우리는 정렬에 도움을 희망의 도시에서 분석 세포 계측법 코어에서 루시 브라운과 알렉산더 Spalla 감사합니다. 이 작품은 건강 (NIH)의 국립 연구소 DAT 지지대에 재생 의학 부여 RB5-07398에 대한 국립 과학 재단 (National Science Foundation) 부여 NSF-DMR-1206121 캘리포니아 연구소 R01DK081587 및 R01DK099734 HTK, 그리고 ADR에 U01DK089533 및 부여에 의해 부분적으로 지원 HTK 푸에르 테 재단과 엘라 피츠 제럴드 재단의 조셉 J. 제이콥스 DAT에 캘리포니아 공과 대학에서 의학 분자 공학 연구소, 이들로부터 감사 인정도 있습니다. 자금 조달 :이 작품은 건강 (NIH)의 국립 연구소에 의해 부분적으로 지원됩니다 HTK에 R01DK081587 및 R01DK099734을 부여하고, ADR에 U01DK089533, 그리고에 재생 의학 부여 RB5-07398에 대한 국립 과학 재단 (National Science Foundation) 부여 NSF-DMR-1206121 캘리포니아 연구소 DAT는 조셉 J. 제이콥스에서 지원DAT에 캘리포니아 공과 대학에서 의학 분자 공학 연구소와 옥스 나드 재단과 HTK에 엘라 피츠 제럴드 재단들도 기꺼이 인정한다. 이 책에서보고 된 연구는 보너스 번호 P30CA33572에서 국립 보건원의 국립 암 연구소 (National Cancer Institute)에 의해 지원되는 분석 세포 계측법 코어 및 광학 현미경 디지털 이미징 코어에서 수행 한 작업을 포함했다. 연구 스폰서 : 스폰서는 데이터의 연구 설계, 수집, 분석 또는 해석에 참여하지 않았다.

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저자는 공개 아무것도 없어.

췌장 식민지 분석 및 단일 콜로니는 여기에 설명 분석 지난 수십 년 23 조혈 전구 세포의 생물학을 해독에서 중요한 역할을 한 메틸 셀룰로오스 함유 조혈 식민지 분석에 의해 영감을했다. 이 분석에서 (그림 5), 췌장 세포로 도금 해리 메틸 셀룰로오스 함유 링, 밀도 또는 내분비 / 선포 식민지 (7)의 형성을 지원하는 적절한 성장 인자와 ECM 단백질과 반 고형 미디어를. ?...

