Name: in vitro pancreas organogenesis from dispersed mouse embryonic progenitors
Uploaded: 2014-07-19T15:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

이 작품은 Det에 FRIE Forskningsråd / Sundhed OG Sygdom에서 유전학 파일럿 상에 NCCR 프론티어, 청소년 당뇨병 연구 재단의 41-2009-775 그랜트 12-126875 순차적으로 투자되었다. 저자는 비디오 촬영을 호스팅 스파 놀리 실험실 감사합니다.

이 프로토콜은 쥐 E10.5에서 파생 된 췌장 organoids 상피 췌장 세포를 해리 성장하는 것을 목표로하고있다. 이 프로토콜은 동물 실험에 대한 윤리적 승인을 필요로합니다. E10.5 마우스 배아에서 지느러미 췌장 버드 1. 해부 <ol><li> 가위로 복부를 열고 배아 일 (E) 10.5 초과 임신 쥐를 희생, 두 자궁 뿔을 제거하고 차가운 인산 완충 생리 식염수 (PBS) 또는 둘 베코 가득 10cm 페트리 접시에 배치 수정 필수적인 매체 (DMEM)을 얼음에 보관. 세포 파종에 희생에서 전체 실험은 세포 손상을 방지하기 위해 60 ~ 90 분에 이루어집니다. </li><li> 작은 가위를 사용하여 개별 배아 세그먼트로 자궁을 분리합니다. 차가운 PBS와 35mm 페트리 접시에 하나의 배아를 전송하고 위의 조명과 해부 현미경을 시각화. 해부 집게와 주변 근육, 탈락과 노른자 SA를 제거C와 배아를 노출합니다. 다시 차가운 DMEM 매체에 배아를 놓습니다. 태아 간단 3 ㎖ 플라스틱 전송 피펫 / 적기를 사용하여 전송 될 수있다. 계속하기 전에 유사한 방식으로 모든 개인 배아를 분리합니다. </li><li> PBS에 깨끗한 35mm 페트리 접시에 배아를 놓습니다. <ol><li> 앞다리를 제거하는 얇은 집게 (0.05 mm 폭)를 사용합니다. 조심스럽게 척수 지역에서 소화 기관 (그림 1)을 분리 개방에 집게를 삽입합니다. 선택 사항 : 더 편리하게, 위를 참조하십시오 아래 노른자 줄기 아래 마음과 꼬리 영역으로, 태아의 상체를 제거합니다. </li><li> 위, 간, 소장을 찾습니다. 집게를 사용하여 소장에 위장에서 소화 기관을 분리하고 (그림 1) 얼음에 차가운 DMEM에 배치합니다. 지느러미 췌장 꽃 봉오리는 위장 등쪽, 후부 (그림 1)가 포함되어 있습니다. </li><li> 시미에있는 모든 개인 위장관 분리계속하기 전에 고맙다 방식. 배아 유전자형을해야 할 경우, 절개시에 꼬리를 수집하고 얼음에 차가운 DMEM과 24 웰 플레이트에서 다른 잘되는 모든 개인 위장관을 유지합니다. </li></ol></li><li> 차가운 PBS에 깨끗한 35mm 페트리 접시에 한 위장관 놓습니다. 이제 해부 현미경 등의 췌장 봉오리를 시각화하고 해부 시야에서 아래에서 조명을 사용합니다. 이러한 조건은 볼륨의 시각화를 최적화합니다. 전해 날카롭게 텅스텐 바늘 또는 20 G 주사기 바늘을 사용하여, 상피 주위 가급적 간엽 가진 췌장 새싹 (도 1)을 격리. <ol><li> 2 ~ 3 분 동안 차가운 디스 파제 용액 (1.25 ㎎ / ㎖)를 포함하는 배양 접시에 고립 된 꽃 봉오리를 전송합니다. 이 시점에서 전송이 가장 편리하게 입 통제 유연한 튜브에 부착 불꽃 뽑아 50 μL 유리 모세관을 수행 할 수 있습니다. 대안 적으로, 그러나 더 RI 함께새싹을 잃을 SK는 적절한 플라스틱 팁을 10 μL 자동 피펫 (Pipetman)를 사용합니다. </li><li> 미세한 제어 췌장 봉오리 흡입과 배출을 수행합니다. 다시 PBS에서 췌장 봉오리를 넣어. 또한 유리 모세관 (그림 1)를 사용하여 바늘과 부드러운 열망을 가진 중간 엽에서 분리 된 췌장 봉오리를 청소합니다. </li><li> 전체 중간 엽을 제거 할 때, 차가운 PBS에서 췌장 싹 씻어; (60 - 웰 미니 트레이 차가운 DMEM 10 μL로 가득) 개인 우물에서 차가운 DMEM 각 봉오리를 전송합니다. 그것은 조직이 매우 끈적하게하는, 디스 파제에서 중간 엽을 제거하지 않는 것이 중요합니다. </li></ol></li></ol> 분산 된 세포의 2. 