Este trabalho foi apoiado pela Escola Pratt de Engenharia da Duke University, a Divisão de Nefrologia da Duke Medical School, um Prêmio de Pesquisa de Uma Cadeira do Departamento de Medicina da Duke University, e um Burroughs Wellcome Fund PDEP Career Transition Ad Hoc Award to S.M.. M.B foi apoiado pelo Programa de Bolsas de Pós-Graduação em Pesquisa da Fundação Nacional de Ciência. Agradecemos ao Laboratório Bursac por nos fornecer generosamente a linha de células-tronco DU11, e o Laboratório Varghese da Universidade duke por compartilhar temporariamente suas instalações de cultura de tecidos com nosso grupo. Esta publicação é dedicada à Profª Laura L. Kiessling, Novartis Professora de Química do Instituto de Tecnologia de Massachusetts, em comemoração aos seus 60anos.

1. Preparação de reagentes
<ol><li>Diluir o suplemento de mídia de cultura celular hiPS (CCM) descontou em meio basal de cultura celular hiPS para obter uma solução 1x de hiPS CCM. NOTA: O suplemento CCM de 5x congelado requer um processo de descongelamento lento, idealmente em 4 °C durante a noite. As alíquotas do CCM 1x hiPS podem ser armazenadas por até 6 meses a -20 °C.</li>
	<li>Preparação da matriz de membrana de porão (BM) placas revestidas de 1 para a cultura celular hiPS: Descongelar matriz BM 1 durante a noite no gelo a 4 °C. Uma vez descongelado, prepare alíquotas com fatores de diluição adequados, conforme sugerido pelo fabricante. NOTA: Normalmente, as alíquotas são preparadas para diluição subsequente em 25 mL de DMEM/F12 frio em um tubo cônico de 50 mL seguido de mistura completa para dissolver completamente a matriz BM 1 e evitar a formação de cristais residuais.</li>
	<li>Transfira 1 mL da solução BM matrix 1 para cada poço de uma placa de 6 poços e incubar a 37 °C por 1-2 h ou a 4 °C pelo mínimo de 24h, envolto em filme de parafina. As placas revestidas da matriz BM 1 podem ser armazenadas a 4 °C por até 2 semanas.</li>
	<li>Preparação da matriz de membrana de porão (BM) 2 - placas revestidas: Diluir quantidades apropriadas de matriz BM 2 em 9 mL destilada estéril para preparar uma concentração final de 5 μg/mL. Adicione 700 μL da solução BM matrix 2 a cada poço de uma placa de 12 poços e incubar a placa em temperatura ambiente por 2h ou durante a noite a 4 °C.</li>
	<li>Prepare 100 soluções de estoque μg/mL cada uma de BMP7, Activin A e VEGF da seguinte forma: reconstitua bmp7 em água destilada estéril contendo 0,1% (wt/vol) BSA e reconstituir Activin A e VEGF separadamente em PBS estéril contendo 0,1% (wt/vol) BSA. Para evitar ciclos frequentes de congelamento, prepare 100 alíquotas de μL de cada solução de estoque e armazene a -20 °C por até 6 meses.</li>
	<li>Prepare uma solução de estoque de 10 mM de Y27632 dissolvendo 10 mg de Y27632 em 3,079 mL de água destilada estéril. Alíquota de 100 μL do estoque e loja a -20 °C por até 6 meses.</li>
	<li>Dissolver 2 mg de CHIR99021 em 143,4 μL de DMSO estéril para preparar uma solução de estoque de 30 mM. Prepare 5 alíquotas de μL e armazene a -20 °C por até 1 mês (ou de acordo com a recomendação do fabricante).</li>
	<li>Dissolva 10 mg de ácido retinóicotrans em DMSO estéril de 3,33 mL. Prepare 500 alíquotas de μL e armazene a -20 °C por até 6 meses.</li>
</ol>2. Preparação de meios de comunicação culturais
<ol><li>Prepare o meio de diferenciação de mesoderm reconstituindo soluções de estoque correspondentes para uma concentração final de 100 ng/mL Activin A, 3 μM CHIR99021, 10 μM Y27632 e 1x suplemento sem soro B27 em um volume apropriado de DMEM/12 com suplemento de glutamina. NOTA: O meio de diferenciação de mesoderm deve ser recém-preparado antes das etapas de diferenciação e em um volume apropriado para a escala do experimento (normalmente, 50 mL de meio é adequado para duas placas de 12 poços).</li>
	<li>Prepare o meio de diferenciação de mesoderme intermediário, reconstituindo soluções de estoque correspondentes para uma concentração final de 100 ng/mL BMP7, 3 μM CHIR99021 e 1x suplemento sem soro B27 no DMEM/F12 com suplemento de glutamina. NOTA: Se necessário, o meio pode ser suplementado com penicilina-estreptomicina de 1% (vol/vol). Ajuste o volume do meio usando DMEM/F12 com suplemento de glutamina. Este meio pode ser preparado em grandes lotes; no entanto, recomenda-se armazenar em alíquotas menores (por exemplo, 45 mL em tubos cônicos de 50 mL) para evitar ciclos repetidos de congelamento. Esta mídia pode ser armazenada a -20 °C por até 3 meses e pode ser descongelada a 4 °C durante a noite antes de ser usada.</li>
