We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

마우스는 ESC에서 마우스 배아 클릭당 비용 (CPC) 1. 유도 <ol><li> 마우스 배아 섬유 아 세포 (MEFs) 세포층을 준비합니다. <ol><li> 따뜻한 MEF 매체 37 ° C에 (DMEM에 10 % FBS). </li><li> 젤라틴 코팅 된 플레이트를 준비합니다. <ol><li> 10cm 접시에 6 웰 플레이트의 우물 또는 5 ㎖에 물에 0.1 % 젤라틴의 1 ML을 추가합니다. </li><li> 적어도 30 분 동안 판 또는 요리 37 ° C에서 또는 실내 온도를 남겨주세요. 사용하기 전에 젤라틴을 열망. </li></ol></li><li> 해동은 부드러운 흔들림으로, 37 ° C의 물을 욕조에 신속하게 MEFs에를 조사. </li><li> 15 ml의 50 ML 원뿔 튜브에 부드럽게 세포를 전송합니다. 예열 MEF 매체의 5 ML을 추가합니다. 천천히 세포를 소용돌이 및 5 분 동안 200 XG에 원심 분리기. </li><li> MEF 매체에서 세포를 재현 탁하고 가능한 세포를 계산합니다. 10cm 접시에서 세포를 2 ml를 6 웰 플레이트에서 웰 당 세포 및 10 ml의 다음 MEF 매체의 볼륨을 사용합니다. </li><li> 6 웰 플레이트에 플레이트 세포 또는 1-2에서 10cm 요리플레이트 / 접시 당 × 10 (6). </li><li> 37 ° C에서 MEF 플레이트 / 접시를 품어, 5 % CO 2 최소 24 시간 이전에 사용할 수 있습니다. 주 : 주보다 오래된 MEF 판 / 요리를 폐기하십시오. </li></ol></li><li> 마우스는 ESC를 준비 <ol><li> 문화 Isl1-CRE; Rosa26YFP / Mef2c-CRE; 90 %의 합류까지 MEFs에에 Rosa26YFP 마우스는 ESC. 사용 ES 세포 배지 410 ml의 DMEM, 75 ㎖의 FBS, 0.1 mM의 MEM NEAA, 2 mM L- 글루타민, 0.1 mM의 피루브산 펜 100 U / 스트렙토 마이신, 및 0.1 밀리미터 β 머 캅토 에탄올로 구성. </li><li> 1.1.2에 설명 된대로 젤라틴 코팅 6 웰 플레이트를 준비합니다. </li><li> 접시에서 대기음 매체와는 DPBS로 세포를 씻어. <ol><li> 기음 DPBS 6 웰 플레이트 잘 하나에 0.4 ml의 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃로, 5 % CO 2에서 세포를 배양한다. 트립신 반응을 중단 1 ml의 예열 ES 매체를 추가합니다. </li></ol></li><li> 수확 세포와 원심 분리기 세포 200 XG와 대기음 매체에서. 재현 탁 된 1 ml의 ES 세포 배지에서 세포와 세포를 카운트. &lt;/ 리&gt; <li> 3 × 104 / ㎠의 밀도에 젤라틴 코팅 플레이트 플레이트는 ESC. 는 ESC는 약 90 % 합류에 도달했을 때 약 2 일 동안 37 ° C, 5 % CO 2에서 배양한다. 매체 일상을 변경합니다. </li><li> 는 ESC가 90 % 합류 경우, 반복 완전히 MEF 피더를 제거하는 1.2.2-1.2.5 단계. </li></ol></li><li> EB 형성에 의해 CPC 분화를 유도 <ol><li> 다음으로 분화 배지 준비 : 36 ml의 IMDM, 12 ml의 햄 F12, 2 mM L- 글루타민, 0.34 ml의 BSA (7.5 %), 0.25 ml의 N2, 0.5 ㎖의 B27, 50 ㎍ / ㎖의 AA, 및 0.45 mM의 1- 티오 글리세롤. </li><li> 기음 세포로부터 중간 DPBS 씻어. 