Os autores gostariam de agradecer a Soon-Cheol Hong (MD, Ph.D., Professor Associado do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade da Coréia) para a prestação de tecido placentário. Este trabalho foi apoiado em parte pelas bolsas (R1211902) da Fundação Nacional de Pesquisa da Coreia (NRF), República da Coreia.

Declaração de ética: O estudo da placenta humana foi realizado de forma prospectiva, com a aprovação do Comitê de Ética em pesquisa com seres humanos da Universidade da Coreia (AN09085-001). Todos os experimentos foram realizados em um livre de germes facilidade Clean Room no Centro Médico da Universidade da Coreia. O design e os procedimentos que utilizam hPSCs experimental foram aprovadas pelo Conselho de Revisão Institucional da Korea University Medical Center (AN12277-003). 1. Preparação de Instrumentos, Meios de Cultura, e pratos <ol><li> Brasão todos os pratos de cultura com 0,1% de gelatina. Autoclave salina tamponada com fosfato (PBS) e fórceps. Esterilizar tesouras cirúrgicas, tubos de microcentrífuga, e gaze a 121 ° C sob £ 15 psi durante mais de 15 min. </li><li> 0,25% de tripsina-etileno amina tetra-acético, ácido preparado pré-aquecido (tripsina-EDTA) e meios de pré-aquecido cultura filtrada (DMEM) contendo 20% de soro fetal de bovino (FBS), 100 U / ml de penicilina e 100 g / ml streptomycantes de iniciar os procedimentos. </li></ol> 2. isolamento de células de placenta humana <ol><li> Obter tecido placentário, após o parto cesariana, de mulheres grávidas saudáveis ​​passando por aborto normal ou terapêutico em 7-32 semanas de gestação após a obtenção do consentimento informado por escrito. </li><li> Já o especialista obstétrica placas corial placenta humana cirurgicamente separados (HPCs) a partir da placenta; incubar estes em 0,25% de tripsina-EDTA a 37 ° C durante 20 minutos, e, em seguida, lava-se com 5 ml de PBS. </li><li> Isolar vilo corial de HPCs usando uma tesoura; mediu as vilosidades e lavá-los três vezes com 1 ml de PBS. Depois de picar a carne, coloque os vilo corial em um tubo de microcentrífuga utilizando uma ponteira; lavar as amostras com 500 ï¿½ de PBS por pipetagem cima e para baixo, e centrifugar tubos de 120 g durante 3 min. Lavar tecidos duas vezes com 500 ul de meio de cultura de pré-aquecido. </li><li> Células em meio de cultura em placas revestidas com gelatina a 37 ° C, em 5% de CO 2 </sub&gt;, e em 95% de umidade. Pré-revestimento de todas as placas com gelatina a 0,1% durante 1 hora a 37 ° C. As colónias de células semelhantes a fibroblastos e vai formar o crescimento aderente cerca de 5-7 dias após o início da cultura no mesmo meio. </li><li> Trocar o meio cada 2-3 dias até a terceira passagem. Durante a cultura, a remoção de detritos de células flutuando no meio de cultura; crescimento de fibroblastos placentários irá anexar à placa. Cultura das células semelhantes a fibroblastos derivadas do HPC-se a 10 de passagem e, em seguida colhem-los para culturas de células estaminais embrionárias humanas após tripsinização e mitomicina-C (10 ug / ml) de tratamento. </li><li> Antes da congelação HPCs como amostras de banco, usar de transcrição reversa-reacção em cadeia da polimerase (RT-PCR) para confirmar que HPCs não estão contaminados com agentes patogénicos que infestam a placenta, incluindo Mycoplasma (M. fermentans, M. hyorhinis, M. arginina, M . orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. neurolyticum, M. hyopneumoniae,e M. capricolum) e as espécies bacterianas, Ureaplasma urealyticum, utilizando um kit de detecção de micoplasma. <ol><li> Seguir as instruções do fabricante para realizar RT-PCR, utilizar todos os iniciadores e as misturas fornecidas no kit. Use os sobrenadantes das células que foram cultivadas por 36 dias