	<li>Prepare o meio de indução de podocito reconstituindo-se a uma concentração final de 100 ng/mL BMP7, 100 ng/mL activin A, 50 ng/mL VEGF, 3 μM CHIR99021, 1x suplemento sem soro B27 e 0,1 μM de ácido retinóico totalmente trans em DMEM/F12 com suplemento de glutamina. Proteger o meio da luz (por exemplo, embrulhando recipiente com papel alumínio). NOTA: Este meio pode ser preparado em lotes grandes e armazenado no escuro a -20 °C por até 3 meses. As alíquotas congeladas devem ser descongeladas durante a noite a 4 °C antes de serem usada.</li>
	<li>Prepare 25 mL de solução neutralizante de trippsina adicionando FBS inativados de calor de 10% (vol/vol) em DMEM/F12 e filtro em condições estéreis.</li>
	<li>Para manutenção pós-diferenciação dos podocitos derivados de células-tronco, prepare o Meio Completo com mídia de manutenção de podocyte adicionando o suplemento ao meio basal de acordo com as diretrizes do fabricante e armazene a 4 °C por até duas semanas.</li>
</ol>3. Cultura celular hiPS livre de alimentadores usando meio de cultura de células hiPS
<ol><li>Aspire a solução residual da matriz BM 1 das placas pré-revestidas e lave os poços 3 vezes com 1 a 2 mL de DMEM/F12 aquecidos.</li>
	<li>Aspirado passou hiPS CCM das células hiPS e enxaguar as células 3 vezes com DMEM/F12 aquecido. Adicione 1 mL de solução de descolamento de células quentes e incubar por 1 min a 37 °C para ajudar a dissociar as células. Realizar a inspeção visual das células sob um microscópio de cultura tecidual e garantir que as bordas das colônias celulares apareçam arredondadas, em seguida, aspire rapidamente a solução de descolamento celular das células (garantindo que as colônias celulares ainda estejam presas à placa, embora vagamente). Enxágue suavemente as células uma vez com DMEM/F12 para remover a solução de descolamento celular.</li>
	<li>Adicione 3 mL de hiPS CCM às células hiPS (em cada poço de uma placa de 6 poços) que foram tratadas com a solução de desapego celular. Raspe colônias usando um levantador de células, e suavemente suspensão de células pipetas para cima e para baixo para desalojar as células frouxamente aderidas. Lave bem a placa para garantir que todas as células sejam colhidas. NOTA: Recomenda-se o uso de uma pipeta de 5 mL para evitar o excesso de tesoura nas células.</li>
	<li>Transfira 0,5 mL da suspensão da célula para cada poço de uma nova placa de 6 poços revestidos de matriz BM com 1 poço contendo 2 mL de ccm hips por poço. Mova a placa na forma de figura oito para distribuir colônias de células dentro de poços e incubar a 37 °C em uma incubadora de 5 % de CO2. Atualize o meio diariamente até que as células estejam prontas para serem passagem ou usadas para o experimento de diferenciação (aproximadamente 70 % de confluência). NOTA: O tamanho ideal da colônia para a passagem de rotina de iPSCs é entre 200-500 μm em condições livres de alimentador usando hiPS CCM (sem inibidor de ROCHA). Se as células forem individualizadas durante o tratamento com enzimas ou tampões de dissociação, o hiPS CCM pode ser suplementado com inibidor de ROCHA (por exemplo, 10 μM Y27632) para melhorar a viabilidade celular.</li>
</ol>4. Diferenciação de células hiPS em células de mesoderme (dias 0-2)
<ol><li>Enquanto as culturas celulares hiPS estão em fase de crescimento exponencial (aproximadamente dentro de 4 dias de cultura após a passagem, e cerca de 70% de confluência), inspecionam visualmente a presença de células espontaneamente diferenciadas dentro e ao redor das bordas das colônias. Se necessário, assepticamente raspar áreas de diferenciação.</li>
	<li>Aspirar hiPS CCM a partir de células hiPS e enxaguar as células 3 vezes com DMEM/F12 quente. Incubar as células com 1 mL de tampão de dissociação celular livre de enzimas por 10 min a 37 °C e verificar a dissociação sob um microscópio. Devido a diferenças inerentes entre diferentes linhas de células hiPS, o tempo real de incubação para o buffer de dissociação celular deve ser determinado para uma determinada linha celular.</li>
