이 작품은 조안나 M. NICOLAY 재단 흑색 종 연구를위한 친교에 의해 루스 L. Kirschstein 국가 연구 서비스 상 F31에서 국립 보건원 (National Institutes of Health)에 의해 지원되었다. 이 작품은 더 NYSTEM 및 트라이 기관 줄기 세포 이니셔티브 (스타 재단)에서 보조금을 통해 지원되었다.

참고 : 여기에 설명 된 멜라닌 세포 프로토콜은 처음 운모 등의 알에 의해 입증되었다. 1. 문화 매체의 제조, 코팅 요리와 hPSCs의 유지 보수 <ol><li> 중간 준비 주 : 저장소를 최대 2 주 동안 어둠 속에서 4 ° C에서 모든 매체. 살균에 대한 모든 매체를 필터링합니다. <ol><li> DMEM / 10 % FBS를 준비합니다. 885 ml의 DMEM, 100 ㎖의 FBS, 10 ml의 펜 / 연쇄상 구균, 5 ml의 L-글루타민을 섞는다. 살균 필터. </li><li> hESC의 매체를 준비합니다. 800 ㎖의 DMEM / F12, 200㎖의 KSR, 5 ㎖ L 글루타민, 10 ㎖ MEM 최소 필수 아미노산 용액, β 머 캅토 에탄올 1 ㎖, 5 ㎖의 펜 / 스트렙토 섞는다. 10 NG / ㎖의 FGF-2 추가 필터링 후. </li><li> KSR-차별화 매체를 준비 : 820 ml의 마네 DMEM, 150 ml의 KSR, 10 ml의 L 글루타민, 10 ml의 펜 / 연쇄상 구균, 10 ml의 MEM 최소 필수 아미노산 용액 1 ml의 β-머 캅토 에탄올을 혼합. 살균 필터. </li><li> N2-차별화 매체를 준비합니다. 12g DMEM / F12 분말 난을 녹여n은 980 ml의 DH 2 O. 1.55 g 포도당, 2g의 중탄산 나트륨 100 mg의 아포 인간 트랜스페린을 추가합니다. 2 ml의 DH 2 O 25 mg의 인간 인슐린와 40 ㎕의 1 N NaOH를 혼합; 용해되면, 매체에 혼합물을 추가합니다. 100 ㎕의 퓨 트레 신 염산염, 60 μL 셀렌, 100 ㎕의 프로게스테론을 추가합니다. 필터링하기 전에 DH 2 O와 1리터에 최종 볼륨을 가져와. </li><li> 전체 멜라닌 세포 매체를 준비합니다. 50 %로 Neurobasal 배지, 30 % 낮은 글루코스 DMEM, 20 %의 결합 MCDB201. 이 추가하려면 : 0.8 % ITS +, 250 nM의 L- 글루타민, 100 μM 아스 코르 산 (L-AA), 50 NG / ㎖ 콜레라 독소, 50 NG / ㎖ SCF, 0.05 μM 덱사메타손, 100 nm의 EDN3, 4 NG / ㎖ FGF2 . 2 % B27 보충, 25 NG / ㎖ BMP4, 3 μM의 CHIR99021, 500 μM의 캠프 : 살균 필터는 남아있는 시약을 추가합니다. </li></ol></li><li> 문화 요리의 코팅 <ol><li> 같은 마트 리겔로 젤라틴 단백질을 사용하여 코팅 수행합니다. 1 ml의 부분으로 개방, 나누어지는 및 동결 마트 리겔에 반복적 인 동결 융해 C를 피하기 위해ycles. 해동 및 19 ml의 DMEM / F12와 함께 1 ml의 냉동 나누어 다시는 일시 중지합니다. 10cm 접시 위에 접시 5 ml의. 실온에서 1 시간 동안 요리를 품어. 즉시 세포를 도금하기 전에 젤라틴 단백질을 기음과 DMEM / F12 씻어. </li><li> 폴리-L ornithin 브롬화 수소 산염 / 마우스 라미닌-I / 피브로넥틴 (PO / 램 / FN)를 사용하여 코팅을 실시하고 있습니다. PBS가 포함 된 코트 요리 15 μg의 / ㎖ 폴리-L ornithin 브롬화 수소 산염. 가습 인큐베이터에서 37 ° C에서 O / N을 품어. 용액을 흡인 및 1 μg의 PBS / ㎖ 마우스 라미닌 2 μg의 I / ㎖의 피브로넥틴을 함유하는 코팅 전에 PBS로 3 회 플레이트를 세척한다. 가습 인큐베이터에서 37 ° C에서 요리 O / N을 품어. <ol><li> 전에 세포를 도금에 솔루션을 기음과 플레이트 조직 문화 후드에 뚜껑없이 완전히 건조 할 수 있습니다. 