Name: a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media
Uploaded: 2015-08-03T15:30:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

저자는 태반 조직을 제공하기 위해 곧-철이 홍콩 (MD, 박사, 부교수, 산부인과학과, 의과 대학 고려 대학교)에 감사의 말씀을 전합니다. 이 작품은 한국의 국립 연구 재단 (NRF), 대한민국에서 보조금 (R1211902)에 의해 부분적으로 지원되었다.

윤리 문 : 인간의 태반 연구는 고려 대학교 (AN09085-001)의 인간 연구를위한 임상 시험 심사위원회의 승인을 전향 적으로 실시 하였다. 모든 실험은 한국 대학 의료 센터에서 클린 무균 룸 시설에서 수행 하였다. 실험 설계 및 hPSCs를 사용 절차는 고려 대학교 병원의 임상 시험 심사위원회 (AN12277-003)에 의해 승인되었다. 악기, 문화 미디어, 그리고 요리 1. 준비 <ol><li> 0.1 % 젤라틴과 코트의 모든 문화 요리. 오토 클레이브 인산염 완충 식염수 (PBS)와 집게. 이상 15 분 동안 15파운드의 PSI 하에서 121 ° C에서 수술 가위, 마이크로 원심 튜브, 멸균 거즈. </li><li> 준비된 미리 예열 0.25 % 트립신 에틸렌 아민 테트라 아세트산 (트립신 EDTA) 및 미리 예열 여과 배양 배지 (DMEM) 20 % 소 태아 혈청 (FBS), 100 U / ㎖ 페니실린을 함유하는, 100 ㎍ / ㎖ streptomyc절차를 시작하기 전에에서. </li></ol> 인간 태반 2. 세포 격리 <ol><li> 서면 동의서를 얻은 후 임신 7-32주에서 정상 또는 치료 낙태를 받고 건강한 임신 한 여성에서 제왕 절개 배달 후 태반 조직을 얻습니다. </li><li> 태반에서 산부인과 전문의 수술 별도의 인간 태반 융모 판 (HPC의)가; 20 분 동안 37 ℃에서 0.25 % 트립신 EDTA에이를 배양 한 다음 5 ml의 PBS로 씻는다. </li><li> 가위를 사용하여 HPC의에서 융모막 융모를 분리; 융모를 말하다 및 1 ㎖의 PBS로 세 번 씻는다. 닦지 후, 피펫 팁을 사용하여 미세 원심 분리 튜브에 융모막 융모를 배치; 아래로 pipetting 및 500 μL PBS로 샘플을 씻어, 3 분 동안 120 XG의 튜브를 원심 분리기. 미리 예열 문화 매체의 500 μL로 두 번 조직을 씻으십시오. </li><li> 37 ° C에서 젤라틴 코팅 접시에 배지에서 배양 세포, 5 % CO 2 </서브&gt;, 95 % 미만 습도. 사전 코트 37 ° C에서 1 시간 동안 0.1 % 젤라틴 모든 판. 섬유 아세포와 같은 세포의 콜로니 형성과 약 5-7일 같은 매체에 문화를 개시 한 후 부착 성장합니다. </li><li> 제 3 통로까지 2-3 일마다 매체를 교환하십시오. 배양 동안에, 배지에 떠있는 세포 파편을 제거하는 단계; 성장하는 태반의 섬유 아세포는 판에 연결됩니다. 문화 후 10 일 통로로 HPC부터 유래 섬유 아세포와 같은 세포는 트립신과 마이 토마 이신 -C (10 μg의 / ㎖) 처리 후 hESC의 배양을 위해 그들을 수확. </li><li> 스톡 시료로서 HPC의 동결 전에, HPC의 일반적 (M.의 fermentans, M.의 hyorhinis, M. 아르기닌, M을 미코 플라스마 포함 태반, 감염 병원체에 오염되지 않은 것을 확인 역전사 - 중합 효소 연쇄 반응 (RT-PCR)을 사용하여 . orale, 엠의 salivarium, M. hominis가, M.의 pulmonis, 엠의 arthritidis, 엠의 neurolyticum, M. hyopneumoniae,그리고 M. capricolum) 및 박테리아 종 Ureaplasma urealyticum의, 마이코 플라스마 검출 키트를 사용하여. <ol><li> , RT-PCR을 수행하기 위해 제조업체의 지침에 따라 키트에 제공된 모든 프라이머 및 이들의 혼합물을 사용합니다. 