	<li>Raspe suavemente o poço com um levantador de células para desalojar células frouxamente aderidas e transferir a suspensão celular para um tubo cônico de 15 mL, seguido de tubulação para cima e para baixo várias vezes para individualizar as células hiPS.<ol>
<li>Leve a suspensão celular para o volume de 15 mL com DMEM/F12 quente e centrífuga por 5 min a 290 x g em temperatura ambiente.</li>
		<li>Aspire suavemente o supernasciente e resuspenque as células com DMEM/F12 quente para mais uma rodada de centrifugação para remover os componentes residuais da matriz BM 1 e tampão de dissociação.</li>
	</ol>
</li>
	<li>Aspire as células supernascidas e resuspendas em 1 mL de meio de indução de mesoderme, conforme descrito acima. Conte o número total de células usando um hemocítômetro ou contador de coulter para determinar o volume apropriado do meio de diferenciação de mesoderme necessário para alcançar uma concentração de 1 x 105 células/mL.</li>
	<li>Aspirar a solução ECM a partir da matriz BM placas revestidas de 2 e enxaguar as placas duas vezes com DMEM/F12 quente. Misture a suspensão da célula hiPS suavemente por pipetar algumas vezes. Transfira 1 mL da suspensão celular para cada poço da matriz BM de 2 placas revestidas de 2 poços e, em seguida, agite suavemente as placas para distribuir as células de forma mais uniforme.</li>
	<li>Incubar a placa a 37 °C em uma incubadora de CO2 de 5%. Refrescar o meio de indução de mesoderm no dia seguinte. NOTA: Após 2 dias, as células de mesoderme derivadas de células hiPS estariam prontas para indução intermediária de mesoderme.</li>
</ol>5. Diferenciação das células de mesoderme derivadas de células hiPS em mesoderme intermediário (dias 2-16)
<ol><li>No dia 2 do protocolo de diferenciação, aspirar meio de indução de mesoderm e reabastecer com 1 mL por meio de indução de mesoderm bem intermediário.</li>
	<li>Atualizar o meio todos os dias para manter um limiar preciso de fatores de crescimento e pequenas moléculas para as células metabolicamente ativas. Se houver crescimento celular substancial e rápido esgotamento dos nutrientes da mídia (indicado pelo amarelamento da mídia), o volume do meio intermediário de diferenciação de mesoderme pode ser aumentado para 1,3 mL por poço das 12 placas do poço.</li>
	<li>Células de cultura por mais 14 dias para obter células intermediárias de mesoderme. Até o dia 16, essas células podem ser criopreservadas para uso posterior.</li>
</ol>6. Diferenciação de células mesoderm intermediárias derivadas de células hiPS em podocitos (dias 16 a 21)
<ol><li>Enxágüe as células intermediárias de mesoderme com DMEM/F12 quente seguido de incubação das células com 0,5 mL por poço de 0,05 % trypsin-EDTA por 3 min a 37 °C. Faça inspeção visual para garantir que as células estejam começando a se dissociar.</li>
	<li>Raspar células usando um levantador de células e pipeta a suspensão celular várias vezes usando uma ponta de pipeta de 1.000 μL para obter células individualizadas (ou pequenas moitas de) células. NOTA: Nesta fase, certifique-se de que as células estão totalmente dissociadas, pois as células agregadas podem não adquirir um fenótipo terminalmente diferenciado dentro da linha do tempo do protocolo.</li>
	<li>Adicione cerca de 2 mL por poço de solução neutralizante de trippsina para parar a atividade de trippsina.</li>
	<li>Transfira as células para um tubo cônico de 50 mL e leve o volume até 50 mL usando DMEM/F12 e, em seguida, centrifugar a suspensão celular por 5 min a 201 x g à temperatura ambiente.</li>
	<li>Aspire as células supernascidas e resuspendas no meio de indução de podocito. Para obter resultados ideais, garanta uma densidade final de semeadura de aproximadamente 100.000 células/poço de uma placa de 12 poços. Adicione a suspensão da célula à matriz BM de placas revestidas de 2 e agite suavemente a placa para ajudar a distribuir as células de forma mais uniforme.</li>
	<li>Incubar células a 37 °C e 5% de CO2 e atualizar o meio diariamente por até 5 dias para obter podocytes até o dia 21. NOTA: Os podócitos derivados de células hiPS resultantes podem ser mantidos na cultura por 2-4 semanas adicionais usando meio completo com mídia de manutenção de podocyte. Uma vez em mídia de manutenção podocyte, as células podem ser alimentadas a cada dois dias e podem ser usadas para estudos subsequentes ou análises a jusante.</li>
</ol>

