Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

이 원고는 토론토 일반과 서양 재단과 밴팅 &amp; 베스트 당뇨병 센터 - 대학 건강 네트워크 대학원 수상에서 자금에 의해 지원되었다.

솔루션 및 미디어 1. 준비 주 : 무균 환경에서 세포 배양에 대한 모든 미디어를 준비합니다. 미디어 만든 즉시 사용할 수 있습니다. 시약 세부 사항은 재료 표에 나와 있습니다. <ol><li> 차별화 미디어 <ol><li> 1 % 글루타민, 2 μM CHIR 99021, (100) NG / ㎖ 티빈 A, 104 M MTG와 RPMI 매체 : 일 0 차별화 미디어를 준비합니다. </li><li> 1 % 글루타민, 100 NG / ㎖ 티빈 A, 104 M MTG, 5 NG / ㎖의 bFGF, 50 μg의 / ㎖ 아스 코르 산으로 RPMI 매체 : 하루 1-2 차별화 미디어를 준비합니다. </li><li> 1 % 글루타민, 1 % B27, 104 M MTG, 0.75 μM Dorsomorphin, 50 NG / ㎖ FGF10로 RPMI 매체 : 하루 3-5 차별화 미디어를 준비합니다. </li><li> 하루 6 ~ 7 차별화 미디어를 준비 : DMEM을 1 % 글루타민, 1 % B27, 50 μg의 / ㎖ 아스 코르 산, 50 NG / ㎖ FGF10, 0.25 μM의 SANT-1, 2 μM 레티노 산, 50 NG / ㎖ 소량으로. 참고 : 레티노산성 마지막 배지에 첨가되어야하며, 용지가 산화 분해 (15)을 방지하기 위해 빛으로부터 보호되어야한다. </li><li> 일 8-12 차별화 미디어를 준비 : DMEM을 1 % 글루타민, 1 % B27, 50 μg의 / ㎖ 아스 코르 산, 50 NG / ㎖ 소량, 100 NG / ㎖ hEGF, 10 mM의 니코틴 아미드와 함께. </li></ol></li><li> PBS에서 10 % FBS, 마이너스 칼슘과 마그네슘 : FACS 버퍼를 준비합니다. </li></ol> 2. HESC 차별화 참고 : HESC는 해동 계대과의 bFGF (16) 보충 KOSR 기반 미디어의 존재에 조사 된 마우스 배아 섬유 아세포에 확장됩니다. 세포가 80-95%의 포화 상태에 도달했을 때 hESCs는 차별화를위한 준비가되어 있습니다. 이 때 식민지 정의 테두리와 &#39;domelike&#39;구조 (그림 1A)와 커야합니다. 모든 세포 배양 평면 바닥 0.1 % 젤라틴으로 코팅 된 조직 배양 처리 된 플레이트에서 수행된다. 일반적으로, 세포 (6)에서 재배되거나각각 2 ml의 또는 1 ㎖의의 매체 볼륨의 12 웰 플레이트. <ol><li> 0 일에서 일 0 차별화 미디어로 KOSR 기반 미디어를 대체하여 차별화를 시작합니다. </li><li> 일 1과 2에서 부드럽게 흡입하기 전에 단일 층에서 죽은 세포를 제거하기 위해 판을 흔들어. 주 1 ~ 2 차별화 미디어로 교체하십시오. </li><li> 3 일에, 유동 세포 계측법을위한 수확 세포. 세포가 90 % 이상 CXCR4 + / CD117 + 표현하면 2.4 단계로 진행합니다. </li><li> 일 3, 5, 부드럽게 이전에 흡입에 단일 층에서 죽은 세포를 제거하기 위해 판을 흔들어. 하루 3-5 차별화 미디어로 교체하십시오. </li><li> 일 6, 7 일, 하루 6 ~ 7 차별화 미디어로 교체합니다. </li><li> 일 8, 10 및 12에서 하루 8-12 차별화 미디어로 교체합니다. </li><li> 날 13 일, 흐름에 대한 수확 세포는 세포 계측법 PDX1과 NKX6-1 식을 결정합니다. </li></ol> 유동 세포 계측법 분석 3. 