도금 및 문화 <ol><li> 세정 10 ㎕의 PBS로 가득 60도 미니 트레이의 원추형의 우물에 불꽃 뽑아 유리 모세관 모든 배아에서 해부 상피를 전송합니다. <ol><li> 트립신 0.05 %의 10 μL에 꽃 봉오리로 이동ND는 4 분 동안 37 ° C에서 배양 할 수 있습니다. 10 μL의 DMEM + 10 % 소 태아 혈청 (FCS)와 우물에 새싹 전송하여 트립신을 불 활성화. </li><li> 피펫 풀러으로 뽑아가는 모세 혈관을 통해 흡인 세포 현탁액을 떼어 놓다. 이것은 세포를 해리 상하 피펫 팅하면서이 단계에서 기포를 방지하는 것이 중요하다. 췌장 organoids 최적 ~ 15 세포의 작은 그룹에서 시작하기 때문에 부분적인 분리는 (그림 1)를 추천합니다. </li></ol></li><li> 개별 처리로 인한 차이를 최소화하기 위해 에펜 도르프 튜브에 몇개의 배아 세포를 해변. 1:3 비율로 냉장 마트 리겔에서 세포 현탁액을 희석. 나누어지는 96 - 웰 플레이트, 8 μL / 잘 또는 영상에 최적화 된 접시에 (아래 참조)이 혼합물. </li><li> 리겔은 두껍게 할 수 있도록 5 분 동안 37 ° C에서 접시를 품어. 선택의 매체의 70 μL로 우물을 채우기 (organoid 또는 구, 탭보기 참조레 1, 2), 37 ° C에서 5 % CO 2, 95 %의 공기를 포함하는 습한 환경에서 떠나 </li><li> 매 4 일째 매체를 교체합니다. 매일 성장하는 췌장 식민지를 모니터링 및 이미징하여 프로세스를 문서화합니다. </li><li> 이전에보고 된 28 또한 작은 분자 또는 단백질, 간섭 실험을 위해이 단계에서 배지에 첨가 할 수있다. </li></ol> 표 1 : Organoid 매체. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 소재의 이름 </td><td> 주식 집중 </td><td> 최종 매체에 집중 </td><td> 주식의 볼륨 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 100 % </td><td> 1 % </td><td> 50 μL </td></tr><tr><td> 녹아웃 혈청 교체 (보충) </td><td> 100 % </td><td> 10 % </td><td> 500 μL </td></tr><tr><td> 2 - 머 캅토 에탄올 </td><td> 14.3 M </TD&gt; <td> 0.1 ㎜ </td><td> 1 μL </td></tr><tr><td> 포르 미리 스테이트 아세테이트 (PMA) </td><td> 16 μM </td><td> 16 nM의 </td><td> 5 μL </td></tr><tr><td> Y-27632 (ROCK 저해제) </td><td> 50 mM의 </td><td> 10 μM </td><td> 1 μL </td></tr><tr><td> EGF </td><td> 50 ㎍ / ㎖의 </td><td> 25 NG / ML </td><td> 2.5 ㎕의 </td></tr><tr><td> 재조합 인간 1 R-spondin </td><td> 250 ㎍ / ㎖의 </td><td> 500 NG / ML </td><td> 10 μL </td></tr><tr><td> - 또는 - </td><td></td><td></td><td></td></tr><tr><td> 재조합 마우스 R-spondin 1 </td><td> 250 ㎍ / ㎖의 </td><td> 500 NG / ML </td><td> 10 μL </td></tr><tr><td> 재조합 인간 FGF1 (aFGF) </td><td> 100 ㎍ / ㎖의 </td><td> 25 ㎍ / ㎖의 </td><td> 1.25 μL </td></tr><tr><td> 헤파린 (Liquemin) </td><td> 2500 U / ㎖ </td><td> 2.5 U / ㎖ </td><td> 2 μL </td></tr><tr><td> 권장binant 인간 FGF10 </td><td> 100 ㎍ / ㎖의 </td><td> 100 NG / ML </td><td> 5 μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 μL </td></tr><tr><td> 합계 </td><td></td><td></td><td> 5,000 μL </td></tr></tbody></table> 표 2 : 구 매체. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 소재의 이름 </td><td> 주식 집중 </td><td> 최종 매체에 집중 </td><td> 주식의 볼륨 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 100 % </td><td> 1 % </td><td> 50 μL </td></tr><tr><td> B27의 X50 (보충) </td><td> 100 % </td><td> 10 % </td><td> 100 μL </td></tr><tr><td> 재조합 인간 FGF2 (bFGF를) </td><td> 100 ㎍ / ㎖의 </td><td> 64 NG / ML </td><td> 3.2 ㎕의 </td></tr><tr><td> Y-27632 (ROCK 저해제) </td><td> 50 mM의 </td><td> 10 μM </td><td> 1 μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8 μL </td></tr><tr><td> 합계 </td><td></td><td></td><td> 5000 μL </td></tr></tbody></table> Organoid 개발의 진행 상황 3. 영상 <ol><li> 이미지 organoids 하나를 매일 또는 시간 경과 현미경 형광 시간 경과 현미경을 사용하여. 시간 경과 영상의 경우, XY (Z) 자동 거꾸로 형광 현미경을 사용합니다. </li><li> 4 - 웰 플레이트 또는 2-5 ML 매체로 채워진 유리 바닥 접시에 3 μL의 예금 작은 방울. 배 장거리 목적으로 이미지. 참고 : 형광 트레이서을 표현 형질 전환 마우스를 사용할 수 있습니다. 예를 들어 영화 1은 Pdx1 - Ngn3-ER TM-IRES-nGFP + 마우스 4에서 organoids의 초기 확장. 핵 GFP 개별 개체 만 유사한 원리는 형광 막과 세포를 추적하는 데 적용 할 수있는 세포를 추적 할 수 있도록 사용자가 그러한MT / MG 마우스 29로. </li><li> 초점이 감도을 방지하고 수동으로 정의 된 위치에 3 일 이상 1 사진 / 시간을 취할 수있는 소프트웨어 제어 자동화를 설정하는 세포를 파종 한 후 시간 경과 영상 3 시간을 시작합니다. 각 위치에 대한, 형광 마커의 발현을보고, 미분 간섭 대비 (DIC) 이미지뿐만 아니라, GFP 신호를 획득. </li></ol> 조직학에 대한 Organoids 4. 복구 <ol><li> 얼음에 96 - 웰 플레이트를 배치하고 얼음 차가운 PBS로 교체, 매체를 제거합니다. 이것은 부분적으로 리겔을 해중합. </li><li> 조심스럽게 전체 아키텍처를 방해하지하기 위해 1000 μL 팁을 사용하여 주변의 리겔을 제거, 각 organoid를 대기음. 얼음 차가운 PBS로 잘 각 organoid을 전송합니다. 얼음에 플레이트를 유지합니다. 리겔 직접 고정도 가능합니다. </li><li> 4 % 파라 포름 알데히드 (PFA)로 15 분 동안 organoid 고쳐 자당에 냉동 보존 및 젤라틴에 포함. 각 처리organoid 이전 (요한슨 등., 2007) 4 기술로 저온 절편 제작 및 조직학에 대한. </li></ol> PCR 및 생화학을위한 Organoids 5. 복구 <ol><li> 얼음에 96 - 웰 플레이트를 놓고 매체를 제거합니다. 세포의 RNA를 안정시키고 보호하기 위해 잘 당 RNAlater의 60 μl를 추가합니다. </li><li> 부분적으로 얼음을 해중합하여 각 아니라 기계적에서 젤을 방해. 전체 아키텍처를 중단 또는 200 μL 팁으로 기계적으로 젤을 방해하여 (리겔 포함) 전체를 잘 복구 할 수 있도록 1000 μL 팁을 사용하여 개별 organoids를 복구 하나. 이는 RNAse 무료 끈적 에펜 도르프 튜브에 잘 콘텐츠를 전송하기 위해 1000 μL 팁을 사용하여 얼음에 보관. </li><li> 60 μL의 RNAlater에 우물을 세척하고 같은 에펜 도르프 튜브에 남아있는 내용을 추가합니다. </li><li> 4 ℃에서 500 ~ 1,000 XG에서 5 분 튜브를 스핀 </li><li> 만 20 ~ 30 μl를 떠나, 뜨는을 제거함께 펠릿과 튜브 RNAlater의. 생화학의 경​​우, 가능한 한 건조 샘플을 저장합니다. </li><li> 즉시 RNAlater에서 샘플을 동결하지 마십시오; 4 ° CO / N에 상점 (RNAlater 철저하게 조직을 침투 할 수 있도록). 조직은 장기 저장을 위해 -20 ° C에 저장할 수 있습니다 나중에 조직의 파괴와 RNA의 소량의 추출을 위해 처리 할 수​​ 있습니다. </li></ol>