마이코 플라즈마 검출 36 일 동안 배양 된 세포의 상층 액을 사용합니다. PCR 혼합물에이 샘플의 3 μl를 추가하고 30 초, 2 분 동안 55 ℃, 1 분 동안 72 ℃에서 각 94 ° C로 구성된 35주기 위해 1 차 PCR의 절차를 수행합니다. </li><li> 조심스럽게 PCR 혼합물에 1 차 PCR 산물의 0.5 μL를 추가하고 이전과 동일한 조건을 이용하여 30 사이클 동안 2 차 PCR 절차를 수행한다. 1 % 아가 로스 겔 전기 영동에 의한 증폭 산물을 분석; 젤에 1 차 및 2 차 PCR 산물을 각각 10 μL를 적용합니다. </li></ol></li><li> HPC의 병원체가 오염 된 것으로 발견되면, 이러한 제거제조업체의 지침에 따라 항생제의 조합 (BM-사이클린)를 사용. 3 일 BM-사이클린 (1) 10 ㎍ / ㎖의를 포함하는 새로운 매체를 추가하고 또 다른 4 일 동안 5 μg의 / ㎖ BM-사이클린 2 세포를 취급합니다. 두 번이 사이클을 반복 한 후 다시 마이코 플라스마 오염 확인합니다. </li></ol> 3. 수확 인간 태반 유래 세포 중간 (hPCCM), 금연실 <ol><li> 오염을 제외한 후, 부착 세포 (85~90% 합류 치료; (1.5 × 107 세포 / 2 시간 30 분 동안 미토 마이신 C (10 μg의 / ㎖)와 함께 플라스크) 및 10 ml의 세포를 3 회 세척 PBS는. 24 시간 동안 마이 토마 이신 C-없이 사전 예열 매체와 세포를 품어. </li><li> 어느 날 치료 후, 10 ml의 배지에서 세포를 배양 (DMEM-F12 20 % 노크 아웃 혈청 교체 [KOSR] 보충, 0.1 MM의 β - 머 캅토 에탄올, 1 % NEAA, 1 % 페니실린 - 스트렙토 마이신). 15 ML 원뿔 관에서 24 시간 배양, 수확 후 CM과0.22 μm의 주사기 필터를 사용하여 필터링 CM. </li><li> 새로운 매체 및 배양의 또 다른 10 ML을 추가합니다. 1 주간 매일 문화에서 모든 상층 액을 수집하고 -80 ° C에서 수확 한 매체를 동결. 반복 동결 - 해동 사이클을 피하십시오. </li></ol> 4. 문화 인간의 배아 줄기 세포 (인간 배아 줄기 세포) 및 다 능성 줄기 세포의 유도 (iPSCs) <ol><li> 인간 배아 줄기 세포주를 확보, (각각 이름 WA01 및 WA09에서 NIH hESC의 레지스트리에 나열된) (H1)과 H9 세포와 유도 만능 줄기 세포 IPSC-1 : 만능 줄기 (포피) -1 IPSC-2 (IISH1i- WiCell 연구소 (매디슨, 위스콘신, 미국)에서 BM). 전지는 제조업체의 지침에 따라 해동 배양해야한다. </li><li> 대조군, 37 ° C에서 널리 사용되는 피더없고 정의 된 무 혈청 배지에서 기저막 매트릭스 코팅 접시에 5 % CO 2에서 배양 세포. 처음 hPSCs의 80-100 덩어리 씨앗 나는N 35-MM 요리. 70-80 %의 합류와 정기적 통로 세포까지이 요리에 세포를 성장 서브 배양, 모든 5-6일 한 번 기계적 또는 효소 적 방법 (스파 아제 (Dispase))를 사용하여. 매체로 두 번 세포를 씻으 1의 비율을 판 : 4. 매일 신선한 매체와 매체를 교체합니다. </li><li> 실험 그룹의 경우, 요리에 hPCCM에서 배양 세포를 기계적으로 또는 효소, 0.1 % 젤라틴으로 미리 코팅 한 번 5 일마다 계대 루틴을 수행합니다. 6-1 : 10 (1)의 범위의 비율로 플레이트 세포. hPSCs는 전송 후 이일 내 젤라틴에 연결합니다. 매일 신선한 매체와 매체를 교체합니다. </li></ol> 인간 만능 줄기 세포의 특성 (5) 참고 : 알칼리 포스 파타 아제 염색, 면역 세포, 정량 실시간 PCR (QRT-PCR), 서부 블로 팅 등의 여러 가지 방법을 사용 hPSCs을 특성화. <ol><li> 면역 형광 염색법 <ol><li> immunofluorescenc 들어E 염색, 문화 hPSCs 4 % 10 분 동안 (W / V) 파라 포름 알데히드와 8 웰 슬라이드 챔버에서 세포를 수정; 그리고, (v / v)의 트리톤 X100 15 분 동안 0.1 %를 함유하는 PBS에서 3 % (v / v)의 정상 말 혈청과 함께 1 시간 동안 세포를 차단 (v / v)의 0.1 %로 세포 투과성이 Tween- (20). 각 단계 사이에 5 분 동안 두 번 PBS를 씻으십시오. </li><li> 4 ° C에서 이차 항체와 O / N : 차 항체 (1,000 일의 10월-4, SSEA4, TRA-1-81, TRA-1-60, 희석)와 세포를 품어. </li><li> 장착 전에, 어두운 곳에서 5 분 동안 4 &#39;, 6-diamidino -2- 페닐 인돌 (DAPI)로 세포를 배양한다. 