기음 DPBS 6 웰 플레이트 잘 하나에 0.4 ml의 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃에서 배양한다. </li><li> 하나의 세포로 세포 클러스터를 분산 아래로 반응 및 피펫 업을 중단하고 1 ml의에게 차별화 매체를 추가합니다. </li><li> 원심 분리기는 ESC 5 분 200 XG에와 매체를 폐기합니다. <ol><li> 재현 탁 1 × 6 &lt;/ SUP&gt; 1 ml의 차별화 매체는 ESC. 제 EB 응집 37 ° C에서 분화 배지에서 1 × 105 세포 / ml의 낮은 농도에서 분리 배양 접시에서 세포를 세포 수 및 배양. </li></ol></li><li> 이틀 EB 형성 한 후, 50 ㎖ 튜브에 요리에서 사채를 전송할 수 있습니다. 1 분 동안 200 XG에 스핀. 매체를 제거하고 칼슘과 마그네슘 2+없이 10 ML의 DPBS로 사채를 씻어. </li><li> 기음 DPBS 1 ml의를 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃에서 배양한다. 반응을 정지 4.5 ml의 차별화 매체를 추가합니다. 피펫 위아래로 하나의 세포로 사채를 해리합니다. </li><li> 5 분 동안 200 XG에 세포를 원심 분리기. 1 ml의 분화 배지에서 대기음 매체에 resuspend 세포. </li><li> 세포 수 및 분화 배지에서 1 × 105 세포 / ㎖로 2 × 106 세포를 희석. 5 NG / ㎖, 0.8 ng를 / ㎖의 최종 농도 매체에 VEGF, BMP4과를 Activin이 추가, 5 NG / ㎖, 각각. Cultu37 ° C에서 두 번째 EB 형성을위한 배양 접시에 세포 재. </li><li> 두 번째 EB 형성 후 40 시간은 50 ㎖ 튜브에 사채를 전송할 수 있습니다. 5 분 동안 37 ° C에서 1 ml의 0.25 % 트립신과 EBS을 (를) 해리, DPBS로 한번 씻어. 피펫 위아래로 하나의 세포로 사채를 해리합니다. </li><li> 5 NG / ㎖ VEGF, 10 NG / ㎖의 bFGF 12.5 NG / ㎖로 FGF10 Stempro-34 배지에서 세포를 재현 탁. 젤라틴 - 코팅 된 플레이트에서 2 × 105 / cm 2에서 셀을 접시. CPC 분리하기 전에 약 32 시간 동안 37 ° C의 배양기에서 문화. </li></ol></li><li> mCPCs를 분리 <ol><li> 대기음 매체와 DPBS 씻어. 5 분 동안 37 ℃에서 배양 플레이트에 0.5 ml의 0.25 % 트립신을 추가합니다. 반응을 정지 4.5 ml의 차별화 매체를 추가합니다. 피펫 위아래로 하나의 세포로 사채를 해리합니다. FACS 정화 2 % FBS / PBS에서 원심 분리기에 resuspend 세포에 의한 세포를 수집합니다. </li><li> 음성 대조군으로 정렬에 미분화는 ESC를 실행합니다. 변경 전압은 앞으로 배설물을 조정합니다터 (FSC)와 측면 산란 (SSC)를 원하는 인구를 선택합니다. 파편과 이중를 제외 측면 분산과 폭 대 앞으로 분산 높이 대 흩어 앞으로 사용합니다. 선택한 하위 집단에서, ESC 컨트롤의 분포에 따라, 세포의자가 형광을 제거하기 히스토그램에 긍정적 인 YFP의 게이트를 그립니다. YFP + 게이팅에서 YFP + CPC를 수집합니다. </li><li> 차별화 매체 (1.3.1) 6 웰 플레이트에서 정제 된 클릭당 비용 (CPC) (YFP + 셀) 플레이트. 심근 및 SMCs에 클릭당 비용 (CPC)의 차별화를위한 37 ° C에서 문화 추가 3~5일. </li></ol></li></ol> 인간는 ESC에서 인간 배아 클릭당 비용 (CPC) 2. 유도 <ol><li> 공급기를 준비 <ol><li> 2 × 104 / ㎠의 밀도로 상기 한 바와 같이 마우스 배아 섬유 아세포 (MEFs에) 세포층을 준비한다. </li></ol></li><li> 인간는 ESC를 유지 <ol><li> CRE; M에 정기적으로 pCAG - 플록스-STOP-플록스-GFP 또는 RFP는 ESC : 인간 ISL1 유지정규 hESC의 매체 (녹아웃 DMEM / F-12 385 ㎖, 넉 아웃 SR 100 ㎖, 2 mM L- 글루타민, 0.1 mM의 NEAA, 0.1 밀리미터 β 머 캅토 에탄올, 10 NG / ㎖ 염기성 FGF)을 사용 EF. 