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S.M. é um autor de uma patente pendente para métodos para a geração de podocitos renais a partir de células-tronco pluripotentes (pedido de patente dos EUA 14/950859). Os demais autores declaram que não têm interesses concorrentes.

Neste relatório, descrevemos um protocolo para a geração de podocitos glomerulares renais a partir de células hiPS. Os podocitos derivados de células hiPS apresentam características morfológicas e moleculares associadas ao fenótipo de podocito renal maduro13. Em publicações anteriores, mostramos que os podocitos derivados de células hiPS podem imitar a estrutura e a função de filtragem seletiva do glomerulus renal quando co-cultivado com células endoteliais microvasculares glomerular...

Os avanços na cultura das células-tronco pluripotentes humanas estão prontos para revolucionar a medicina regenerativa, a modelagem de doenças e o rastreamento de medicamentos, fornecendo aos pesquisadores uma fonte renovável e escalável de material biológico que pode ser projetado para obter quase qualquer tipo de célula dentro do corpo humano1. Essa estratégia é especialmente útil para o derivado de tipos de células especializadas e funcionais que, de outra forma, seriam difíceis de obter. As células-tronco pluripotentes induzidas por humanos (hiPS)células 2,3,4,5 são particularmente atraentes devido à sua origem celular somática e ao potencial que representam para a medicina personalizada. No entanto, o desenvolvimento de métodos para derivar outras linhagens celulares de células hiPS permanece desafiador devido ao uso frequente de condições culturais mal definidas que levam à baixa eficiência e geração não específica de populações de células heterogênios6,7.
Apresentado aqui é um método para a derivação de podocitos renais maduros de células hiPS com especificidade e alta eficiência em condições quimicamente definidas. Ao considerar os papéis de múltiplos fatores dentro do microambiente celular, desenvolveu-se uma estratégia de diferenciação de células-tronco que envolveu a otimização de fatores solúveis apresentados no meio da cultura celular, bem como fatores insolúveis, como componentes da matriz extracelular ou substratos adesivos. Dada a importância da sinalização integrin no desenvolvimento e função podocyte, a expressão de receptores integrin na superfície celular foi inicialmente examinada. β1 integrins foram altamente expressos não apenas em células hiPS, mas também em seus derivados, incluindo mesoderme e células intermediárias de mesoderme8,9,10. Experimentos subsequentes confirmaram que ligantes que se ligam a β1 integrinos (incluindo laminina 511 ou fragmento de laminina 511-E8) suportam a adesão e diferenciação das células hiPS em podocitos quando usados em conjunto com a mídia indutiva solúvel descrita abaixo.
A indução do compromisso de linhagem celular foi iniciada pela primeira vez confirmando que as células hiPS cultivadas nas superfícies revestidas de laminina por dois dias na presença de um meio contendo Activin A, CHIR99021 e Y27632 O inibidor de rocha pode se diferenciar em células que expressam os primeiros marcadores de mesoderme HAND1, goosecoid e brachyury8,11. O tratamento das células de mesoderme por 14 dias com um meio suplementado com proteína morfogenética óssea 7 (BMP-7) e CHIR99021 possibilitou a derivação de células intermediárias de mesoderme que expressavam os marcadores celulares nefrão-progenitor do Tumor 1 (WT1), proteína relacionada 1 (OSR1)8,11, e gene de caixa emparelhada 2 proteína (PAX2)12. Para derivar os podocitos glomerulares renais maduros, as células mesoderm intermediárias foram tratadas por 4-5 dias com um novo meio composto por BMP-7, Activin A, fator de crescimento endotelial vascular (VEGF), ácido retinóicototalmente trans e CHIR99021. A citometria de fluxo e a imunossuagem foram utilizadas para confirmar que >90% das células resultantes apresentavam as características moleculares, morfológicas e funcionais do podocito renal maduro8,11,13. Essas características incluem o desenvolvimento de processos de pé primário e secundário; a expressão de genes específicos de linhagem podocyte, incluindo SYNPO, PODXL, MAF, EFNB28 e a expressão de proteínas incluindo podocina, nefrina e WT114,15,16. Além disso, verificou-se que os podocitos derivados de células hiPS podem ser mantidos na cultura por até quatro semanas in vitro usando um meiocomercialmentedisponível 8,11 que fornece uma flexibilidade adicional no tempo de experimentos a jusante. Para obter mais informações sobre os painéis de citometria de fluxo utilizados para determinar a pureza dos hiPS-podocytes, consulte nossa publicação anterior11.