para a detecção de micoplasma. Adicionar 3 mL destas amostras para as misturas de PCR e executar o procedimento 1 r PCR durante 35 ciclos, cada um consistindo em 94 ° C durante 30 seg, 55 ° C durante 2 min, e 72 ° C durante 1 min. </li><li> Adiciona-se cuidadosamente 0,5 mL do produto de PCR 1 r-se à mistura de PCR e executar o procedimento 2 nd PCR durante 30 ciclos utilizando as mesmas condições que anteriormente. Analisar os produtos amplificados por electroforese em gel de 1% de agarose; aplicam-se 10 ul de cada um dos 1 e 2 de produtos de PCR para os géis. </li></ol></li><li> Se HPCs são encontrados para ser contaminados com agentes patogénicos, eliminar estesusando uma combinação de antibióticos (BM-Ciclina), seguindo as instruções do fabricante. Adicionar um novo meio que contém 10 ug / ml de BM-ciclina 1 durante 3 dias, e, em seguida, tratar as células com 5 ug / ml de BM-Ciclina 2 durante mais 4 dias. Repita este ciclo duas vezes e, em seguida, verificar se há contaminação por micoplasma novamente. </li></ol> 3. Células de colheita Placenta Humana derivados de meio condicionado (hPCCM) <ol><li> Após a exclusão de contaminação, tratamento das células ligadas (85-90% de confluência; (1,5 x 10 7 células / frasco) com mitomicina C (10 ug / ml) durante 2 horas e 30 minutos, e lavaram-se as células três vezes com 10 ml PBS. Incubam-se as células com meio de pré-aquecido, sem a mitomicina-C durante 24 h. </li><li> Um dia após o tratamento, incubar as células em 10 ml de meio (DMEM-F12 suplementado com 20% de Knock-Out substituição Serum [KOSR], 0,1 mM de β-mercaptoetanol, 1% de NEAA, e 1% de penicilina-estreptomicina). Após 24 h de incubação, a colheita CM num tubo cónico de 15 ml efiltrar o CM através de um filtro de seringa de 0,22? m. </li><li> Adicionar 10 mililitros de novo meio e incubação. Recolhe todos os sobrenadantes a partir das culturas a cada dia durante 1 semana e congelar a forma colhidos a -80 ° C. Evitar congelar-descongelar repetidamente. </li></ol> 4. Cultura de células estaminais embrionárias humanas (hESCs) e indução de células-tronco pluripotentes (iPSCs) <ol><li> Obter linhas de células estaminais embrionárias humanas, as células H1 e H9 (listados no registro do NIH hESC sob o nome WA01 e WA09, respectivamente) e induzida por células-tronco pluripotentes iPSC-1: iPS (prepúcio) -1 e iPSC-2 (IISH1i- BM), do Instituto de Investigação WiCell (Madison, WI, EUA). As células devem ser descongeladas e cultivadas seguindo as instruções do fabricante. </li><li> Para o grupo de controlo, as células em pratos de cultura de Basement Membrane Matrix-revestidas em meio de cultura definido isento de soro, isento de alimentador e amplamente usado, a 37 ° C e em 5% de CO 2. Inicialmente, sementes 80-100 aglomerados de hPSCs in pratos de 35 mm. Cultivar células em esses pratos até 70-80% de confluência e rotineiramente células de passagem para a sub-cultura de uma vez a cada 5-6 dias, utilizando métodos mecânicos ou enzimáticos (Dispase). Lavar as células duas vezes com meio e placa-los em uma proporção de 1: 4. Substitua o meio com meio fresco todos os dias. </li><li> Para os grupos experimentais, as células de cultura em hPCCM sobre pratos pré-revestidas com gelatina a 0,1% e executar passagens em rotina uma vez a cada 5 dias, quer mecanicamente ou enzimaticamente. As células em placas a proporções na gama de 1: 6-1: 10. hPSCs anexar a gelatina dentro de 2 dias após a transferência. Substitua o meio com meio fresco diariamente. </li></ol> 5. Caracterização de células estaminais pluripotentes humanas NOTA: Caracterizar hPSCs usando vários métodos, tais como a coloração da fosfatase alcalina, imunocitoquímica,-PCR quantitativo em tempo real (qRT-PCR), e transferência de