심장 분화 이전에, 적어도 2 개의 통로의 피더 자유로운 상태는 ESC 인간 옮긴다. </li></ol></li><li> 피더 무료 조건에서 인간는 ESC를 준비 <ol><li> 인간는 ESC 문화 코팅 된 플레이트를 준비합니다. <ol><li> 4 ° C에서 하룻밤 리겔을 녹여. 얼음에 50 ML 튜브를 넣고 튜브에 30 ml의 차가운 DMEM / F12 배지를 추가합니다. </li><li> 차가운 매체에 1 ml의 마트 리겔을 넣고 잘 섞는다. 10cm 접시에 6 웰 플레이트의 우물 또는 5 ㎖에 1 ml의 차가운 리겔 - DMEM / 12 매체를 추가합니다. </li><li> 2 시간 동안 판 / 4 ° C에서 하룻밤 요리 또는 실내 온도를 남겨주세요. 대기음 매체를 사용하기 전에. </li></ol></li><li> MEF 플레이트에서 대기음 미디어와 DPBS 인간의는 ESC를 씻어 : 인간 플레이트 상에는 ESC를 분할합니다. 이어서 6- 웰 플레이트의 웰에 1 ㎖의 디스 파제 (1 mg / ㎖)을 추가한다. 37 ° C에서 세포를 품어인큐베이터는 식민지의 가장자리가 분리 시작할 때까지. </li><li> 디스 파제 솔루션을 제거합니다. 다음 2.5 ml에 추가 / 잘 mTeSR 매체 또는 MEF-조정 배지 당 판에 두 번 DPBS 씻어. </li><li> 작은 덩어​​리로 인간의 ESC 식민지 아래 휴식을 셀 스크레이퍼를 사용하여 세포를 긁고 조심스럽게 피펫합니다. </li><li> ESC 덩어리를 전송하고 1을 희석 : 3 코팅 된 판에. </li><li> 2 ㎖ / 잘 mTeSR 매체 또는 MEF 에어컨 매체를 추가하고 매일 매체를 변경합니다. </li><li> mTeSR 매체 또는 적어도 두 구절 코팅 접시에 MEF 에어컨 매체 문화 인류는 ESC. </li></ol></li><li> 인간는 ESC에서 CPC의 분화를 유도 <ol><li> 세포가 ~ 90 % 컨 플루 언트 될 때, 배지를 흡인하고 DPBS로 헹군다. 그런 다음 20 분 동안 37 ℃에서 0.5 ㎎ / ㎖의 인간 배아 줄기 스파 아제 (Dispase)를 배양한다. </li><li> 셀 리프터와 접시에서 세포를 제거, 두 번 DPBS와 인간 배아 줄기 린스, 다음 DMEM / F12는 18 % FBS, 0.1 mM의 NEAA, 2 mM의를 포함하는 (분화 배지에서 세포 클램프를 재현 탁L- 글루타민, 0.1 MM의 β-머 캅토 에탄올 및 50 ㎍ / ml의 아스 코르 빈산 산). </li><li> EB 형성을위한 6 자 초저 부착 판에 세포를 전송합니다. </li><li> 다음날 차별화 매체를 변경합니다. </li><li> 3 일마다 중간 교체 및 현탁 배양에 사채을 유지한다. </li></ol></li><li> HCPCS를 분리 <ol><li> 더 이상 인간의 CPC 격리를위한 차별화의 날 (9)보다 사채를 수집하지 않습니다. </li><li> 대기음은 중간 두 번 DPBS로 사채를 씻어. 5 분 동안 37 ° C에서 6 웰 배양 플레이트의 각 웰에 0.5 ml의 0.25 % 트립신을 추가한다. </li><li> 반응을 정지 분화 배지의 동일한 볼륨. 피펫 위아래로 하나의 세포로 사채를 해리합니다. 2 % FBS / DPBS에 5 분에 resuspend 세포 200 XG에서 원심 분리하여 세포를 수집합니다. 40 μm의 셀 스트레이너 및 FACS 정제 RFP + CPC 세포와 필터 세포로 1.4.3에 설명. </li><li> 플레이트 geltin 코팅 된 플레이트 상에 HCPCS 정렬. 7 분화 배지에서 배양 HCPCS-9 일 심근 세포 및 평활근 세포로 분화한다. 칠일 도금 후, 문화 DMEM / F12 배지에서 5 % 녹아웃 SR과 세포를 분화. </li></ol></li></ol>