판은 약 10 ~ 15 분 동안 건조하도록 허용합니다. 참고 : 결정 구조가 눈에 의해 표면에 나타날 때 요리 건조하고 세포 도금 준비가되어 있습니다. 작은지면ES는 PBS만큼 액체가 증발하지 않기 때문에, 2 주 동안 인큐베이터에서 LAM / FN 함유하여 유지할 수있다. 판은 몇 시간 동안 실온에서 건조하게 유지 될 수있다. </li></ol></li></ol></li><li> hPSCs의 유지 보수 참고 : hPSCs는 hESC의 매체에서 0.1 % 젤라틴 및 mitotically 불 활성화 된 마우스 배아 섬유 아 세포 (MEFs)에 유지된다. 셀마다 6~8일 분할한다. <ol><li> 코트 5 분 동안 실온에서 PBS에서 0.1 % 젤라틴으로 10cm 접시. </li><li> 37 ° C의 물을 욕조에 신속하게 냉동 MEFs에 해동. </li><li> 젤라틴을 기음과 세포를 접시. DMEM / 10 % FBS에서 ~ 50,000 세포 / ㎠의 밀도로 플레이트 MEFs에. 이전 hPSCs를 추가로 37 ° CO / N에서 MEFs에 품어. </li><li> 준비 MEF 플레이트에서 DMEM / 10 % FBS를 대기음, PBS로 접시를 씻어 hPSCs 10 ml의 hESC의 매체를 추가합니다. 유로 (1)의 비율로 hPSCs : 이전에 계대 밀도에 따라 5/10. 참고 : hPSCs가 해동 또는 단일 세포로 계대되는 경우, SUP제 1 통로까지 10 μM Y-27632 디 하이드로 클로라이드 (ROCKi)와 영양 보충제. </li><li> ml의 10 신선한 hESC의 중간 매일 세포를 공급. </li><li> 분화는 분화 된 세포, 불규칙한 경계, 또는 투명 센터 (28)를 포함 할 나타나는 만능 식민지를 제거 시작하기 전에. 기계적시키다 및 해부 현미경으로 층류 후드 피펫 불규칙 콜로니 대기음. </li></ol></li></ol> 차별화를위한 hPSCs 2. 도금 참고 : 차별화 조건이 10cm 요리에 대해 설명합니다. <ol><li> 단계 1.3.1에 ​​설명 된대로 분화를 시작하기 전에 10cm 마트 리겔 요리를 준비합니다. </li><li> 차별화를위한 도금 hPSCs는 통과를 위해 준비 밀도 (~ 80 %의 합류), hESC의 매체를 대기음 PBS로 세척하고 세포를 0.05 % 트립신 EDTA 3 ㎖를 추가로 hPSCs의 접시로 시작합니다. </li><li> 적극적으로 접시 수평 보링을 흔들MEFs에 단일 세포로 들어 올립니다 때까지 y를 2 분 동안 현미경으로 시각화있다. hPSC 식민지는 식민지로 부착 된 상태를 유지해야합니다. </li><li> MEFs에이 올린 후 트립신을 대기음하지만 hPSCs의 콜로니를 분리하기 전에. </li><li> 추가 판에 10 μM Y-27632 디 히드로 클로라이드를 포함 hESC의 중간 10 ㎖와 식민지 이상까지 피펫 팅에 의해 아래로 세포를 분리. </li><li> 2.1 단계에서 제조 된 10cm 접시에서 마트 리겔 솔루션을 기음과 덩어리를 제거하는 DMEM / F12 씻어. 마트 리겔 플레이트 상에 2 비율 : 1의 hPSCs 플레이트. 10ml의 최대 10 μM Y-27632 디 하이드로 클로라이드를 포함 hESC의 매체를 추가합니다. 37 CO / N에 품어. 참고 :이 도금 / ㎠에서 약 10 만 셀을 초래한다. </li></ol> 신경 분화 3. 유도 참고 : hPSCs 80 %의 합류 때 분화 (0 일) 시작되어야한다. 세포 hESC의 매체로 매일 공급 될 수있다분화를 개시 할 때까지 10 μM Y-27632 디 히드로 클로라이드를 포함. <ol><li> 제 0 일과 1 일에, 100 나노 LDN193189 10 μM SB431542 함유 KSR 분화 배지 10 mL로 세포를 공급. </li><li> 제 2 일에, 100 나노 LDN193189 10 μM SB431542 3 μM의 CHIR99021 함유 KSR 분화 배지 10 mL로 세포를 공급. </li><li> 제 3 일에 10 μM SB431542 3 μM의 CHIR99021 함유 KSR 분화 배지 10 mL로 세포를 공급. </li><li> 4 일 및 5 피드 15 75 % KSR 분화 배지 ㎖의 3 μM의 CHIR99021 함유 25 % N2 분화 배지 세포. 