이 작품은 그랜트를 시작 NUS에 의해 (R-397-000-192-133) 및 ETPL 간격 기금 (R-397-000-198-592)를 지원합니다. GS는 NUS 연구 학자이다. 저자는 셀의 미세화에 그녀의 기술 지원을위한 박사 Jiangwa 싱에게 감사의 말씀을 전합니다.

참고 :이 프로토콜은 PDMS 스텐실을 사용하여 hESC의 라인의 레이저 절단 및 미세 가공, H9 1,000 μm의 패턴 PDMS 스텐실의 제조에 대해 설명합니다. 1. 디자인 및 미세화를위한 PDMS 스텐실의 제작 <ol><li> 원하는 형상과 크기 (예를 들면 1000 ㎛의 원) 및 컴퓨터 지원 설계 소프트웨어 (10)를 이용하여 공판 가스켓의 ​​관통 구멍 스텐실 시트 디자인. </li><li> 120-150 μm의 두께 폴리 디메틸 실록산 (PDMS) 시트는 각각 CO 2 레이저 커터 (10)를 사용하여 2mm의 스텐실 시트 및 가스켓을 레이저 컷. </li><li> 미세화 용 PDMS 스텐실을 얻었다 3-4 시간 동안 60 ° C에서 액체 비경 PDMS와 베이크하여 PDMS 스텐실 시트와 함께 PDMS 개스킷 접착. </li><li> 사용 전에 오븐에서 30 분간 건조하고, 120 ℃에서 오토 클레이 빙하여 PDMS 스텐실 소독. </li></ol> 2. hESCs는 홈페이지보수 <ol><li> 문화 hESC의의 37 ° C에서 1 × hESC의 자격을 갖춘 지하 막 매트릭스 코팅 된 세포 배양 접시에 피더 - 무료 유지 보수 매체에 선 및 5 % CO 2. </li><li> 항로가 약 70 %의 디스 파제 치료 37 ° C에서 3 분 배양하여 합류 기계적으로 100 ~ 200 μm의 덩어리로 hESC의 식민지를 해리있는 hESCs는. 6 분할 비율 : 1에서 기저막 매트릭스 코팅 조직 문화 폴리스티렌에 성장 지역의 9.6 cm 2 당 30 ~ 50 덩어리의 밀도에 hESCs는 대단히 짧은 플레이트. </li></ol> 인간 배아 줄기 세포 (3) 스텐실 미세화 <ol><li> 패턴 셀하기 전에 준비 <ol><li> 페트리 접시에 초순수 700 ㎕의 70 % 분석 등급의 에탄올을 분배하고 상단에 스텐실을 배치하여 60mm 페트리 접시 상에 PDMS 스텐실을 밀봉합니다. </li><li> t 밤새도록 바이오 안전성 캐비닛 내부의 페트리 접시를 놓습니다그는 건조 에탄올. </li><li> 더 거품이 스텐실은 페트리 접시와 좋은 물개를 형성하도록 스텐실 아래에 존재하지 않는 확인하십시오. 이는 ECM 코팅 용액의 누출을 방지한다. 이 세포 파종을위한 준비가 될 때까지 무균 조건 하에서 배양 접시 및 저장 인감. </li><li> 분석의 인증서에 따라 hESC의 자격을 갖춘 기저막 행렬의 분취 량을 준비합니다. 영양 혼합물 F-12 (DMEM / F12) 제조 업체의 제품 시트에 따라 각각 나누어지는 수익률 1 둘 베코의 변형 이글 중간의 25 ㎖에 희석 × hESC의 자격을 갖춘 지하 막 매트릭스 솔루션이 있는지 확인하십시오. </li><li> 1.5 × hESC의 정규화 기저막 매트릭스 용액을 만들기 위해 DMEM / F12 16.7 ㎖ 중에 hESC의 정규화 기저막 매트릭스의 한 분취 량을 첨가하여 ECM 코팅 용액을 제조 하였다. 겔화를 방지하기 위해 얼음 모든 ECM 코팅액 유지. </li><li> 10 μM의 ROCK 저해제 (Y27632)와 hESC의 유지 보수 매체를 보충합니다. </li></ol></li><li><strong&gt;는 스텐실 기판 상에 ECM 코팅 <ol><li> 90 초 동안 100 플라즈마 2 호와 페트리 접시를 취급합니다. 불임을 보장하기 위해, 단지 이전에 플라즈마 처리에 플라즈마 챔버 내부의 페트리 접시 덮개를 열고 신속하게 처리 완료 후 페트리 접시를 다시 커버. 이 표면 습윤을 촉진하고 ECM 코팅 용액의 첨가시에 관통 구멍 미세 패턴에서 기포 형성을 방지한다. </li><li> 전체 스텐실 다루 1.5 × hESC의 자격을 갖춘 지하 막 매트릭스 솔루션의 450 μl를 추가합니다. 