western. <ol><li> Imunofluorescência <ol><li> Para immunofluorescence de coloração, hPSCs cultura e fixar as células em câmaras de 8 poços de deslizamento com 4% (w / v) de paraformaldeído durante 10 min; em seguida, as células permeabilizadas com 0,1% (v / v) de Triton-X100 durante 15 min, e bloquear as células durante 1 h com 3% (v / v) de soro de cavalo normal em PBS contendo 0,1% (v / v) de Tween- 20. Lave PBS duas vezes durante 5 minutos entre cada passo. </li><li> Incubar as células com o anticorpo primário (Oct-4, SSEA4, TRA-1-81, TRA-1-60, diluição de 1: 1000) O / N a 4 ° C e com o anticorpo secundário. </li><li> Antes da montagem, incubar as células com 4 &#39;, 6-diamidino-2-fenilindole (DAPI) durante 5 min no escuro. Lavar as células rigorosamente três vezes durante 5 min cada e secos completamente, as células no escuro. Preservar as células na fluorescência meio de montagem e observá-los sob um microscópio de fluorescência. </li></ol></li><li> qRT-PCR <ol><li> Isolar o ARN total a partir de células utilizando um kit RNeasy Mini acordo com as especificações do fabricante e quantificar o ARN extraído utilizando um Espectrofotómetro Nano gota. </li&gt; <li> Sintetizar ADNc por adição de 2 ug de ARN total para a 20 ul de mistura de reacção contendo o oligo (dT) e iniciadores de transcriptase reversa Superscript II; siga as instruções do fabricante no presente processo. </li><li> Amplificar o cDNA sintetizado com um sistema iQ Bio-Rad iCycler, usando iQ SYBR Green qPCR Master Mix e as sequências de primers descritos na Tabela Suplementar S1. Normalizar os valores de limiar de ciclo dos genes de interesse para os da desidrogenase do gliceraldeído-3-fosfato (GAPDH). </li></ol></li><li> A fosfatase alcalina (AP) coloração <ol><li> Detectar a atividade AP usando o celular ES Caracterização Kit, de acordo com o protocolo do fabricante. </li><li> No dia 5, aspirar a mídia e corrigir os hPSCs com paraformaldeído a 4% em PBS durante 12 min. Não overfix; que fixa as células durante mais de 2 minutos resultará na inactivação de fosfatase alcalina. Aspirar o fixador e lavar com 1x Rinse Buffer. Nãopermitir que os poços sequem entre etapas. </li><li> Adicionar solução de coloração suficiente para cobrir cada poço (1 ml por poço de um prato de 35 mm). Incubar as placas no escuro à temperatura ambiente durante 15 min. </li><li> Após a incubação, lavar pratos com 1x lavagem com tampão. Células de cobertura com 1x PBS para evitar a secagem e examinar e imagem das células marcadas utilizando um microscópio Olympus. </li></ol></li><li> Western Blot <ol><li> Realizar a análise Western blot, conforme descrito anteriormente em 9. Resumidamente, a determinação das concentrações de proteína em lisados ​​celulares. Resolver quantidades iguais de proteína sobre uma electroforese em gel de poliacrilamida-sulfato (SDS-PAGE) em gel de dodecilsulfato de sódio e transferir as proteínas para uma membrana de fluoreto de polivinilideno (PVDF). </li><li> Bloquear a blot e sonda que O / N com anticorpos primários diferentes a 4 ° C; subsequentemente incubar a mancha com anticorpos secundários conjugados com HRP durante 1 hora. Lava-se a membrana três vezes durante 10 min de cada vez entre incubações. Detectar sinais usando um reagente ECL. </li&gt; </ol></li><li> Curto Repita Tandem (STR) Análise <ol><li> hPSCs foram colhidas a partir do controle e os grupos experimentais na mesma passagem. O ADN genómico foi extraído usando um kit de ADN Micro, de acordo com as instruções do fabricante. </li><li> Amplificar o ADN genómico de 16 loci genéticos diferentes usando o Kit AMPF / STR Amplificação por PCR, de acordo com as especificações do fabricante e electroforese capilar foi realizada utilizando um analisador genético. </li></ol></li></ol>