참고 : 부동 세포의 관점에서 세포 죽음의 많은 양을보고 놀라지 마십시오. 이는 정상적인 예상 할 수 있습니다. </li><li> 일 6 7 15 50 % 공급 KSR 분화 배지 ㎖의 모두 3 μM C​​HIR99021 25 NG / ㎖ BMP4, 100 nM의 EDN3 함유 50 % N2 분화 배지 세포. </li><li> 일 8 일및 공급 구 (20)의 25 % KSR 분화 배지 ㎖의 모두 3 μM C​​HIR99021 25 NG / ㎖ BMP4, 100 nM의 EDN3 함유 75 % N2 분화 배지 세포. </li><li> 하루 11 세포의 재 도금 1.2.2에 표시된 일 9, 10 일 PO / 램 / FN 요리를 준비합니다. </li><li> 3 μM C​​HIR99021 25 NG / ㎖ BMP4, 100 nM의 EDN3 함유 N2 분화 배지 20 ㎖와 10 일째 공급 세포. </li></ol> NC 사양에 대한 방울 4. Replating <ol><li> 하루 11 대기음 램에 / 완전히 준비 PO / LAM / FN 판과 건조로부터 FN. </li><li> 하루 11 세포에서 매체를 제거 PBS로 세척 및 10cm 접시 당 Accutase로 4 ml의 세포 분리 솔루션을 추가 할 수 있습니다. 37 ° C에서 25 분 동안 품어. </li><li> 접시에 전체 멜라닌 세포 매체의 5 ML을 추가하고 모든 세포가 판을 해제 할 때까지 10 ml를 피펫으로 수동으로 피펫 팅에 의해 아래로 세포를 재현 탁. 15 ML 튜브에 현탁액을 전송합니다. </li><li> 스핀다운 200 XG에서 5 분 동안 세포와 멜라노 전체 배지 용액에 2 × 106 세포에서 세포를 재현 탁. 혈구 또는 이에 상응하는 기술에 트리 판 블루 배제를 사용하여 세포를 계산합니다. </li><li> 건조 PO / 램 / FN 10cm 요리 상 (그들을 건드리지 않고) 서로 가까이 판 (10) μL 방울. 플레이트가 충분히 건조되지 않은 경우, 물방울이 잘 에지를 정의 실행하지 않은 것이다. 참고 :이 확장을위한 요리 내 공간을 유지하면서 세포를 replating 때 중요한 세포에 대한 고밀도 지역의 환경을 만듭니다. </li><li> 천천히 (첨부 된 세포를 방해하지) 전체 멜라닌 세포 배지 10 ㎖를 추가, 방울 세포가 부착 할 수 있도록 10 ~ 20 분 동안 실온에서 방치한다. 인큐베이터에 접시를 이동합니다. </li></ol> 5. 멜라닌 세포 전구 세포를 확장 <ol><li> 모든 2~3일 전체 멜라닌 세포 배지로 먹이를 계속합니다. 참고 : 색소 침착이 w 볼 수 있도록 시작해야첫 주 말까지 세포의 클러스터를 ithin와 두 번째 주에 의해 명확하게된다. 이들 초기의 작은, 어두운 클러스터를 식별하기 위해 종이로 흰색 배경 위에 접시를 볼 수 있습니다. 전체 판 (그림 2B 참조) 균일하게 착색 될 때까지 세포는 시간이 지남에 따라 점차적으로 착색 될 것입니다. </li><li> ~ 1의 비율로 한번에 주 통로 셀 6 (액적으로서 도금이 단계에서 더 이상 필요하지 않다). 세포를 떼어 놓다 및 전체 멜라닌 세포 배지에서 다시 도금하기 전에 일반로 Neurobasal 매체로 두 번 씻어 Accutase를 사용합니다. PO / LAM / FN 접시에 세포를 유지한다. 참고 : 우리는 전체 멜라닌 세포 매체는 유지하고 hESC의 및 IPSC 파생 된 멜라닌 세포 확장을위한 가장 적합한 것으로 나타났습니다. 그러나, 세포는 시판 M254 배지에서 배양 간략 수 있지만 간단 미디어 죽어 플레이트를 올려 셀의 위험을 증가시킨다. </li></ol>