이러한 파라 필름 등 자기 융착 필름으로 페트리 접시를 밀봉 마르지 용액을 방지하기 위해, 사용하기 전에 37 ℃에서 5 시간 동안 배양한다. </li></ol></li><li> 스텐실 기판 상에 시드 hESCs는 <ol><li> 둥근 단단히 packe의 전형적인 hESC의 형태의 손실을 보여주는 차별화 된 셀 영역을 식별하는 6 웰 플레이트에 hESC의 식민지를 검사합니다 (예를 들어, 손실D 상피 형태, 눈에 띄는 핵소체 높은 핵 / 세포질 비율). 진공 흡입기를 사용하여 차별화 된 영역을 제거합니다. </li><li> 물론 당 2 ml의 DMEM / F12로 두 번 씻으십시오. </li><li> 6 웰 플레이트의 웰 당, 같은 Accutase 등의 소화 효소, 1 ㎖를 추가하고 8 분 동안 37 ° C에서 품어. 기판에서 모든 식민지를 분리 부드럽게 판을 누릅니다. </li><li> 소화 효소 1 ㎖ 당 DMEM / F12의 최소 4 ㎖로 잘 각각 씻어 15 ML 원뿔 튜브에 세포 현탁액을 수집합니다. </li><li> 실온에서 3 분 동안 200 × g에서 세포 현탁액을 원심 분리기. </li><li> 대기음은 상층 액을 제거하고 세포를 다시 일시 중단 ROCK 저해제 (ROCKi)로 보충 hESC의 유지 보수 매체의 400 μl를 추가합니다. 세포 현탁액을 위로 피펫 부드럽게 아래로 3 회는 하나의 세포로 덩어리를 중단 할 수 있습니다. </li><li> 단일 세포 현탁액을 잘 혼합하고 DMEM / F12 (1시 20분 희석) 190 μL로 세포 샘플의 10 μl를 희석. hemocytome를 사용하여운이 스톡 세포 현탁액의 세포 밀도를 결정한다. </li><li> 주어진 스텐실에 필요한 세포 파종 밀도를 계산합니다. 참고 : 예를 들어, 실험적 단셀 합류 단층 약 / mm이 4,444 세포 구하는 세포 접종 밀도를 결정했다. 따라서, 450mm (2)의 영역과 400 μL의 시딩 부피 스텐실는 2 백만 세포 / 400 μL의 밀도가되도록 세포 현탁액을 요구할 것이다. </li><li> ROCKi 보충 hESC의 배양 배지에 필요한 파종 밀도 (단계 3.3.8 참고 참조) 주식 세포 현탁액을 희석. </li><li> 각 공판으로 셀의 필요한 수를 포함하는 세포 현탁액의 지정된 양을 첨가하고 세포를 정착 할 수 있도록 실온에서 5 분 동안 후드 흐트러 페트리 접시를 떠난다. </li><li> 인큐베이터로 배양 접시를 전송하고 세포 부착을 허용하기 위해 1 시간 동안 품어. 동안 배양 접시 수준을 유지하기 위해주의세포가 스텐실에서 단일 층으로 남아 있도록 프로세스를 전송합니다. </li></ol></li><li> 스텐실 제거 및 패터닝되지 않은 기판의 보호 <ol><li> 세포가 제대로 하부 기판에 부착되어 있는지 확인하기 위해 현미경 페트리 접시를 검사합니다. </li><li> 스텐실에서 세포 현탁액을 멀리 대기음. </li><li> 스텐실을 둘러싸는 영역에 DMEM / F12에서 0.5 % 세포 배양 호환 비이 온성 계면 활성제를 2 ㎖ / 접시를 추가한다. </li><li> 부드럽게 스텐실을 벗​​겨 멸균 포셉 한 쌍을 사용합니다. 스텐실이 마르지 세포 않도록 박리 같은 비이 온성 계면 활성제 용액을 주위에 소용돌이. 시각 페트리 접시에 미세 패턴 세포를 관찰합니다. </li><li> 37 ° C에서 10 분 동안 0.5 % 비이 온성 계면 활성제 DMEM / F12 용액 중의 미세 패턴 세포를 인큐베이션. </li><li> 멀리 대기음 비 이온 계면 활성제-DMEM / F12 솔루션입니다. 2 ㎖ / DMEM / F12의 접시로 3 회 반복한다. </li><li> hESC의 2 ㎖ / 요리 추가유지 매체 ROCKi 보충하고 밤새 37 ° C에서 배양한다. </li><li> 하룻밤 동안 배양 후, ROCKi 보충 흡 hESC의 유지 배지, DMEM / F12로 한번 씻어 2 ㎖ / 100 NG / ㎖ 티빈 25 NG / ㎖ BMP4 10 NG / ㎖ FGF2 보충 분화 배지 접시를 추가 mesoendoderm 분화를 유도 . </li><li> 위상차 이미지를 이용하여 미세 패턴을 평가하기 이전에 8 바와 같이 각 패턴에 대한 평균 Brachyury (T) 강도 프로파일을 계산한다. </li></ol></li></ol>