췌장은 세 가지 주요 세포 계통으로 구성된다; 선포 세포는 소화 효소가, 덕트가 병원균을 방어하고 장내에 소화 효소를 수송하는 점액 분비 분비 및 내분비 세포는 포도당 항상성을 유지하는 인슐린과 글루카곤을 포함한 호르몬을 분비. 췌장의 배아 발달 동안 초기 도관 세포는 성체 동물의 1,2- pancreata에서 세 계통 상승을 제공 할 수있는 트라이 강력한 선조 세포의 근원이다. 골수 줄기 세포와 같은 성체 줄기 세포 및 선조 세포는 이미 성공적으로 각종 질병 (3)을 치료하는 데 사용되기 때문에, 성인 췌장 줄기 및 전구 세포를 찾는데 큰 관심이있다. 분리 및 성인 췌장 줄기 및 전구 세포의 조작이 가능하다면,이 세포는 인슐린 분비 세포가자가 면역에 의해 파괴되는 제 1 형 당뇨병 등의 질병을 치료하는 데 사용할 수 있습니다. 생체 CRE-LOX 계보 - 추적 기술을,이나 다와 동료 마커, 탄산 탈수 효소 II 표지 성인 쥐 도관 세포, 세 췌장 계통 (4)을 야기 할 수 있음을 보여 주었다. 그러나, 이러한 HNF1b 5 Sox9이 다른 도관 마커를 이용하면, 도관 세포는 성체 생쥐에서 베타 세포의 주요 원인없는 것으로 결론 지어졌다. 몇 년 전에, 우리는 전술 논쟁의 원인은 필요한 자기 갱신 멀티 혈통 분화 개의 조건을 측정하는데 사용될 수있는 적절한 분석 도구의 필드 -6,7-에서 부족에 기인 할 수 있음을 제안 줄기 세포를 정의합니다. 언급 된 생체 CRE-LOX 계보 - 추적 기술위의 인구 수준에서 조상 - 자손 관계에 대한 증거를 제공 할 수 있습니다. 그러나,이 혈통 추적 기술은 단일 전구 세포는 자기 갱신과 여러 계통으로 분화 할 수 있는지 여부를 식별하기 위해 힘 제한된다. 여러 모노 강력한 조상, 다른 혈통 잠재력이 각각 함께 분석 하였다 경우, 그들은 집단적으로 멀티 혈통 분화 능력을 가지고 나타날 수 있기 때문에 단일 세포 분석은 중요하다. 또한, 줄기 세포는 일반적으로 성숙한 기관의 작은 집단이다. 작은 세포 집단의 활성은 주요 집단에 의해 마스크 될 수있다. 따라서, 모집단 연구에서 부정 결과가 반드시 줄기 세포의 유무를 나타내지 않는다. 마지막으로, CRE-LOX 계보 추적은 현재 자기 갱신의 측정을 허용하지 않습니다. 췌장 전구 세포 생물학, 식민지의 분야에서 기술 격차를 해결하는 시작하려면 7-11 또는 12-15 organoid </su3D 문화 시스템을 사용하여 페이지&gt; 분석 고안되었다. 췌장 전구 세포에 대한 두 식민지 분석은 우리의 실험실에서 개발 된 하나는 쥐의 세포 외 기질 단백질 (ECM) (방법 및 장비 표 참조)의 상업 준비를 포함, 다른 하나는 라미닌 하이드로 겔, 정의 된 인공 ECM 단백질 7-11 포함되어 있습니다. 전구 세포는 반고체 배지 함유 메틸 혼합한다. 메틸 목재 섬유로 제조 된 생물학적 불활성 점성 재료이고, 통상적으로 조혈 콜로니 분석 (16)에 사용되어왔다. 그들은 다시 집합체 수 없도록 메틸 - 함유 반고체 배지 단일 전구 세포의 이동을 제한한다. 