세척 세포 엄격에게 어둠 속에서 각각의 건전지 완전히 5 분 3 회. 형광 장착 배지에서 세포를 유지하고, 형광 현미경 하에서이를 관찰한다. </li></ol></li><li> QRT-PCR <ol><li> 제조 회사의 사양에 따라 분리하고, RNeasy 미니 키트를 사용하여 세포로부터 총 RNA를 분리하고 나노 드롭 분광 광도계를 사용하여 추출한 RNA를 정량화. </리&gt; <li> 20 ㎕의 반응 혼합물을 함유하는 올리고으로 총 2 μg의 RNA를 첨가하여 cDNA를 합성 (DT) 프라이머 및 슈퍼 스크립트 II 역전사 효소는; 이 과정에서 제조업체의 지침을 따르십시오. </li><li> iQ를 SYBR 그린 qPCR의 매스터 믹스와 기업 S1 표에 기재된 프라이머 서열을 이용하여, 바이오 래드 iCycler IQ 시스템으로 합성 된 cDNA를 증폭. 글리 세르 알데히드 -3- 포스페이트 탈수소 효소 (GAPDH)들에 관심 유전자의 사이클 임계 값들을 정규화. </li></ol></li><li> 알카라인 포스파타제 (AP) 염색 <ol><li> 제조사의 프로토콜에 따라, ES 세포 특성 분석 키트를 사용하여 AP의 활동을 감지. </li><li> 5 일에서 미디어를 대기음 12 분 동안 PBS에서 4 % 파라 포름 알데히드와 hPSCs를 해결. overfix하지 마십시오 이상 2 분 동안 세포를 고정하는 알칼리 포스 파타 아제의 비활성화가 발생합니다. 정착액을 기음과 1X 린스 버퍼 씻어. 하지 마라우물 단계 사이에 건조 할 수 있습니다. </li><li> 각 웰 (35mm 접시 잘 당 1 ㎖)에 충분한 염색 액을 추가합니다. 실온에서 15 분 동안 어두운 데에서 인큐베이션 플레이트. </li><li> 배양 후, 1X 린스 버퍼과 설거지. 1X PBS와 표지 세포의 건조를 방지하고 올림푸스 현미경을 사용하여 염색 된 세포를 조사하고 이미지. </li></ol></li><li> 서쪽 오점 <ol><li> 이전에 9에 설명 된대로 웨스턴 블롯 분석을 수행합니다. 간략하게, 세포 용 해물에서의 단백질 농도를 결정한다. 소듐 도데 실 설페이트 - 폴리 아크릴 아미드 겔 전기 영동 (SDS-PAGE) 겔상의 단백질의 동량을 해결하고, 폴리 불화 비닐 리덴 (PVDF) 막에 단백질을 옮긴다. </li><li> 오점을 차단하고 4 ° C에서 다른 1 차 항체 / O, N 그것을 조사; 이후 1 시간 동안 HRP - 복합 이차 항체와 얼룩을 품어. 10 분 배양 사이의 때마다 막 세 번 씻으십시오. ECL 시약을 이용하여 신호를 검출. </li&gt; </ol></li><li> 짧은 탠덤 반복 (STR) 분석 <ol><li> hPSCs는 같은 통로에서 제어 및 실험 그룹에서 수확했다. 게놈 DNA는 제조자의 지시에 따라서, DNA 마이크로 키트를 사용하여 추출 하였다. </li><li> 제조자의 명세 및 모세관 전기 영동에있어서, AmpF / STR PCR 증폭 키트를 사용하여 16 개의 서로 다른 유전자좌에 대한 게놈 DNA를 증폭하는 유전 분석기를 사용하여 수행 하였다. </li></ol></li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+supports+undifferentiated+human+embryonic+stem+cell+growth+without+conditioned+medium.">Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.</a> Stem Cells. 23 (3), 315-323 (2005).</li><li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+differentiation+of+stem+cells+mesenchymal+progenitors+by+inhibition+of+TGF-beta/activin/nodal+using+SB-431542.">Enhanced differentiation of stem cells mesenchymal progenitors by inhibition of TGF-beta/activin/nodal using SB-431542.</a> J Bone Miner Res. 25 (6), 1216-1233 (2010).</li></ol>