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저자는 더 경쟁 재정적 이해 관계가 없습니다.

미세 패턴 스텐실의 제작 스텐실 미세화 틈새 매개 분화 패턴을 조사하기위한 hPSC의 미세 패턴을 생성 할 수있는 이상적인 방법을 제공한다. 이러한 마이크로 콘택트 인쇄, 포토 패터닝과 같은 다른 미세 가공 기술 위에 스텐실 패턴의 주요 장점은 표면 개질을 필요로하지 않고, 종래의 FPC 기판의 기판 상에 구현 될 수 있다는 것이다. 따라서, 다른 hPSC 라?...

배아 줄기 세포 (hESCs는)와 유도 만능 줄기 세포 (hiPSCs)를 포함하여 인간 만능 줄기 세포 (hPSCs)는, 널리 모든 세포 계통으로 인해 분화 가능성의 정상 및 병에 걸린 기관 형성의 실험 모델링뿐만 아니라 재생 의학에 이용된다 삼 배엽 1, 2. hPSCs의 분화 운명은 물리적 단서 3-5에 의해 매개 분비 또는 1 신호 분비뿐만 아니라 mechanotransduction 과정을 조절할 수 지역의 환경 요인에 매우 민감하다. 셀 미세화 공간적 예컨대 세포 - 세포 상호 작용 (6) 및 세포 - 매트릭스 상호 작용 (3)로서, 로컬 셀룰러 미세 환경을 제어하는 수단으로 세포 집단의 형상과 위치를 조직하기 위해 개발 된 기술의 집합을 포함한다. hPSCs의 맥락에서, 셀의 미세화가 틈새 - 의존 방식으로 유의 한 통찰력을 얻기 위해 사용되어왔다엔트 분비 신호는 초기 배아 분화 패턴 (6)에 hESC의 다 능성 분화 결정 (7)과 조직을 변조한다. 2D 및 3D 미세 패턴 hPSCs 차례로 세 개의 배엽으로 분화 8,9 결정 영향 다세포 패턴의 콜로니의 크기를 제어하기 위해 사용되어왔다. 우리는 인테그린-E-cadherin의 누화 세포 운명 얼룩이 열을 일으킬 수 있는지 조사하는 hPSC 콜로니에서 세포 - 세포 및 세포 - 기질 상호 작용의 정도를 조절하는 다세포 hPSC의 미세 패턴을 채용 하였다. 조직 또는 장기 개발 과정에서 성장 인자와 호르몬의 효과를 연구하기 위해 개발 질병 (11)에 대한 약물 독성 검사를위한 실험 모델로 hPSCs의 다세포 미세 패턴의 적용으로 위의 보고서 열려있는 새로운 길에서 데모, 그리고의 형성을 해명하기 조직 패턴. 세포 micropatter의 수많은Falconnet 등의 검토로 닝 기술이 개발되어왔다. 등. (12) 그러나 마이크로 접촉이 7,8,13 인쇄 등 소수에 불과는 마이크로 웰 문화 14, 15, 포토 패터닝은 6 microstencils (16)이 성공적으로 hPSCs로 구현되었다. 미세화의 hPSCs와 도전은 자신의 취약점과 세포 부착과 생존을위한 특정 세포 외 기질 (ECM) 및 성장 조건의 엄격한 요구 사항에 자리 잡고 있습니다. 2D hPSC 패턴의 경우, 마이크로 컨택트 프린팅 조직 배양 및 유리 기판 (13)에 hPSC에 미세 패턴을 생성하기위한 가장 일반적인 방법 중의 하나이다. 상기 방법은 마트 리겔 라미닌 및 기저막 매트릭스를 포함 hPSC 배양에서 사용 패턴 ECM 공통으로 사용될 수있다. 