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The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

심장 질환은 세계에서 가장 주요 원인 오늘날 남아 사망률은 지난 20 년 (미국 심장 협회)에서 이뤄지지 않고있다. 효과적으로 방지 또는 심부전를 취소하는 신규 한 치료 전략의 개발에 대한 중요한 필요가있다. 한가지 유망한 전략은 줄기 세포 생물학 (1)의 급속한 발전 다음 세포 기반 치료이다. 이와 관련, 다 능성 클릭당 비용 (CPC)으로 인해 증식하지만 심장 혈통 차별화하기 위해 최선을 다하고 자신의 능력으로 치료를위한 우수한 셀 소스 수 있습니다. 따라서, 효율적이고 강력한 방법은 생성과 심장 세포 치료제 연구를위한 매우 중요하다 클릭당 비용 (CPC)을 분리합니다. 이 프로토콜은 초기 배 발생 및 방법는 ESC에서 자신의 세대에서 확인 된 배아 클릭당 비용 (CPC)에 초점을 맞추고있다. 다양한 클릭당 비용 (CPC)에도 뼈 된 marrows 2에서, 배아와 성인 마음에서 고립되었다. 배아 개발하는 동안, 뼈 morphoge마그네틱 단백질 (BMP 형식)은, 날개 형 MMTV 통합 사이트 가족 (Wnts)와 마디 신호는 Mesp1 + 능성 중배엽 3의 헌신을 유도한다. Mesp1 + 세포는 배아의 CPC 사로 분화. 이 클릭당 비용 (CPC)은 일반적으로 NK2의 호 메오 박스 (5) (Nkx2-5가), ISL LIM의 호 메오 박스 (1) (Isl1가), T-상자 5 (Tbx5) 및 심근 증강 인자 2C (Mef2c가), 차 및 제 마음 필드를 형성, HCN4으로 표시하고 있습니다 cardiogenesis ~ 10시 심장의 주요 부분에 기여하고 있습니다. Nkx2-5 + 및 Isl1 + / Mef2c + 두 클릭당 비용 (CPC)가 심근 세포로 분화 할 수있는, 평활근 세포 (SMCs) 및 내피 세포 5-8. 따라서 이들의 CPC 심장 혈관계뿐만 아니라 심장 조직을 야기하고 심장 세포 기반 치료를위한 이상적인 셀 소스있는 것이다. 결과적으로, 시험 관내에서 CPC를 생성하는 심혈관 연구에서 주요 연구 초점이되어왔다. 는 ESC 무제한 확장 용량을 가지고 있기 때문에ND 포배기에서 ICM 세포를 나타내고, 천연 배아 배아 다음의 CPC는 ESC로의 분화는 CPC를 획득하기 위해 논리적이고 효과적인 방법으로 간주된다. 는 ESC에서 클릭당 비용 (CPC)을 얻을 수있는 한 넓게 적용 방법은 사채 11로는 ESC를 집계하는 것입니다. 분화 효율을 향상시키기 위해, 심장 개발 기술에 기초하여 정의 화학적 및 성장 인자 12-14 사용되고있다. 그러나, 현장에서 널리 허용되는 명확한 CPC 마커, 특히 아니 세포 표면 마커는 없다. 이 문제를 해결하기는 ESC는 Isl1 + 또는 Mef2c + 클릭당 비용 (CPC) 및 Cre 호텔 /에 loxP 시스템을 사용하여 형광 기자들과 이들의 유도체를 표시하도록 설계되었습니다. CRE 재조합 효소는 Isl1 / Mef2c 프로모터 / 인핸서의 제어에 노크한다. 구성 적 프로모터에 의해 구동되는 형광 단백질의 변성 또는 RFP YFP 유전자 CRE 재조합 효소와 플록스 정지 코돈의 절단에 의해 활성화 될 수있다(ISL1 : CRE;-pCAG - 플록스-STOP-플록스 GFP 또는 RFP / Isl1-CRE; Rosa26YFP / Mef2c-CRE; Rosa26YFP) 5,6. 는 ESC가 두 번째 심장 필드 클릭당 비용 (CPC)로 분화되면, Isl1 / Mef2c 프로모터 / 증강 구동 CRE는 형광 기자를 활성화하고 클릭당 비용 (CPC)은 FACS 정화에 의해 강화 될 수있다. 간략하게, EB 집계 방법은 ESC의 분화를 시작하는 데 사용된다. 분화 효율을 향상시키기 위해, 분화 세포는 아스코르브 산 (AA) 및 BMP4, 티빈 13,15 VEGF와 같은 성장 인자로 처리된다. 이 프로토콜은 마우스와 인간는 ESC를 모두 사용하여 강력하고 효율적인 CPC 차별화 할 수 있습니다.

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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.

이 프로토콜은 여러 ES 세포주에서 클릭당 비용 (CPC)의 유도를 보여줍니다. 는 ESC는 클릭당 비용 (CPC)로 분화하는 교환 사채를 형성하기 위해 집계됩니다. 는 ESC 정기적으로 MEF 피더 (그림 1A, F)가 유지 관리 및 공급 장치는 차별화 전에 제거됩니다. 분화 배지에서 EB를 (도 1b, G)는 ESC에 응집하면, EB를 형성 제는 마우스 세포주 (도 1C)에서 중배엽 분화를 향상 BMP4 ...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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 ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Research

JoVE Journal

Developmental Biology

8.1K 조회수.  University of Michigan. 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.

비디오: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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 ...

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

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

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

말초 혈액 조혈 전구 세포에서 인간 신경 줄기 세포의 직접 유도

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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

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

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

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

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

배아 줄기 세포와 조직의 심장 차별화의 성적인 규정

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

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

수평 한 전체적인 거치 : 피부의 두껍고, 3 차원 조직 단면도를위한 비발 한 가공 그리고 화상 진찰 의정서

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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 ...

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

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

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

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

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

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

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

파생 및 개 난소 중간 엽 줄기 세포의 분화

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

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 ...