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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/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>1mg/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>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</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&#160;</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&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</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 ...

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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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

동축 노즐을 사용하여 다 능성 줄기 세포의 알긴산 캡슐화

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

연구

발생학

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

기본 및 전임상 연구 밀폐 세포 배양 시스템에서 인간 다 능성 및 신경 줄기 세포를 배양

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

마이크로 전극 배열에 네트워크 활동을 측정하기위한 유도 다 능성 줄기 세포의 빠른 신경 세포의 분화

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

인간의 배아 또는 유도 만능 줄기 세포에서 망막 안료 상피 세포의 급속 한, 감독 차별화

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

약물 발견에 대 한 만능 줄기 세포 유래 Hepatocyte 같은 세포를 유도 하는 인간을 사용 하 여

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

가족성 콜레스테롤 혈 증 인간의 간 공상 마우스 모델을 사용 하 여 유도 만능 줄기 세포 유래 Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.