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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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=high-content+screening+for+novel+pigmentation+regulators+using+a+keratinocyte/melanocyte+co-culture+system.">high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.</a> Exp dermatol. 23, 125-129 (2014).</li></ol>

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><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>&amp;beta;-Mercaptoethanol</td><td>Gibco-Life Technologies</td><td>21985-023</td><td>10 mg/ml</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-bp</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris-R&amp;amp;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 &amp;mu;M</td></tr><tr><td>DMEM - Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco-Life Technologies</td><td>11985-092</td><td></td></tr><tr><td>DMEM/F12 - Dulbecco&#039;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 &amp;mu;M</td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>356008</td><td>200 &amp;mu;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&amp;nbsp;</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&amp;amp;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&amp;nbsp;</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td>0.032 g in 100 ml 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&amp;amp;D Systems</td><td>233-FB-001MG/CF</td><td>10 mg/ml</td></tr><tr><td>SB431542</td><td>Tocris-R&amp;amp;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)&amp;nbsp;</td><td>Peprotech Inc.</td><td>300-07</td><td>50 &amp;mu;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&amp;amp;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

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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

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 ...

연구

발생학

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

인간의 흑색 종 종양 침윤 림프구에서 유도 만능 줄기 세포의 생성

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

신경과학

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

피더 및 제노 프리 정의 상태에서 인간 다능성 줄기 세포에서 혈생 내피 분화

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

피더가 없고 화학적으로 정의된 배양 조건 하에서 인간 다능성 줄기 세포를 사용하여 후갱이온 교감 뉴런의 효율적인 분화

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

완료 녹아웃 세럼 교체 피더 - 무료 매체를 사용하여 인간의 IPS 세포 피더 - 자유 적응력, 문화와 Passaging

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

생물학

Isolating Primary Melanocyte-like Cells from the Mouse Heart

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

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

마우스의 심장에서 차 멜라닌 세포와 같은 세포를 분리

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

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

인간의 Cardiomyocytes의 생성 : 피더없는 인간 유도 만능 줄기 세포에서 분화 프로토콜

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...