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The authors have nothing to disclose.

시험 관내에서 기능적인 베타 세포의 대규모 생산은 여전히 하나 비효율적이다. 이 도전적인 상황에서, 발달 생물학 연구 기능의 베타 세포의 분화에 필요한 정확한 신호를 해독하는 데 도움이 될 수 있습니다. 이 프로토콜은 체외에서 배아의 췌장 전구 세포의 유지 보수, 확장과 차별화 할 수 있습니다. 이것은 다른 내분비 호르몬을 공동으로 표현하지 않는 인슐린을 생산?...

장기 문화는 생체 내 연구에서 복잡하지만 관련성이 높은 사이의 간격 및 세포 라인 모델의 편리하지만 대략적인 시뮬레이션을 다리에 유용한 모델을 제공합니다. 췌장의 경우, 내분비와 외분비 세포 시뮬레이션 세포주가 변형 되더라도 췌장 전구 세포로 완전히 동등한 어떠한 세포주도 없다. 성인 전체 췌장을 배양 할 수 없습니다; 절연 내분비 아일렛 세포 증식없이 몇 주 동안 유지 될 수 있으며, 조직 조각은 몇 시간에서 5 시험 관내에서 유지 될 수있다. 배아의 췌장 문화가 널리 개발을 연구뿐만 아니라 사용되었습니다뿐만 아니라 8을 처리하는 이미지로, 상피 - 간엽 상호 작용 4,6,7을 조사하기 위해 화학적으로 그 9 방해 할 수 있습니다. 두 기관 배양 방법이 주로 사용된다 : 첫 번째는 conve입니다 피브로넥틴 코팅 플레이트 2에 췌장 꽃 봉오리를 배양에 구성촬상 목적 더욱 편리 할; 두 번째 옵션은 문화에 가장 형태 형성을 유지 공기 - 액체 인터페이스 3,4에서 필터에 대한 기관입니다. 매우 유용하지만, 이러한 방법은 평탄화의 어느 정도 이어질; 정상적인 발달에 비해 시작 인구가 췌장 세포 및 중간 엽 세포의 모든 유형을 포함하는 복잡한 같이 선조의 팽창은 매우 제한된다. 문화 능력과 분산 일차 전지를 확장 계보 관계를 연구하고 고립 된 세포 유형 (10)의 고유 특성을 발견 할 가치가있다. 스기야마 등. 11 췌장 전구 세포 및 피더 레이어에 문화에 3 ~ 5 일 일부 기능 문자를 유지 내분비 전구 세포를 유지할 수 있습니다. 조상의 자연 / 줄기 세포하지만 neurosphere를 12, 13 mammospheres에 가깝다 Pancreatospheres는 성인 독도 및 유관 세포에서 확장되었습니다이러한 분야를 생성하는 것은 명확하지 않다. 또, 생리 현상과는 대조적으로, pancreatospheres 일부 뉴런에게 14,15을 함유 하였다. 분야는 최근 배아 췌장 전구 세포 (16, 17)과 좋은 시조 확장 이후의 분화와 pancreata 18 재생에서 생산하지만, 형태 형성을 요점을 되풀이하는 데 실패했다. 소형 기관에 자체 조직으로 분산 종종 정의 세포에서 3D 모델은 최근 번성하고 소장 (19, 20), 배 (21), 간 (22), 전립선 (23)과 기관 등의 여러 장기의 개발 또는 성인 매출을 시뮬레이션 한 24. 광섬유 컵 25, 26 또는 뇌 부위 (27)의 경우와 같이 몇몇 경우에, 형태 형성 발달과 분화는, ES 세포로부터 3D로 효과적으로 요약되었다. 여기, 우리 DES그들은 차별화 자체 구성 할 수 있습니다 3D 리겔 비계에서 해리 다 능성 췌장 전구 세포를 확장하는 방법을 cribe.