그러나, 상기 매체가 전구 세포의 성장과 3 차원 공간에서 세포의 콜로니로 분화 할 수 있도록 충분히 부드러운 것이다. 혈액학의 전통에 따라, 세포의 식민지에 상승을 제공 할 수있는 선수는 췌장 전구 세포는 N이었다아메드 췌장 집락 형성 단위 (PCFU). 뮤린 ECM 함유 콜로니 에세이에서 성장시 PCFUs는 &quot;링&quot;콜로니 7 명명 낭성 콜로니를 야기. 의 Wnt 작용제를 첨가하면, R-spondin1는 쥐 ECM 함유 문화로, 일부 링 식민지는 &quot;밀도&quot;식민지 7 차례. 이 기사에서는 쥐 ECM 문화에서 자란 식민지의 두 가지 유형은 모두 &quot;반지 / 밀도&quot;식민지라고합니다. 벨소리 / 고밀도 콜로니가 단일 세포 현탁액 내로 해리 라미닌 하이드로 겔을 함유 배양에 재 플레이 팅하는 경우 &quot;내분비 / 선포&quot;7 콜로니를 형성한다. 하나의 식민지 분석을 사용하여, 9 쥐의 췌장, 중 성인에서 (2-4개월 된) 7,11 또는 젊은 (1 주령) 링 / 밀도 및 내분비 / 선포 식민지의 대부분, 세 가지를 모두 표현하는 것으로 나타났습니다 혈통 마커. 이것은 원래 PCFUs 대부분 트라이 강력한 것을 시사. 쥐 ECM 함유 식민지 분석에서, 성인 쥐 PCFUs 견고하게 자기 갱신과 문화 7 11 주 동안 약 50 만 회를 확장합니다. 라미닌 하이드로 겔의 존재, 쥐 PCFUs는 내분비 및 선포 세포로 우선적으로 분화하도록 권장하고 유관 계통 7,9,11 너무 적은 반면 쥐 ECM은 우선적으로, 내분비 및 선포 계통을 통해 도관 세포의 분화를 지원합니다. 중요한 것은, 인슐린 + 글루카곤 - 모노 - 호르몬 세포 라미닌 하이드로 배양 생성 기능 성숙 시사 7,9 관내에서 자극 글루코오스에 반응의 인슐린을 분비한다. 7,9 잠재적 인 트라이 혈통 차별화와 개별 PCFUs의 자기 갱신 (11)는 콜로니 형성을 위해 잘 당 하나의 세포를 배양, 단일 셀의 미세 조작, 즉에 의해 확인된다. 함께, 이러한 결과는 자기 갱신, 트리 poten 존재한다는 증거를 제시t, 3D 문화 활동을 보여 출생 후의 쥐의 췌장 전구와 같은 세포. 이 문서에서 설명하는 쥐 PCFU 분석은 쥐의 배아 줄기 세포 (mESCs) 17에서 차별화 된 전구 세포 위해 설계된 이전의 식민지 분석에서 파생됩니다. 즉, 프로토콜은 다른 조브 간행물 (18)에 자세히 설명되어 있습니다. 배양 성분 성인 PCFUs 대한 뮤린 ECM 함유 콜로니 에세이를 수행하는데 필요한 기술은 MESC - 유래 전구 세포 (17, 18)와 동일하다. 따라서, 이러한 분석의 측면은 여기서 반복되지 않을 것이다; 대신 다음 절차는 해결 될 것입니다 : 1) 성인 췌장의 해리 성인 마우스 7, 2) 정렬 된 세포의 단일 세포 조작, 3) 단일 식민지에서 PCFUs을 풍요롭게 CD133 + Sox9 / EGFP + 도관 세포를 정렬 미세 유체 QRT-PCR 및 전체 마운트 면역 염색 및 콜로니 4) 분리를 사용하여 분석단일 세포 현탁액과로의 쥐 ECM 또는 라미닌 하이드로 겔 식민지 분석에 재 도금.