<ol>
	<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=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat Biotechnol. 19 (10), 971-974 (2001).</li><li>Amit, M. C., Shakiri, V., Margulets, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itskovitz-Eldor+Feeder+layer+and+serum-free+culture+of+human+embryonic+stem+cells.">Itskovitz-Eldor Feeder layer and serum-free culture of human embryonic stem cells.</a> Biol Reprod. 70 (3), 837-845 (2004).</li><li>Nagaoka, M., Si-Tayeb, K., Akaike, T., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+pluripotent+stem+cells+using+completely+defined+conditions+on+a+recombinant+E-cadherin+substratum.">Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.</a> BMC Dev Biol. 10, 60 (2010).</li><li>Jin, S., Yao, H., Weber, J. L., Melkoumian, Z. K., Ye, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic,+xeno-free+peptide+surface+for+expansion+and+directed+differentiation+of+human+induced+pluripotent+stem+cells.">A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells.</a> PLoS One. 7 (11), e50880 (2012).</li><li>Xu, R. 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. 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Os autores indicam que não há conflitos de interesse.

Este modelo foi desenvolvido para propagar com sucesso hPSCs, mantendo ao mesmo tempo as suas características, em condições humanizadas de cultura isento de alimentação, sem a adição de factores de crescimento exógenos, tais como recombinantes de bFGF ou insulina, permitindo a manipulação de hPSCs num microambiente humanizado. Suplementação exógena bFGF é comum e a aplicação de substratos, tais como Matrigel ou laminina é essencial para a cultura livre de alimentador de hPSCs 14. <p class...

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.

Uma vantagem deste método de propagação HPSC é que ele utiliza componentes segregadas a partir de células humanas. Um passo mais ciritical no protocolo é o isolamento e cultura de células derivadas da placenta humana. Isto requer a dissecção precisa e de uma parte exacta da placa HPC vilosidades. A Figura 1 mostra o procedimento para o isolamento de células derivadas da placenta humana. Este processo é simples e conveniente para o isolamento de células, e pode ser facilmente realizada dentro...

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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.

A Cultura Modelo Novel para pluripotentes estaminais humanas Propagação celular em gelatina no Media Placenta condicionado

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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Research

JoVE Journal

Developmental Biology

7.8K visualizações.  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.

Vídeo: 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.

Encapsulation alginato de pluripotentes células estaminais utilizando um bocal Co-axial

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

Biologia do Desenvolvimento

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.

Cultura pluripotentes humanas e células-tronco neurais em um sistema de cultura celular fechado de Pesquisa Básica e pré-clínica

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.

Derivação sem alimentador de melanócitos a partir de células-tronco pluripotentes humanas

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.

Produção em larga escala de cardiomiócitos a partir de células-tronco pluripotentes humanas utilizando um altamente reprodutível pequeno-Based Molecule Protocolo de Diferenciação

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.

Rápido Neuronal diferenciação de células estaminais pluripotentes induzidas para medir a atividade de rede na Arrays Micro-eletrodo

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.

Uma nova técnica de transplante de almofada de gordura mamária para visualizar a geração de vasos de células vasculares endoteliais vasculares

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.

Rápida, dirigida a diferenciação de células epiteliais de pigmento Retinal de células-tronco humanas embrionárias ou induzida de Pluripotent

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.

Usar humanos induzida pluripotentes células-tronco derivadas hepatócitos células para descoberta de drogas

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.

Um modelo de Mouse quimérico fígado humano de hipercolesterolemia familiar usando hepatócitos de derivadas de células-tronco pluripotentes induzidas

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.