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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=KIT+expression+reveals+a+population+of+precursor+melanocytes+in+human+skin.">KIT expression reveals a population of precursor melanocytes in human skin.</a> J invest dermatol. 106, 967-971 (1996).</li><li>Li, 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=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J cell sci. 123, 853-860 (2010).</li><li>Nishimura, E. 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저자는 공개 충돌하는 이익을 더이 없습니다.

hPSCs에서 멜라닌의 성공적인 분화 다음 방법이 고려되어야한다. 무엇보다도, 항상 무균 배양 조건에서 동작하는 것이 필수적이다. 또한,이 다 능성 완전히 분화 hPSCs 시작하는 것이 중요하다; 시작 인구가 분화 세포를 포함하는 경우 수익률은 변함없이 오염 물질이 멜라닌 세포 지향 할 수 없으며 더욱 제대로 분화 세포를 방해 할 수 있으므로 삭제합니다. 세포가 만능 줄기 ?...

인간 다 능성 줄기 세포 (hPSCs)은 질병 모델링, 약물 스크리닝, 세포 대체 요법 1-6 대한 확장 형태로 정상적인 분화를 모방하는 플랫폼을 제공한다. 특히 관심, hPSCs은 분리하기 어렵거나 환자 샘플이 부족하다 희귀 / 과도 세포 유형 공부 길을 엽니 다. 또한, 유도 만능 줄기 세포 (iPSCs)는 고유의 메커니즘 1,2,7-11을 해명하기 위해 환자 특정 방식으로 개발 및 질병 모델링을 연구하는 연구자 수 있습니다. hPSCs에서 멜라닌 세포의 분화 이전에 발행 된 프로토콜은 차별화 6 주 정도가 필요하며 L-Wnt3a 세포 (12)로부터의 조건화 된 배지로 배양 세포를 포함한다. 이 프로토콜은 처음 운모 등 제시.과 설명 여기에 3 주 색소 세포를 생산하고 모호함과 컨디셔닝 매체와 관련된 불일치를 제거합니다. 멜라닌 세포 아칸소신경 크레스트, 척추 동물에 고유 한 세포의 이주 인구에서 파생 된 전자. 신경 크레스트 낭배 동안 정의 신경 및 비 신경 외배엽 간의 접경 신경 판의 가장자리 세포 집단을 나타내고있다. neurulation 동안, 신경 조직은 신경 튜브 13, 14의 결과 지느러미 중간 선에서 수렴 신경 주름을 형성 신경 판에서 진화. 신경 능선 세포는 척색 맞은 편에 신경 튜브의 지붕 판에서 등장하고 분화 된 세포의 다양한 인구 증가를 제공 멀리 이주하기 전에 중간 엽 전이에 상피를 받아야. 크레스트 세포의 운명은 배아 체 축을 따라 지붕 판의 해부학 적 위치에 의해 부분적으로 정의된다. 신경 능선 세포 유도체 모두 중배엽 계통의 특성 (평활근 세포, 골아 세포, 지방 세포, 연골 세포) 및 외배엽 세포를 포함한다 (멜라닌 세포, 슈반 CELL 학생, 뉴런) 14. 신경 크레스트 줄기 세포는 전사 인자 SOX10을 상향 조절 및 P75 및 HNK1에 대한 항체로 정렬 형광 활성화 세포에 의해 분리 될 수있다. 멜라닌 세포가 멜라닌 단계를 통과 6,21 MITF은 멜라닌 세포 개발의 마스터 레귤레이터 및 전사 인자가 멜라닌 세포 개발의 많은 부분을 제어해야하는 KIT와 MITF (소안 구증 관련 전사 인자)를 상향 조절 될 운명 신경 능선 세포 22- 24. 인간 melanoblasts는 머리 팽창 또는 표피에서 각질 형성 세포에 의해 둘러싸여 성숙, 멜라닌 색소에 대한 전구체로서 기능 할 (색소 형성 단위) 중있는 표피의 기저층에 이동한다. 