수확 세포 <ol><li> 와 세포를 떼어 놓다제조 업체의 프로토콜과 3 분 동안 37 ° C에서 부화에 따라 1 배 상업 트립신 솔루션입니다. </li><li> 30 μL의 DNase의의 I. 1,000 μL FACS 버퍼에 단일 세포를 상용 트립신 용액을 제거하고 재현 탁 </li><li> 미세 원심 분리 관에 35 ㎛의 나일론 메쉬 셀 스트레이너 및 전달을 이용하여 필터 셀. </li><li> 5 분 455 XG에 원심 분리기 세포. 어느 라이브 또는 고정 염색 세포를 준비합니다. </li></ol> 유동 세포 계측법 4. 염색 <ol><li> 3 일에 라이브 염색에 대비 흐름 <ol><li> 1000 μL FACS 버퍼에 재현 탁 세포. 전송 200 μL (모두 흠 염색 샘플) 96- 웰 플레이트의 웰에 두 개 (약 1-3 × 105 세포). </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> APC 및 CD117 : 차 복합 항체, CXCR4으로 얼룩 PE는 3 (M의 aterials 표 참조)실온에서 0 분 빛으로부터 보호. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> 100 μL FACS 버퍼에 재현 탁 샘플. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> 1 ml의 마이크로 테스트 튜브 또는 5 ㎖ 둥근 바닥 튜브 300-500 μL FACS 완충액에서 총 부피 각각의 샘플 및 전송을 재현 탁. </li><li> (17) 흐름 cytometer에서 실행 샘플. 샘플을 즉시 실행할 수없는 경우, 4 ℃에서 보관 빛으로부터 보호. </li></ol></li><li> 하루 (13)에 고정 염색에 대비 흐름 <ol><li> 4 ℃에서 24 시간 동안 상업 고정 / permeabilization 솔루션에서 세포 펠렛을 Resuspend. </li><li> 5 분 동안 455 XG에서 샘플을 원심 분리기. 400 μL 파마 / 세척 용액을 Resuspend. </li><li> (TW)에 샘플 (약 0.5 × 10 6 세포)의 200 μl를 전송(IgG의 제어 및 PDX1 / NKX6-1 염색 용) 96 웰 플레이트의 O를하는 것은 우물. 2 분 동안 931 XG에 원심 분리기. </li><li> 안티 PDX1 및 안티 NKX6-1 차 또는 이소 제어 항체를 포함하는 100 ㎕의 파마 / 세척 용액의 세포 펠렛을 재현 탁 (재료 표 참조). 4 ℃에서 밤새 품어. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. 100 μL 파마 / 세척 용액을 Resuspend 샘플. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> 빛으로부터 보호 실온에서 1 시간 동안 이차 항체를 포함하는 100 μL 파마 / 워시 샘플을 재현 탁. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> 100 μL 파마 / 세척 용액에 재현 탁 샘플. </li><li> 2 분 동안 931 XG에 접시를 원심 분리기. 접시를 반전하여 상층 액을 제거합니다. </li><li> 대표단계 4.2.9 및 4.2.10을 먹는다. </li><li> 1 ml의 마이크로 테스트 튜브 또는 5 ㎖ 둥근 바닥 튜브 300-500 μL FACS 완충액에서 총 부피 각각의 샘플 및 전송을 재현 탁. </li><li> (17) 흐름 cytometer에서 실행 샘플. 샘플을 즉시 실행할 수없는 경우, 4 ℃에서 보관 빛으로부터 보호. </li></ol></li></ol>

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저자는 공개 아무것도 없어.