그러나, 일반적으로 폴리 D 라이신의 도움 두 단계의 코팅 공정이 필요하며,이 hPSCs 6,13-에 부착 안정 ECM의 미세 패턴을 특정 불활성 대기 습도 조건을 필요. 각각의 미세 가공 방법의 가장 중요한 고려 사항은 주변에 비특이적 세포 부착을 최소화하면서 표면 개질 체제 원하는 기하학적 해상도 hPSC 접착 ECM 패턴을 생성 할 수 있는지 여부이다. 여기서는 hPSCs가 부착하기위한 종래 접착제 ECM 패턴의 발생을 추가로 표면 개질 단계없이 hPSC에 미세 패턴을 생성하기위한 간단한 방법으로서 공판 미세화의 사용을보고한다. 셀 스텐실 관통 구멍 물리적 ECM 코팅이어서 hPSCs 시드를 포함하는 세포 배양 용 기재 상에 밀봉 크기 밀리미터 미크론으로, 얇은 막, 예를 들면, 폴리 디메틸 실록산 (PDMS) 시트를 포함한다. 스텐실 패턴은 물리적으로 hPSC가 기본 ECM 코팅 된 기판에 직접 액세스하여 첨부 할 수 있습니다 위치를 억제하여 작동,이 방법은 hPSC 문화를 지원할 수있는 다양한 기판과 호환됩니다. 유일한 requirement는 스텐실 재료의 선택은 기판과 가역적 시일을 형성 할 수 있다는 것이다. 이 기판은 기존의 조직 문화 폴리스티렌 (FPC 기판) (17), 리간드 결합 된 기판 (18)뿐만 아니라 가변 강성과 탄성 기판을 포함한다 (예., PDMS) 19. 이 방법은 또한, 적절한 부착 및 hPSCs 분화를 허용하는 그러한 비트로 넥틴 (VTN 또는 단백질), 라미닌 및 기저막 매트릭스 (예, 마트 리겔과 Geltrax)로 상이한 ECM의 코팅을 허용한다. 특정 hPSC 라인 그러므로 우리는 최적화 전송할 수 있습니다 ECM-기판 구성은 최적의 세포 - 매트릭스 접착, 생존 및 분화에 대한 미세 가공 스텐실합니다. 최근, 유사한 방법이 또한 폴리 (메틸 메타 크릴 레이트)를 사용하여 미세화 hESCs는 (PMMA) 공판 마이크로 어레이 간 (16)에 의해 분화를 유도하는 것으로보고되고있다. 셀 스텐실은 다른 재료로 제조 될 수 충족 포함루게릭 병 (20, 21), 폴리 (p- 자일 릴렌) 폴리머 22, 23, PMMA (16)과 가장 일반적으로, PDMS 24-28. 실리콘 및 폴리 (P-크실렌) 폴리머 스텐실은 생물학적 사용자에게 자신의 접근성을 제한하는 특수 장비 20-23로 관통 구멍의 직접 에칭이 필요합니다. PDMS 스텐실은 통상적으로 3 μm 인 2,000 ㎛의 범위 11,26-29 필요한 피처 크기에 따라 다른 방법에 의해 제조 될 수있다. 작은 특성이 요구되는 경우, 얇은 스텐실 시트는 미세 패턴 (28)의 릴리프를 포함하는 미세 가공 된 실리콘 템플릿에서 프레스 성형 PDMS 예비 중합체가 생성 될 수있다. 기능&gt; 1000 μm의 들면, CO 2 레이저 커터 직접 스텐실 제조 동안에 프리 캐스트 PDMS 시트의 패턴을 절단하는 쉽고 저렴한 방법을 제공한다. PDMS 스텐실의 재활용도 충분한 일관성 일련의 실험을 수행하도록 경제적한다. (10) 결과 방법을 조사하기 위해 E-cadherin의 응집력 hESC의 식민지 내의 유착을 중재.