저자는 관심의 충돌을 표시하지 않습니다.

이 모델은, 예컨대의 bFGF 또는 인슐린과 같은 외인성 재조합 성장 인자 첨가없이 인간화 피더가없는 배양 조건에서의 특성을 유지 인간화 미세 환경에서 hPSCs의 조작을 가능하게하면서 성공적 hPSCs를 전파하기 위해 개발되었다. 외인성 bFGF를 보충 일반적입니다 및 마트 리겔 또는 라미닌 등 하층의 응용 프로그램은 hPSCs (14)의 공급이없는 배양을 위해 필수적이다. 이 ?...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table><tbody><tr><td>Mitomycin-C</td><td>Sigma-Aldrich Corporation</td><td>M4287</td><td>10 &amp;mu;g/ml</td></tr><tr><td>Mycoplasma detection kit</td><td>TaKaRa Bio Inc.</td><td>#6601</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>#354277</td><td></td></tr><tr><td>mTeSR1</td><td>STEMCELL Technologies Inc.</td><td>#05850</td><td></td></tr><tr><td>Dispase</td><td>Worthington Biochemical Corporation</td><td>LS02100</td><td>1 mg/ml</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich Corporation</td><td>G2500</td><td>0.10%</td></tr><tr><td>BM-Cyclin</td><td>Roche</td><td>799 050</td><td>10 &amp;mu;g/ml</td></tr><tr><td>RNeasy mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Nano Drop Spectrophotometer</td><td>Thermo Fisher Scientific Inc</td><td></td><td></td></tr><tr><td>iQ SYBR Green qPCR Master Mix&amp;nbsp;</td><td>Bio-Rad Laboratories</td><td>#170-8882AP</td><td></td></tr><tr><td>ES Cell Characterization kit</td><td>Chemicon International, Inc.,</td><td>SCR001</td><td></td></tr><tr><td>Power cDNA Synthesis Kit&amp;nbsp;</td><td>iNtRON Biotechnology</td><td>25011</td><td></td></tr><tr><td>QIAamp&amp;reg; DNA Micro kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>AmpF/STR&amp;reg; Identifiler&amp;reg; PCR Amplification kit</td><td>Applied Biosystems Inc.</td><td>4322288</td><td></td></tr><tr><td>Applied Biosystems&amp;reg; 3130xl Genetic Analyzer&amp;nbsp;</td><td>Applied Biosystems Inc.</td><td></td><td></td></tr></tbody></table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

이 hPSC 전파 방법의 이점은 인간 세포로부터 분비 된 요소를 사용한다는 것이다. 프로토콜에서 가장 ciritical 단계는 인간 태반 유래 세포의 분리 및 문화입니다. 이 요구 및 HPC 판 융모의 정확한 부분에서 정확한 해부. 한 인간 태반 유래 세포의 분리를위한 절차를 보여줍니다. 이 과정은 간단하고 세포의 분리를위한 편리하고 용이하게 1 시간 내에 수행 될 수있다. ?...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

소설 문화 모델은 인간 다 능성에 대한 태반 에어컨 미디어에서 젤라틴에 세포 전파 줄기

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

a-novel-culture-model-for-human-pluripotent-stem-cell-propagation-on

Research

JoVE Journal

Developmental Biology

7.8K 조회수.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

비디오: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

로봇 액체 처리 시스템을 사용하여 확장 가능한 96 웰 IPSC 문화 기반 플레이트 및 생산

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

연구

발생학

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

생체공학

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

신경과학

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

인간의 태반과 탈락 오르간 문화는 산모 - 태아 인터페이스에서 감염을 연구하는

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

면역학 및 감염병학

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

폴 리 비닐 알코올-Co-Itaconic 산 Hydrogels 이종 없는 조건 하에서 다양 한 강성에 인간의 Pluripotent 줄기 세포 문화

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

자동 배양 시스템을 사용한 유도 만능 줄기세포 분화

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

저자 스포트라이트: 재현성 향상을 위한 iPSC 문화 자동화 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

인간 유도 만능 줄기 세포의 유지 및 분화를 위한 자동 배양 시스템

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

iPSC에서 효율적인 인간 장 오가노이드 생성을 위한 매트릭스 비교

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

저자 스포트라이트: iPSC를 사용한 약물 개발을 위한 오가노이드 생성 발전

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

인간 줄기세포 유지와 장 오가노이드 생성을 위한 기저막 매트릭스의 비교 연구

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

소설 문화 모델은 인간 다 능성에 대한 태반 에어컨 미디어에서 젤라틴에 세포 전파 줄기

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인간 줄기세포 유지와 장 오가노이드 생성을 위한 기저막 매트릭스의 비교 연구