성공적으로 시험 관내에서 hPSCs에서 NKX6-1 + 췌장 전구 세포를 생성하는 췌장 개발 과정에서 중요한 발달 단계를 관리하는 특정 신호 전달 경로의 정확한 규정을 포함 hPSCs의 높은 품질의 문화와 감독 분화의 사용에 의존한다. 이전에 다음과 같은 고려가 이루어져야 NKX6-1 효율적인 생성을 위해, 3이 나타내는 바와 같이 프로토콜 hPSC 라인의 다양한 걸쳐 NKX6-1의 강력?...

당뇨병의 유병률은 증가하고 캐나다 당뇨병 협회에 따르면, 제 1 형 당뇨병 (T1D) 1을 가진이 개인의 5 ~ 10 %로, 캐나다에서 만 11 이상의 개인이 당뇨병 또는 당뇨병 진단 전의 것으로 추정된다. T1D는 랑게르한스섬 내에 위치 인슐린 생성 β 세포의 파괴에 의해 야기 된자가 면역 질환이다. 현재 T1D 함께 사는 사람들은 인슐린이의 외인성 소스를 필요로한다. 인슐린 치료에서의 진보에도 불구하고, T1D 환자는 혈당 수치를 조절하는데 어려움이 있고 하이포 고혈당 모두 고생 계속합니다. 치료의 유망한 형태는 T1D에서 정상 혈당이 생체 내 및 시험 관내 3 모두 β 세포의 인슐린 생산의 무제한 공급을 생성하는데 사용될 수있는 인간 배아 줄기 세포 (hESCs는)의 사용이다 복원 <s클래스 = &quot;외부 참조&quot;&gt; 4, 5, 6, 7까지. hESCs는 차별화하는 β-같이하는 세포는 가능한 제 2 형 당뇨병에 대한 새로운 치료 표적의 식별을 허용, 시험 관내에서 당뇨병을 연구하고 T1D 환자에 이식 세포를 제공하기 위해 만들 수 있습니다. 체외에서 hESCs는에서 인슐린 생산 세포를 생성에서 가장 성공적인 시도는 췌장 개발 4, 5시에 발생하는 배아 이벤트를 요점을 되풀이하는 것입니다. 이것은 정확하게 현상 췌장의 주요 단계를 모델링 별개의 신호 경로의 조작을 포함한다. 췌장 개발은 CXCR4 및 CD117 (C-KIT) 8, 9의 발현을 특징으로 최종 내배엽의 유도로 시작합니다. definit의 정확한 조절필자 내배엽 조직은 다음 전방 - - 후방 및 복부 - 등의 패턴 거쳐 창자 관의 형성이 필요합니다. 지느러미와 복부 췌장 싹이 췌장 개발 (10)에 필요한 췌장과 십이지장 호 메오 박스 유전자 (Pdx1)를 표현하는 후방 foregut의 영역에서 등장. 지느러미와 복부 싹 퓨즈는 광범위한 상피 리모델링 및 확장 (11)를 거쳐 췌장을 형성한다. 내분비 및 외분비 혈통에 의지가 표현하는 다 능성 전구 세포 (MPC와)의 생성을 동반, 다른 사람의 사이에서, 전사는 Pdx1, Nkx6.1 및 Ptf1a 12, 13 요인. 내분비 및 유관 세포가 될 것이다 MPC와는 Ptf1a 식을 줄이면서 Nkx6-1을 표현하는 것을 계속한다. 이에 대하여, 외분비 혈통 세포는 expressio 잃게됩니다Nkx6-1의 n 및 Ptf1a 식 (12)을 유지한다. 전사 인자 Nkx6-1 특히 세포와 β 내분비 전구 세포의 분화 과정, 췌장 발달에 중요한 역할을한다. 이전 췌장 개발 14 중 β 세포 장애가 형성 Nkx6-1 결과, 결실 한 바와 같이. 따라서, 생체 외 및 생체 내 인슐린 생성 β 세포를 발생시키는 Nkx6-1의 효율적인 도입을 필요로한다. 우리는 최근에 효율적으로 hPSCs에서 PDX1 + / NKX6-1 + 췌장 전구 세포를 생성 할 수있는 프로토콜을 개발했다. 이 hPSC 파생 췌장 전구 세포는 면역 결핍 마우스 3에 이식에 따라 성숙 β 세포를 생성합니다. 1) 최종 내배엽 유도 : 분화 프로토콜 4로 분할 될 수 특징 스테이지2) 후방 foregut 패터닝, 3) 췌장 사양 및 4) NKX6-1 유도. 여기서 우리는 분화 방향의 각 단계에 대한 상세한 설명을 제공한다.