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td>
		</tr>
		<tr>
			<td>Growth Factors and Media Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>72262</td>
			<td>Stem Cell Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>17504044</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>04-0004</td>
			<td>Stemgent</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete Medium Kit with CultureBoost-R</td>
			<td>4Z0-500-R</td>
			<td>Cell Systems</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>12634028</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>10565042</td>
			<td>Thermo/Life Technologies</td>
			<td>DMEM/F12 with glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>10082147</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin A</td>
			<td>PHC9564</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>PHC9544</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>PHC9394</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>05850</td>
			<td>Stem Cell Technologies</td>
			<td>hiPS cell culture media (CCM)</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin, liquid (100&#215;)</td>
			<td>15140-163</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 ROCK inhibitor</td>
			<td>1254</td>
			<td>Tocris</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</td>
			<td>ab20680</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>GP-N2</td>
			<td>Progen</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>AF1759</td>
			<td>R&#38;D Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX2</td>
			<td>71-6000</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>MAB4234</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>N-892012</td>
			<td>Iwai North America</td>
			<td>Basement membrane (BM) matrix 2</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>354277</td>
			<td>BD Biosciences</td>
			<td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Enzymes and Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A1110501</td>
			<td>Thermo/Life Technologies</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>A9418</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D2438</td>
			<td>Sigma-Aldrich</td>
			<td>DMSO is toxic. Should be handled in chemical safety hood</td>
		</tr>
		<tr>
			<td>Enzyme-free cell dissociation buffer, Hank&#8217;s balanced salt</td>
			<td>13150016</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>97065-058</td>
			<td>VWR</td>
			<td>Ethanol is flammable and toxic</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>431097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>28906</td>
			<td>Thermo/Life Technologies</td>
			<td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>14190-250</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Distilled Water</td>
			<td>15230162</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>97062-208</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>25300-120</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>29442-462</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>B99516</td>
			<td>Beckman Coulter</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>352097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>352098</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes</td>
			<td>3395465</td>
			<td>Thermo/Life Technologies</td>
			<td>For storing frozen aliquots</td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>AMF7000</td>
			<td>Thermo/Life Technologies</td>
			<td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>100503-092</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>51030403</td>
			<td>Thermo/Life Technologies</td>
			<td>For the routine culture and maintenace of iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>40101-346</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>21905-049</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small</td>
			<td>21905-026</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>P1096-FR</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>P1125</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>P1126</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>P1121</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>P1122</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, individually wrapped</td>
			<td>356551</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, individually wrapped</td>
			<td>356525</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, individually wrapped</td>
			<td>356543</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#956;m, PES</td>
			<td>SCGP00525</td>
			<td>EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes</td>
			<td>1138W14</td>
			<td>Thomas Scientific</td>
			<td>For aliquoting growth factors</td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated six-well plates</td>
			<td>353046</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR white techuni lab coat</td>
			<td>10141-342</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>3008</td>
			<td>Corning</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

