li><li> 이 출시 된 후 세포를 스핀 다운. B27, VEGF-A (50 NG / ㎖) 및 SB-431542 (10 μM)의 2 % 용액이 보충-MV EGM2 배지로 배지를 교체; 매 2 일 매체를 새로 고칩니다. </li><li> 100 U와 / ㎖ 콜라게나 제 IV를 세포를 떼어 놓다 약 10 일 후 (~ 분화가 시작된 후 하루 14 예), 시간을 분리유동 세포 계측법을 통한 분화 세포 집단에서 IPSC의 EC-EC는 특정 마커 단백질의 발현을 분석하여 (예를 들어, CD31, CD144) 3. </li><li> 설립 프로토콜 (3)을 통해 고립 hiPSC-되는 EC를 확장합니다. </li></ol></li></ol> 3. hiPSC-SMCs에 hiPSCs 차별화 <ol><li> 젤라틴 단백질 혼합물로 코팅 된 플레이트 상에 hiPSCs 종자, 문화 mTeSR1 배지에서 세포를 5 % CO 2, 37 ℃에서 하루 중 변화와 합류 할 때까지 (~ 2 일간). </li><li> 3 일간 RPMI1640 배지 및 2 % B27 플러스 인슐린에 CHIR99021 (5 μM) 및 BMP-4 (10 ng의 / ㎖) 세포를 배양하여 혈통 중배엽 세포로의 분화를 시작. </li><li> 주로 합성 표현형과 hiPSC-SMCs을 생성하려면 : <ol><li> 문화 VEGF-A (25 NG / ㎖), RPMI1640 내에서 섬유 아세포 성장 인자 β (FGFβ 5 NG / ㎖)을 세포<html> dium, 2 %의 B27 마이너스 인슐린 5 % CO 2에서 4 일 동안 37 ° C. </li><li> 문화 VEGF-A의 셀 (25 NG / ㎖) FGFβ 5 % CO 2 및 37 RPMI1640 배지 (5 NG / ㎖)에 2 % B27 플러스 인슐린 2 일 동안 C를 °. </li><li> 문화 혈소판 유래 성장 인자 β (PDGFβ 10 NG / ㎖), TGFβ, 5 % CO 2 및 37 RPMI1640 (3 NG / ㎖), 2 % B27 플러스 인슐린 세포 4 일 동안 C를 °. </li></ol></li><li> 주로 수축 표현형과 hiPSC-SMCs을 생성하려면 : <ol><li> 문화 4 RPMI1640에서 VEGF-A와 일 (25 NG / ㎖) 및 FGFβ (5 NG / ㎖) 및 2 % B27 마이너스 인슐린에 대한 세포. </li><li> RPMI1640 배양하고, 2 % B27 플러스 인슐린 PDGFβ (5 NG / mL) 및 TGFβ (2.5 NG / ㎖) 7 일 동안 세포. </li></ol></li><li> 6 ~ 일 대사 RPMI1640 배지를 함유하는 락트산 (4 mM)을 상기 세포를 배양하여 최종 hiPSC SMC-집단을 정제. </li></ol>jove_title &quot;&gt; 4. IGF-포함하는 마이크로 스피어 만들기 <ol><li> 열 올리브 오일 수조에서 45 ° C까지. </li><li> 열 50 ° C로 10 % 젤라틴 용액 5 ㎖. </li><li> 올리브 오일, 볶음에 젤라틴을 추가하고 물을 욕조에 얼음을 추가하여 5 ° C에 다음 급속 냉각. </li><li> 스물 다섯 분 후, 마이크로 스피어의 형성을 유도하기 위해 올리브 오일에 냉장 (4 ℃로) 아세톤을 추가합니다. </li><li> 도 5에서의 온도를 유지 1 시간 동안 ° C; 다음 올리브 오일을 제거하기 위해 미리 냉각 아세톤으로 그들에게 5 번 세척, 미세을 수집하고, 4 ℃에서 자연 건조로 할 수 있습니다. </li><li> 가교 결합을 유도 밤새 4 ℃에서 70 % 에탄올 및 0.25 %의 글루 타르 알데히드의 용액에 미소 구를 재현 탁하고 100mM의 글리신과 혼합하여 중화. </li><li> H를 30 분 동안 5 μg의 IGF와 0.1 % 소 혈청 알부민을 함유하는 2 O 증류수 15 ㎕의 5 mg의 미세 구를 혼합하여 미립자로로드 IGF. </li></ol> 5. 세포를 손상의 사이트를 통해 패치를 작성 및 주입 <ol><li> 1 ml의 최소 필수 중간 (MEM)에 함께 hiPSC 파생 SMCs (100 만)의 CM (200 만), 및되는 EC (1000000)를 일시 중단합니다. </li><li> 1 ㎖의 피브리노겐 용액 (25 ㎎ / ㎖)에 5 ㎎ 미소 일시 중단. </li><li> 셀 - 함유 용액, 피브리노겐 / 미세 용액과 다른 주사기 및 트롬빈 용액 1 ㎖와 함께 제 주사기 한 주사기를 채우기 (80 NIH 단위 / ㎖, 2 ㎕의 400 mM의 CaCl2를 200 mM의 ε 아미노 카프로 보충 산). </li><li> 수술이 이전에 설명 된 바와 같이 관상 동맥을 내림차순 왼쪽 전방의 결찰을 통해 돼지 심장에서 심근 경색을 유도한다. 참고 : 간단한, 여성 요크셔 돼지에서 (~ 13kg이, 연령 45 일)을 Telazol / 크 실라 (4.4 ㎎ / ㎏)의 근육 내 주사로 진정되고, 이소 플루 란 (0.5 %)와 전신 마취하에 삽관. 4 번째 intercostal 개흉술을 수행하고 노출 관상 동맥을 내림차순 앞쪽에 남아 있습니다. 관상 동맥을 60 분간 결찰하여 가려 짐 후 봉합사를 제거한다. 직접 전기 세동은 심실 세동의 경우에서 수행된다. </li><li> 돼지 마음의 경색 영역의 심장 외막에 멸균 플라스틱 링 (~ 2.5 cm)을 배치하고 동시에 링에 미세 / 피브리노겐과 트롬빈 솔루션을 주입. </li><li> 혼합물 (~ 30 초)을 고화하도록 한 다음 세포를 심근 경색으로 응고 된 혼합물을 통해 세포 함유 주사기의 바늘을 삽입하고 삽입. 3-0 또는 2-0 Monocryl 봉합이 동물의 크기에 따라와 근육 층, 피하 조직, 그리고 가슴 벽을 닫습니다. 부 프레 노르 핀 0.01-0.02 밀리그램 / kg 근육 내 주사는 8 시간마다 수술 후 처음 3 일 동안 통증을 제어하는 ​​데 사용된다. </li><li> 심장 자기 공명를 사용하여 심장 기능을 평가수술 한 주일 수술 2, 3, 4 이후 사주 전 영상 (MRI). 최종 MRI 연구 후에, 동물을 희생하고 조직 학적 및 분자 분석을 2, 3, 4의 마음을 처리한다. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Qu, Z., 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=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142 (5), 1257-1267 (1998).</li><li>Tang, X. 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=Intracoronary+administration+of+cardiac+progenitor+cells+alleviates+left+ventricular+dysfunction+in+rats+with+a+30-day-old+infarction.">Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction.</a> Circulation. 121 (2), 293-305 (2010).</li><li>Zeng, 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=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Davis, M. E., 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=Local+myocardial+insulin-like+growth+factor+1+(IGF-1)+delivery+with+biotinylated+peptide+nanofibers+improves+cell+therapy+for+myocardial+infarction.">Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction.</a> Proc Natl Acad Sci U S A. 103 (21), 8155-8160 (2006).</li><li>Li, 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=Overexpression+of+insulin-like+growth+factor-1+in+mice+protects+from+myocyte+death+after+infarction,+attenuating+ventricular+dilation,+wall+stress,+and+cardiac+hypertrophy.">Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.</a> J Clin Invest. 100 (8), 1991-1999 (1997).</li><li>Wang, L., Ma, W., Markovich, R., Chen, J. 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hiPSC-CM이 향상된 수율 / 순도 의 CM으로 인간의 줄기 세포를 분화에 대한 기존의 프로토콜들은 종종 낮은 수율과 순도에 의해 제한된다 예를 들어, hESC의-CM이 단지 35-66% 느린 마이 오신 중쇄 또는 cTnT의 6 표현 퍼콜 분리 및 심장 몸 형성을 통해 획득했습니다. 분화 hiPSC-CM 인구의 순도는 대략의 프로모터에 결합되어 리포터 유전자의 발현을 위해 선택함으로써 ?...