<table><tbody><tr><td>&lt;strong&gt;Media and cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Thioglycerol (MTG)</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC/CF&amp;nbsp;</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Buffer</td><td>BD Bioscience</td><td>554722</td><td></td></tr><tr><td>BD Perm/Wash buffer, 1x</td><td>BD Bioscience</td><td>554723</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>CHIR990210</td><td>Tocris</td><td>4423a</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DNase I</td><td>VWR</td><td>80510-412&amp;nbsp;</td><td></td></tr><tr><td>Dorsomorphin</td><td>Sigma</td><td>P5499</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D</td><td>236-EG</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>88150</td><td></td></tr><tr><td>FGF10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>NO636</td><td></td></tr><tr><td>NOGGIN</td><td>R&amp;amp;D</td><td>3344-NG</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875</td><td></td></tr><tr><td>SANT-1</td><td>Tocris</td><td>1974</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), phenol red</td><td>Life Technologies</td><td>12605-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies for flow cytometry (working dilutions)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD117 PE (1:100)</td><td>Life Technologies</td><td>CD11705</td><td></td></tr><tr><td>CXCR4 APC (1:50)</td><td>BD&amp;nbsp; Bioscience</td><td>551966</td><td></td></tr><tr><td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td><td>Life Technologies</td><td>A-31571</td><td></td></tr><tr><td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.</td><td>705-546-147</td><td></td></tr><tr><td>Isotype Control Mouse IgG&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.&amp;nbsp;</td><td>015-000-003</td><td></td></tr><tr><td>Isotype Control Goat IgG</td><td>R&amp;amp;D&amp;nbsp;&amp;nbsp;</td><td>AB-108-C</td><td></td></tr><tr><td>NKX6-1 (1:2,000)</td><td>DSHB</td><td>F55A10</td><td></td></tr><tr><td>PDX1 (1:100)</td><td>R&amp;amp;D</td><td>AF2419</td><td></td></tr></tbody></table>

efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors

다 능성 줄기 세포는 자기를 갱신하고의 연구 및 미래 당뇨병 치료에 사용될 수있는 췌장 선조 세포의 생성을위한 매력적인 소스 만드는 다중 계통으로 분화하는 능력을 가지고있다. 이 문서에서는 인간 배아 줄기 세포 (hESCs는)에서 췌장 전구 세포를 생성하도록 설계된 4 단계의 분화 프로토콜을 설명합니다. 이 프로토콜은, 인간 다 능성 줄기 세포 (hPSC) 라인의 수에 적용 할 수있다. 췌장 전구 세포를 생성하기 위해 취해진 방식은 정확하게 췌장 개발의 주요 단계를 모델링 hESCs는 구별한다. 이것은 티빈 A, 염기성 섬유 아세포 성장 인자 (bFGF에) 및 CHIR990210의 존재 하에서 세포를 배양함으로써 달성되는 최종 내배엽의 유도와 함께 시작된다. 섬유 아세포 성장 인자 10 (FGF10) 및 Dorsomorphin와 또한 차별화 및 패터닝 후방 foregut를 닮은 세포를 생성합니다. 레틴의 추가OIC 산, 소량는 SANT-1과 FGF10은 췌장 내배엽의 특성 세포로 후방 foregut 세포를 구별합니다. 마지막으로, 표피 성장 인자 (EGF)의 조합은, 니코틴 및 소량은 PDX1 + / NKX6-1 + 세포의 효율적인 생성을 이끈다. 유동 세포 계측법 췌장 개발의 주요 단계에서 특이 마커의 발현을 확인하기 위해 수행된다. 스테이지 (4)의 끝에 PDX1 + / + 췌장 선조 NKX6-1은 면역 결핍 마우스에 이식시 성숙한 β 세포를 발생시킬 수 있으며 상기 체외 인슐린 생성 세포를 생성하기 위해 구별된다. 따라서, PDX1의 효율적인 생성 + / NKX6-1 + 췌장 전구 세포,이 프로토콜에서 입증 된 바와 같이,이 시험 관내에서 인간의 췌장 개발을 연구하기위한 플랫폼을 제공하기 때문에 매우 중요하고 분화 잠재력 세포의 공급원을 제공한다 EV 수 β 세포entually 당뇨병 치료에 이용 될 수있다.