<table><tbody><tr><td>Murine ECM proteins (Matrigel)</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>354230</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Laminin Hydrogel</td><td>Provided by David Tirrell (Pasadena, CA USA)</td><td></td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Methylcellulose</td><td>Shinetsu Chemical (Tokyo, Japan)</td><td></td><td>1,500 centipoise (dynamic viscosity unit equal to 15 g/cm/sec) (high viscosity)</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Mediatech (Manassas, VA, USA)</td><td>21-031-CV</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td></td></tr><tr><td>50 ml Flacon Conical vial</td><td>Corning Inc. (Corning, NY, USA)</td><td>352070</td><td></td></tr><tr><td>100 mm x 20 mm Suspension culture dish</td><td>Corning Inc. (Corning, NY, USA)</td><td>430591</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td></td></tr><tr><td>DNase1</td><td>Calbiochem (Darmstadt, Germany)</td><td>260913</td><td></td></tr><tr><td>Collagenase B</td><td>Roche (CH-4070, Basel, Schweiz, Switzerland)</td><td>11088831001</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Anti-mouse CD16/32</td><td>Biolegend (San Diego, CA, USA)</td><td>101310</td><td>low endotoxin, azide free</td></tr><tr><td>PE-Cy7 Rat IgG2a &amp;kappa; Isotype Control</td><td>Biolegend (San Diego, CA, USA)</td><td>400522</td><td></td></tr><tr><td>Rat IgG1 &amp;kappa; Isotype Control</td><td>eBioscience (San Diego, CA, USA)</td><td>13-4301-82</td><td></td></tr><tr><td>Anti-CD133-Biotin</td><td>eBioscience (San Diego, CA, USA)</td><td>13-1331-82</td><td></td></tr><tr><td>Anti-CD71-PE-Cy7</td><td>Biolegend (San Diego, CA, USA)</td><td>113812</td><td></td></tr><tr><td>Streptavidin-Allophycocyanin</td><td>Biolegend (San Diego, CA, USA)</td><td>405207</td><td></td></tr><tr><td>4&#039;,6-Diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>3571</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Anti-mucin 1</td><td>Thermo Fisher Scientific (Waltham, MA USA)</td><td>HM-1630-P1</td><td></td></tr><tr><td>Dylight 649 Goat anti-Armenian Hamster</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>127-495-160</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12</td><td>Mediatech(Manassas, VA, USA)</td><td>10-092-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Tissure Culture Biologicals (Long Beach, CA, USA)</td><td>101</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Tris Ethylenediaminetetraacetic acid</td><td>TEKnova (Hollister, CA, USA)</td><td>T0221</td><td></td></tr><tr><td>Rneasy Micro Kit</td><td>Qiagen (Venlo, Netherlands)</td><td>74004</td><td></td></tr><tr><td>QuantiTec Reverse Transcription Kit</td><td>Qiagen (Venlo, Netherlands)</td><td>205310</td><td></td></tr><tr><td>CellsDirect One-Step qRT-PCR Kit</td><td>Ambion/Invitrogen(Grand Island, NY, USA)</td><td>11753-100</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Santa Cruz Bio (Santa Cruz, CA, USA)</td><td>SC-281692</td><td></td></tr><tr><td>Goat serum</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>005-000-121</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Donkey serum</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>0017-000-121</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Triton X-100</td><td>Sigma (St. Louis, MO, USA)</td><td>T9284</td><td></td></tr><tr><td>Trypsin</td><td>Sigma (St. Louis, MO, USA)</td><td>T-4799</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>15575-020</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies (Waltham, MA, USA)</td><td>25200-056</td><td></td></tr><tr><td>Sterile Water</td><td>Gibco (Grand Island, NY, USA)</td><td>15230-147</td><td>Molecular biology grade</td></tr><tr><td>Pasteur Pipette</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>13-678-8B</td><td></td></tr><tr><td>40 &amp;mu;m Filter Mesh</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filter mesh</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>08-771-2</td><td></td></tr><tr><td>TC Plate 96 Well Suspension</td><td>Sarstedt</td><td>83.3924 (Previously 83.1835)</td><td></td></tr><tr><td>1 cc Syringe</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>309659</td><td></td></tr><tr><td>10 cc Syringe</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>301604</td><td></td></tr><tr><td>48.48 Dyanmic Array Chip</td><td>Fluidigm (San Francisco, CA, USA)</td><td>BMK-M-48.48</td><td></td></tr><tr><td>Fluidigm GE 48.48 Dynamic Array Sample &amp;amp; Assay Loading Reagent Kit</td><td>Fluidigm (San Francisco, CA, USA)</td><td>85000800</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems (Grand Island, NY, USA)</td><td>4304437</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate</td><td>Sigma (St. Louis, MO, USA)</td><td>P7949</td><td></td></tr><tr><td>Glass Bottom Dish</td><td>MatTek (Ashland, MA, USA)</td><td>P35G-1.5-14-C</td><td>35 mm Petri dish with glass bottom</td></tr><tr><td>Mouth Piece/Rubber Tubing</td><td>Renova Life Inc. (College Park, MD, USA)</td><td>MP-SET</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma (St. Louis, MO, USA)</td><td>N0636</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Vascular Endothelial Growth Factor</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>293-VE</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Activin B</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>659-AB</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Extendin 4</td><td>Sigma (St. Louis, MO, USA)</td><td>E7144</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Rspondin-1</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>3474-RS</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Falcon 5 ml Polystyrene Round-Bottom Tube</td><td>Corning Inc. (Corning, NY, USA)</td><td>352054</td><td></td></tr><tr><td>PrecisionGlide Needle 18 G x1 1/2</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>305196</td><td></td></tr><tr><td>PrecisionGlide Needle 16 G x 1 1/2</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>305198</td><td></td></tr><tr><td>Costar Ultra-Low Attachment Surface 24 well flat bottom plate&amp;nbsp;</td><td>Corning Inc. (Corning, NY, USA)</td><td>3473</td><td></td></tr><tr><td>Costar 96 Black Well Plate</td><td>Corning Inc. (Corning, NY, USA)</td><td>3603</td><td>Flat, clear bottom with lid.&amp;nbsp; Black polystyrene TC-treated microplates</td></tr><tr><td>Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope</td><td>Carl Zeiss AG (Oberkochen, Germany)</td><td></td><td></td></tr><tr><td>Biomark HD&amp;nbsp;</td><td>Fluidigm (San Francisco, CA, USA)</td><td></td><td></td></tr><tr><td>Aria Special Order Research Product Cell Sorter</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td></td><td></td></tr><tr><td>PBS/BSA</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)</td></tr><tr><td>PBS/BSA/Dnase1</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)&lt;br/&gt;
Dnase 1 (2,400 Units/ml per pancreas)</td></tr></tbody></table>