A doença renal afeta mais de 10% da população global e custa bilhões de dólares em gastos federais. As formas mais graves de doença renal e eventual insuficiência renal em estágio terminal são frequentemente causadas pelo dano aos podocitos glomerulares, que são as células epiteliais altamente especializadas que funcionam junto com células endoteliais e a membrana do porão glomerular para formar a barreira de filtragem do rim. Os avanços na medicina renal têm sido dificultados pela limitada disponibilidade de tecidos primários e pela falta de métodos robustos para a derivação de células renais humanas funcionais, como os podocitos. A capacidade de derivar podocitos de fontes renováveis, como células-tronco, poderia ajudar a avançar a compreensão atual dos mecanismos de desenvolvimento renal humano e doenças, bem como fornecer novas ferramentas para a descoberta terapêutica. O objetivo deste protocolo foi desenvolver um método para derivar podocitos maduros e pós-mitóticos de células-tronco pluripotentes induzidas por humanos (hiPS) com alta eficiência e especificidade, e sob condições quimicamente definidas. Os podócitos derivados de células hiPS produzidos por este método expressam marcadores específicos de linhagem (incluindo nefrina, podocina e tumor 1 de Wilm) e exibem as características morfológicas especializadas (incluindo processos de pé primário e secundário) associadas a podocitos maduros e funcionais. Curiosamente, essas características especializadas estão notavelmente ausentes na linha celular podocito imortalizada amplamente utilizada no campo, o que sugere que o protocolo descrito aqui produz podocitos renais humanos que têm um fenótipo de desenvolvimento mais maduro do que as linhas celulares podocyte existentes normalmente usadas para estudar biologia renal humana.

O objetivo deste protocolo foi demonstrar que podocitos humanos maduros podem ser derivados de células hiPS em condições quimicamente definidas. Os dados apresentados neste manuscrito foram gerados por meio da linha de células DU11 hiPS17, que foram testadas pela primeira vez, e constataram estar livres de mycoplasma. A análise cromossômica também foi realizada, e as células foram encontradas karyotipicamente normais. Começando com as células hiPS DU11 indiferenciadas, a estratégia de d...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Apresentado aqui é um protocolo quimicamente definido para a derivação de podocitos renais humanos de células-tronco pluripotentes induzidas com alta eficiência (>90%) e independente de manipulações genéticas ou seleção de subpopulação. Este protocolo produz o tipo de célula desejada dentro de 26 dias e pode ser útil para testes de nefrocoxicidade e modelagem de doenças.

Diferenciação guiada de podocitos renais maduros de células-tronco pluripotentes induzidas humanas sob condições quimicamente definidas

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

guided-differentiation-mature-kidney-podocytes-from-human-induced

Research

JoVE Journal

Bioengineering

4.4K visualizações.  Duke University. Apresentado aqui é um protocolo quimicamente definido para a derivação de podocitos renais humanos de células-tronco pluripotentes induzidas com alta eficiência (>90%) e independente de manipulações genéticas ou seleção de subpopulação. Este protocolo produz o tipo de célula desejada dentro de 26 dias e pode ser útil para testes de nefrocoxicidade e modelagem de doenças.

Vídeo: Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Desenvolvimento, expansão e<em&gt; In vivo</em&gt; Monitorização das células NK humanas de células estaminais embrionárias humanas (hESCs) e e induziu células-tronco pluripotentes (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Bioengenharia

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplante de Progenitores Miogênicos derivados de células-tronco pluripotentes induzidos em modelos de regeneração muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

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.

Cultura de células-tronco pluripotentes humanas em polivinil álcool-Co-itacónico ácido hidrogel com rigidez diferentes condições Xeno-livre

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

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

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

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

Isolamento de fibroblastos dérmicos humanos adultos da pele abdominal e geração de células-tronco pluripotentes induzidas usando um método não integrador

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

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Geração escalável de organoides cerebelares maduros de células-tronco pluripotentes humanas e caracterização por imunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medição potencial de ação óptica unicelular em cardiomiócitos derivados de células-tronco pluripotentes induzidos por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tecido conjuntivo de engenharia humana derivada do fibroblasto para aplicações de triagem

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chip de glomérulo renal isogênico projetado a partir de células-tronco pluripotentes induzidas por humanos

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

Geração de Organoides de Vasos Sanguíneos Humanos a partir de Células-Tronco Pluripotentes

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...