들이 환자의 면역계에 의해 거부되지 않은 셀의 잠재적으로 무한 범위의 수로 구분 될 수 있기 때문에 유도 인간 다 능성 줄기 세포 (hiPSCs)는 재생 세포 치료에 가장 유망한 제제들이다. (SMCs을하지만, 자기 복제 및 분화 능력은 또한 종양 형성을 초래할 수 있으며, 결과적으로, hiPSCs 완전히 같은 심근 (CMS), 내피 세포 (EC에) 및 평활근 세포와 같은 특정 세포 유형으로 분화 될 필요 ), 투여 전. 세포 투여 간단하고 가장 일반적인 방법 중 하나는 직접 심근 내 주입이지만, 네이티브 심근 조직으로 이식 접목되는 세포의 수는 매우 낮다. 이 감소의 대부분은 허혈성 조직의 세포 독성 환경에 기인한다; 그러나, 생쥐 배아 줄기 세포 (는 ESC)가 있었을 때 손상되지 않은 마음의 심근에 직접 주입 O투여 된 세포의 상당한 비율이 그들이하는 동안 생성 된 높은 압력에 의해 바늘 트랙을 통해 압박했다 아마도 때문에 관리 사이트를 종료 제안 3-5 시간 1 일 동안 유지되었다 전달 된 5 백만 셀 할수 있답니다는 ~ 40 % 심근 수축. 여기, 우리는 hiPSC 유래 심근 세포 (hiPSC-CMS) 2, 내피 세포 (hiPSC-되는 EC) 3, 평활근 세포 (SMCs)을 4 생성 소설과 실질적으로 더 효율적인 방법을 제시한다. 특히,이 hiPSC-SMC 프로토콜은 주로 합성 또는 수축 SMC 표현형으로 세포를 연출하여 체세포 SMCs 5에서 관찰 형태 학적 및 기능적 특성의 넓은 범위를 모방 처음이다. 우리는 또한 사이토 카인 함유 피브린 (P)을 생성하여 주사 세포의 생착 속도를 향상 세포 전달 방법을 제공하는주사 부위를 통해 ATCH. 패치는 적어도 3 일에 걸쳐 인슐린 유사 성장 인자 (IGF)를 해제함으로써, 심근 종료로부터 세포를 방지 바늘 트랙 및 세포 생존을 밀봉함으로써, 두 세포 유지를 향상시키기 위해 나타난다.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 monofilament suture</td>
			<td>Ethilon</td>
			<td>627H</td>
			<td></td>
		</tr>
	</tbody>
</table>