in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas

줄기 성인 췌장 전구 세포가 제 1 형 당뇨병 환자를 치료하기위한 치료제 베타 - 유사 세포의 잠재적 인 원인이 될 수있다. 줄기 및 전구 세포를 성인 췌장에 존재하는지 그러나, 아직 알려져 있지 않다. 성인 쥐 계통 추적 CRE-LOX를 사용하여 연구 전략 지원 또는 중 베타 세포 덕트 성인 췌장 전구 세포가 존재할 수있다 추정 위치에서 생성 될 수 있다는 생각을 반박한다는 결과를 수득 하였다. 이러한 생체 CRE-LOX 계보-추적 방법은, 그러나, 자기 갱신과 줄기 세포를 정의하는 데 필요한 멀티 혈통 분화 두 가지 기준의 질문에 대답 할 수 없습니다. 이 기술 격차를 해결하기 위해, 우리는 췌장 전구 세포에 대한 3 차원 식민지 분석을 고안했다. 곧 우리의 초기 발행 후, 다른 연구소는 독립적으로 organoid 분석라는 비슷하지만 동일하지, 방법을 개발했다. organoid 검정에 비해, 우리의 방법을 사용낮은 농도에서 세포 외 기질 단백질의 포함을 허용 점성 용액을 형성하는 메틸. 많은 성인 장기 줄기 세포의 경우, 상기 메틸 - 함유 분석은 쉽게 검출 및 선조 작은 서브 모집단을 구성 할 때 중요한 단일 세포 수준에서 선조 세포의 분석을 허용한다. 함께, 여러 실험실에서 결과는 마우스의 췌장 전구와 같은 세포의 체외 자기 갱신 및 멀티 혈통 분화에 보여줍니다. 현재의 프로토콜을 마우스 췌장 전구 세포의 특징을 두 메틸 계 콜로니 검정을 기술; 하나는 라미닌 하이드로 알려진 뮤린 세포 외 기질 단백질의 상업적 제조 및 다른 인공 세포 외 기질 단백질을 포함한다. 여기에 도시 된 기술은 1)은 췌장의 해리 CD133 + Sox9 / EGFP의 정렬 성체 생쥐 + 도관 셀은 S 2) 단일 세포 조작이다orted 세포, 3) 단일 콜로니는 자기 갱신 또는 차별화를 평가하기 위해 보조 식민지 분석에 미세 유체 QRT-PCR 및 전체 마운트 면역 염색 및 단일 세포 현탁액 및 재 도금에 차 식민지 4) 분리를 사용하여 분석한다.

성인 췌장 전구 세포는 형광 활성화 세포 분류 (도 1)에 의해 농축 될 수있다. ~ Sox9 20 75킬로바이트 상류 ~ 150킬로바이트 하류 서열을 여기서 사용 Sox9 / EGFP 유전자 변형 마우스 선 먼저 GENSAT 뇌 아틀라스 프로젝트 (19)의 결과로 생성하고, EGFP 리포터를 함유하는 세균 인공 염색체가 제어 . 이러한 쥐, EGFP 효율적 특히 22 췌장 관 라벨. 췌?...

Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

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

자기 갱신 성인 쥐의 췌장으로부터 분리 전구 세포의 분화를 검출하는 시험 관내 콜로니 검정 고안되어있다. 이러한 분석에서, 췌장 전구 세포가 메틸 - 함유 반고체 배지에서 3 차원 공간에서 세포 콜로니를 야기. 단일 세포와 개별 식민지의 특성을 처리하기위한 프로토콜이 설명되어 있습니다.

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. ...

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Developmental Biology

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

9.4K 조회수.  Beckman Research Institute of City of Hope.  자기 갱신 성인 쥐의 췌장으로부터 분리 전구 세포의 분화를 검출하는 시험 관내 콜로니 검정 고안되어있다. 이러한 분석에서, 췌장 전구 세포가 메틸 - 함유 반고체 배지에서 3 차원 공간에서 세포 콜로니를 야기. 단일 세포와 개별 식민지의 특성을 처리하기위한 프로토콜이 설명되어 있습니다. 

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

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

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 ...

발생학

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 ...

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 ...

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

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.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

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 ...

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

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