<table><tbody><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>KnockOut Serum replacement (supplement)</td><td>Gibco</td><td>10828-028</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma Aldrich</td><td>3148-25ML</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Phorbol Myristate Acetate (PMA)</td><td>Calbiotech</td><td>524400-1MG</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Y-27632 (ROCK inhibitor)</td><td>Sigma Aldrich</td><td>ab120129</td><td>Stock kept at -20 &amp;deg;C. Attention! Stability/source is a frequent source of problems.</td></tr><tr><td>EGF</td><td>Sigma Aldrich</td><td>E9644-2MG</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human R-spondin 1</td><td>R&amp;amp;D</td><td>4645-RS-025/CF</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Mouse R-spondin 1</td><td>R&amp;amp;D</td><td>3474-RS-050</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF1 (aFGF)</td><td>R&amp;amp;D</td><td>232-FA-025</td><td>Stock kept at -80 &amp;deg;C - do not include to increase beta cell production</td></tr><tr><td>Heparin (Liquemin)</td><td>Drossapharm</td><td></td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF10</td><td>R&amp;amp;D</td><td>345-FG-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>21331-020</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>B27 x50 (supplement)</td><td>Gibco</td><td>17504-044</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF2 (bFGF)</td><td>R&amp;amp;D</td><td>233-FB-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>RNAlater - RNA stabilizing reagent</td><td>Qiagen</td><td>76104</td><td>Store at RT</td></tr><tr><td>Dispase&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4818-2MG</td><td>Working concentration: 1.25 mg/ml. Stock kept at -20 &amp;deg;C</td></tr><tr><td>BSA for reconstitution</td><td>Milipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/item/81-068-3&quot;&gt;81-068&lt;/a&gt;</td><td>For reconstituition of cytokines&amp;nbsp; - stock kept at -20 &amp;deg;C</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Gibco</td><td>16141079</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>60-well MicroWell trays</td><td>Sigma Aldrich</td><td>M0815-100EA</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>95-well plates F bottom</td><td>Greiner Bio</td><td>6555180</td><td></td></tr><tr><td>Glas bottom plates</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Disposal glass micropipettes</td><td>Blaubrand</td><td>708745</td><td></td></tr><tr><td>Microscope</td><td></td><td></td><td>Cell&amp;#174; imaging station (motorized inverted Olympus IX81 stand) equipped with a Hamamatsu ORCA ER B7W camera and the Ludin Cube and Box.&lt;br /&gt;
Leica DMI6000 B stand surrounded with a Ludin Cube and Box, equipped with a Leica DFC365 FX camera and the AF6000 Expert/Matrix software command interface.</td></tr><tr><td>Objective</td><td></td><td></td><td>Olympus UPLAN FL NA 0.30 air 9.50 mm 10X long distance;&lt;br /&gt;
Leica HC PL FLUOTAR NA 0.30 air 11.0 mm 10X long distance</td></tr></tbody></table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

3D 리겔에서 해리와 시드 E10.5 지느러미 췌장 전구 세포는 췌장 개발을 요점을 되풀이하다. 조상은 쉽게 형광 기자와 올 수 있습니다. 우리의 경우 우리는 (그림 2) Neurog3 4 활성화하지 않고 타목시펜의 부재에 따라서 Pdx1 프로모터 (Pdx1 - Ngn3-ER TM-nGFP) (동영상 1)에 의해 제어되는 핵 GFP 단백질을 발현하는 형질 전환 마우스를 사용. organoid 매체?...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

이 프로토콜에 기재된 삼차원 배양법 분지 기관에 자신의 상당한 팽창, 분화 및 형태 발생을 포함한 분산 배아 마우스 췌장 전구 세포로부터 췌장 개발 되풀이되었습니다. 이 방법은 영상, 틈새 시장의 기능 장애 및 조작에 대한 의무입니다.

분산 된 마우스 배아 전구 세포에서 생체 췌장 기관 형성에

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K 조회수.  Swiss Institute for Experimental Cancer Research.  이 프로토콜에 기재된 삼차원 배양법 분지 기관에 자신의 상당한 팽창, 분화 및 형태 발생을 포함한 분산 배아 마우스 췌장 전구 세포로부터 췌장 개발 되풀이되었습니다. 이 방법은 영상, 틈새 시장의 기능 장애 및 조작에 대한 의무입니다. 

비디오: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

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. ...

연구

발생학

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

신생아 마우스 췌장의 기능 독도의 효과적인 분리

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

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

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

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

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

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 ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

의학

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

비 계, 3 차원 인슐린 표현 Pancreatoids 마우스 췌 장 창시자에 체 외에서 의 세대

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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 ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

분산 된 마우스 배아 전구 세포에서 생체 췌장 기관 형성에

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