pluripotent stem cell derived cardiac cells for myocardial repair

인간 유도 만능 줄기 세포 (hiPSCs)이 완전히 심근 (hiPSC-CMS), 내피 세포 (hiPSC-되는 EC), 및 평활근 세포 (SMCs)에 hiPSCs 차별화에 대한 관리 전에 특정 세포 유형,하지만 기존의 프로토콜로 분화해야합니다 자주 낮은 수율, 순도, 및 / 또는 가난한 표현형의 안정성에 의해 제한. 여기서는 신규 hiPSC-CM이, -ECs를 생성하기위한 프로토콜 및 -SMCs 실질적으로 종래의 방법보다 더 효율적이며, 물론 투여의 사이트를 통해 생성 된 사이토 카인 - 함유 패치 세포 주입을 조합하는 방법을 제시한다. 패치는 장기간 동안 인슐린 유사 성장 인자 (IGF)를 해제함으로써, 심근에서 압착되는 세포 않도록 바늘 트랙 및 세포 생존을 밀봉함으로써, 주입 된 세포의 보존을 모두 개선한다. 심근 허혈 재관류 손상의 돼지 모델에서 생착 속도가 클 때 두 배보다 더이었다세포는 세포 및 패치 모두 시토 킨 - 함유 패치없이 셀 비교 패치 및 치료 투여 아니라 단독 세포, 심장 기능 및 경색 크기에서 상당한 개선 연관되었다.

차별화 된 hiPSC-CM이, -ECs 및 -SMCs의 특성 hiPSCs의 차동 용량 4, 2 (3)을 평가했다. 세포 계측법 최종 hiPSC-CM 인구의 순도가 90 % (그림 1A, 1B, 패널 B1)를 초과 할 수 있음을 시사 심장 트로포 닌 T (cTnT를) 식의 분석 흐름. 거의 모든 셀 느린 미...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

우리는 심근 세포, 내피 세포, 평활근 세포 및 패치 - 매개 사이토 카인 배달 세포 주입을 조합함으로써 이식 된 세포의 생착을 향상 배송 방법으로 인간 유도 만능 줄기 세포를 구별하기위한 세 가지 신규하고보다 효율적인 프로토콜을 제안한다.

다 능성 줄기 세포는 심근 수리를위한 심장 세포를 유도

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

pluripotent-stem-cell-derived-cardiac-cells-for-myocardial-repair

Research

JoVE Journal

Developmental Biology

8.2K 조회수.  University of Alabama at Birmingham.  우리는 심근 세포, 내피 세포, 평활근 세포 및 패치 - 매개 사이토 카인 배달 세포 주입을 조합함으로써 이식 된 세포의 생착을 향상 배송 방법으로 인간 유도 만능 줄기 세포를 구별하기위한 세 가지 신규하고보다 효율적인 프로토콜을 제안한다. 

비디오: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Myh6 발기인 - 선택과 함께 ES-세포 프로그래밍을 바탕으로 설치류 심장 페이스 메이커 세포 집합체의 생성

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

연구

발생학

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

헤어 유래 각질 세포를 사용하여 통합없는 인간 유도 만능 줄기 세포의 생성

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

트랜스포존 시스템을 사용하여 마우스 양수 세포로부터 다 능성 줄기 세포의 제조

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

제대혈 유래 다 능성 줄기 세포로부터 연골 형성 펠렛 형성

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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 ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

약물 발견에 대 한 만능 줄기 세포 유래 Hepatocyte 같은 세포를 유도 하는 인간을 사용 하 여

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

가족성 콜레스테롤 혈 증 인간의 간 공상 마우스 모델을 사용 하 여 유도 만능 줄기 세포 유래 Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...