착색 멜라닌 세포에 melanoblasts의 분화와 성숙은 머리 전구의 식민지와 표현 멜라닌 생성 경로 (TYRP1, TYR, OCA2와 함께 수반 발생PMEL) 25, 26. 환자에서 인간의 멜라닌 세포와 melanoblasts을 분리하는 것은 비용이 어렵고 수량에 제한입니다. 이 프로토콜은 셀 정렬없이 잘 정의 된, 신속하고, 재생 가능한 확장 성, 저렴한 방법으로 멜라닌 세포 또는 멜라닌 세포 전구체에 hPSCs을 (유도 또는 배아) 구별하기 위해 연구자 수 있습니다. 프로토콜은 색소 질환 환자 iPSCs 차별화 때 질병 특정한 결함을 식별하는 데 이전에 사용 하였다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

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.

이 프로토콜은 체외 피더없이, 비용 효율적이고 재현 방식 hPSCs에서 완전히 착색 된, 성숙한 멜라닌 세포를 유도하는 방법을 제공한다. hPSC 유래 멜라닌 세포에 대해 이전에 확립 팡 등. 프로토콜 달리 명시된 프로토콜 조정 배지를 필요 시간 요건을 감소하지 않는다. 팡 등. 프로토콜은 WNT3A 생산 쥐의 세포 라인에서 조절 된 매체를 사용하고 색소 침착 ...

Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

이 작품은 신경 능선을 통해 인간 만능 줄기 세포에서 색소, 성숙한 멜라닌 세포를 생산하고 공급이없는 25 일간의 프로토콜을 사용하여 중간 단계 멜라닌하는 체외 분화 프로토콜을 설명합니다.

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

feeder-free-derivation-melanocytes-from-human-pluripotent-stem

Research

JoVE Journal

Developmental Biology

10.2K 조회수.  Memorial Sloan-Kettering Cancer Center.  이 작품은 신경 능선을 통해 인간 만능 줄기 세포에서 색소, 성숙한 멜라닌 세포를 생산하고 공급이없는 25 일간의 프로토콜을 사용하여 중간 단계 멜라닌하는 체외 분화 프로토콜을 설명합니다. 

비디오: Feeder-free Derivation of Melanocytes from Human Pluripotent 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 ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

인간 유도 만능 줄기 세포에서 파생 된 피라미드 뉴런에서 돌기 쪽의 3 차원 정량화

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

피더가없는 조건에 센다이 바이러스를 사용하여 인간 말초 T 세포로부터 유도 된 다 능성 줄기 세포의 생성

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

정의 및 간세포와 같은 세포의 확장 성 생성 인간 다 능성 줄기 세포에서

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Laminin 521 매트릭스를 이용한 인간 유래 다 능성 줄기 세포의 통합 된 자유 유도

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

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.

인간 다능성 줄기 세포를 간 세포로 효율적으로 분화

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...