<table><tbody><tr><td>&amp;nbsp;2 mm thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.005</td><td>Used to form reservoir for stencil</td></tr><tr><td>120-150 &amp;mu;m thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.040</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>60 mm Petri dish</td><td>Nunc Nunclon Delta</td><td>150326</td><td>Substrate for micropatterning</td></tr><tr><td>Accutase</td><td>Accutase, Merck Millipore, Singapore</td><td>SCR005</td><td>Enzyme to break H9 cells into single cells</td></tr><tr><td>Activin&amp;nbsp;&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-AC-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>BMP4&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-BP-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>Plasma system&amp;nbsp;</td><td>Femto Science, Korea</td><td>CUTE-MP</td><td>For plasma oxidation of stencil</td></tr><tr><td>Dispase</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>7923</td><td>Enzyme used to weaken the cell-ECM adhesion during passaging</td></tr><tr><td>DMEM/F12</td><td>GIBCO, USA</td><td>11330032</td><td>Basal medium for H9 cells</td></tr><tr><td>FGF2</td><td>R&amp;amp;D Systems, Singapore</td><td>233&amp;ndash;FB&amp;ndash;025</td><td>Growth factor for H9 differentiation</td></tr><tr><td>H9 cell line</td><td>WiCell Research Institute, Inc., USA</td><td>WA09</td><td>Human embryonic stem cells</td></tr><tr><td>hESC-qualified basement membrane matrix</td><td>Matrigel, BD Biosciences, Singapore</td><td>354277</td><td>Extra-cellular matrix coating to support growth of H9 cells</td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems, Singapore</td><td>DMi1</td><td>For capturing bright-field images</td></tr><tr><td>Laser cutter</td><td>Epilog Helix 24 Laser System</td><td></td><td>Used to generate through holes in PDMS sheet</td></tr><tr><td>mTeSR&lt;sup&gt;&amp;trade;&lt;/sup&gt;1 medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5850</td><td>Maintainence medium for H9 cells</td></tr><tr><td>PDMS&amp;nbsp;</td><td>SYLGARD&amp;reg; 184, Dow Corning Co., USA</td><td>3097358-1004</td><td>Used for sticking the PDMS stencil and reservior</td></tr><tr><td>ROCKi Y27632</td><td>Calbiochem, Merck Millipore, Singapore</td><td>688000</td><td>Maintains H9 cells as single cells&amp;nbsp;</td></tr><tr><td>STEMdiff&lt;sup&gt;&amp;trade;&lt;/sup&gt; APEL&lt;sup&gt;&amp;trade;&lt;/sup&gt; medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5210</td><td>Differentiation medium for H9 cells</td></tr><tr><td>Polyethylene terephthalate film</td><td>SureMark Singapore</td><td>SQ-6633</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>Cell culture compatible non-ionic surfactant</td><td>Pluronic acid F-127, Sigma, Singapore</td><td>P2443</td><td>Passivating reagent to repel cell adhesion in non-micropatterned substrates</td></tr></tbody></table>

stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

이 논문은 1000 ㎛의 특징을 생성하는 레이저 커터를 사용하여 셀 공판의 제조를 설명한다. 스텐실은 두 부분으로 구성되었다 : 얇은 스텐실 시트의 관통 구멍을 포함하는 미세 패턴 (두께 약 100 ~ 200 μm의) 및 ECM 코팅 용액 또는 세포 현탁액을 포함하는 PDMS 개스킷. 여기서, 127 μm의 두께 2mm 시판 PDMS 시트 각각 스텐실 시트 개스킷을 사용 하였다. 예컨대 스핀 코팅과 같은 제?...

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Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

인간 만능 줄기 세포 (hPSCs)는 차별화 별개의 조직 패턴으로 자기 조직하는 고유 능력을 가지고; 이 공간 환경 그라데이션의 표현을 필요로하지만. 우리 hPSC 분화 패턴을 제어하기위한 생화학 적 및 기계적 그라디언트를 생성하는 간단하고 강력한 방법으로서 공판 미세 패턴을 제시한다.

차별화 운명의 공간 조직을 프로빙에 대한 인간 다 능성 줄기 세포의 스텐실 미세화

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

stencil-micropatterning-human-pluripotent-stem-cells-for-probing

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.4K 조회수.  National University of Singapore.  인간 만능 줄기 세포 (hPSCs)는 차별화 별개의 조직 패턴으로 자기 조직하는 고유 능력을 가지고; 이 공간 환경 그라데이션의 표현을 필요로하지만. 우리 hPSC 분화 패턴을 제어하기위한 생화학 적 및 기계적 그라디언트를 생성하는 간단하고 강력한 방법으로서 공판 미세 패턴을 제시한다. 

비디오: Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

인간 다 능성 줄기 세포의 서스펜션 기반의 심장 차별화를위한 마이크로 웰의 집계 크기 최적화

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

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발생학

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

기본 및 확실 한 조 혈 창시자 인간 만능 줄기 세포에서의 감독

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

향상 된 Multilineage 차별화 힘으로 순진한 같은 상태로 기존의 인간의 Pluripotent 줄기 세포의 화학 귀선

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

마이크로패턴과 정량적 이미징을 사용하여 포유류 세포의 응급 공간 조직 매핑

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

레이저 컷 스텐실을 사용한 간단한 리소그래피 없는 단일 셀 마이크로패터닝

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

기형 종의 형성에 의해 인간 배아 줄기 세포의 운명 매핑

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

생물학

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2D 및 3D 인간 유도 만능 줄기 세포 기반 모델은 신코르티컬 개발 중 1 차용성 개입을 해부합니다.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

조직 수준 역학을 제어하기 위해 준수 기판에 인간 배아 줄기 세포 식민지의 기하학적 패터닝

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

생체공학

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

비통합 방법을 사용하여 복부 피부에서 성인 인간 진피 섬유 아세포의 분리 및 유도 만능 줄기 세포의 생성

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

<meta charset="utf8"></head><body>이미지 기반 ML 전략을 통한 만능줄기세포 분화 모니터링

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

<meta charset="utf8"></head><body>저자 스포트라이트: Enhancing PSC-to-Functional Cell Differentiation Using ML Models Based on Live-Cell Bright-field Imaging (라이브 셀 명시야 이미징 기반 ML 모델을 사용하여 PSC-기능성 세포 분화 향상)

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

만능줄기세포 분화를 모니터링하기 위한 살아있는 세포 이미지 기반 기계 학습 전략

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...