도 1a에 개략적으로 도시 된 바와 같이, 췌장 전구 세포의 효율적 생성, 분화 프로토콜 중 특정 신호 분자의 정확한 첨가하여 미분화 된 세포의 적절한 성장 및 유지에 의존한다. 0 일에서 미분화 세포 80-95% 합류를해야하며, 식민지 정의 가장자리 (그림 1A)를 가져야한다. 세포 사멸이 단계에서 매우 일반적이기 때문에 1 단계 동안, 미디어 가능성이 ...

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

여기에서는 시험 관내 NKX6-1 + 췌장 전구 세포에 인간 배아 줄기 세포가 분화하는 4 단째의 프로토콜을 설명한다. 이 프로토콜은, 인간 다 능성 줄기 세포의 종류에 적용될 수있다.

능성의 효율적인 분화 NKX6-1에 줄기 세포<sup&gt; +</sup&gt; 췌장 전구 세포

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

efficient-differentiation-pluripotent-stem-cells-to-nkx6-1-pancreatic

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JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

7.9K 조회수.  University Health Network.  여기에서는 시험 관내 NKX6-1 + 췌장 전구 세포에 인간 배아 줄기 세포가 분화하는 4 단째의 프로토콜을 설명한다. 이 프로토콜은, 인간 다 능성 줄기 세포의 종류에 적용될 수있다. 

비디오: Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

인간의 췌장 개발에 리니지의 결정 요인을 들여다보기위한 게놈 편집 및 hPSCs의 감독 차별화

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

연구

유전학

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

인간 배아 줄기 세포로부터 생성 된 내배엽 세포의 정제를위한 신속하고 효율적인 방법

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

발생학

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

체외 식민지 분석 실험에서 트라이 강력한 전구 세포를 특성화를위한 성인 쥐 췌장에서 분리

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

효율적인 세대의 췌 장/십이지 Homeobox 단백질 1+ 뒤 Foregut/췌 장 창시자 접착 문화에 hPSCs에서

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

2D 배양 시스템에서 인간 만능줄기세포를 췌장 베타세포 전구체로 분화

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

인간 만능 줄기세포를 인슐린 생산 섬 클러스터로 분화(Differentiation of Human Pluripotent Stem Cells into Insulin-producing Islet Clusters)

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

생물학

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

인간 배아의 내피 세포 공동 배양 Mediates의 성숙은 감독 차별화 접근법에서 췌장 인슐린 생산 세포로 세포를 줄기

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

시험관 내 췌장 혈통을 향한 인간 치과 펄프 줄기 세포의 유도

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

생체공학

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

정의된 분화 시스템을 사용하여 만능 줄기 세포에서 간 선조 사양

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

능성의 효율적인 분화 NKX6-1에 줄기 세포

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