The authors have no conflicts of interest to disclose.

1. O enriquecimento de células progenitoras hematopoiéticas a partir de Sangue Total Adulto NOTA: progenitoras hematopoéticas ou células CD34 + podem ser purificados a partir de células mononucleares do sangue periférico (PBMCs) derivadas a partir de uma variedade de fontes, incluindo o sangue do cordão umbilical, material de leucoferese e sangue total usando métodos baseados em gradiente de densidade de centrifugação. O método está aqui usa sangue total como um exemplo. <ol><li> Isolamento de PBMC a partir de Sangue Total: <ol><li> Adicionar 4,5 ml de meio de separação de linfócitos para o tubo SepMate pipetando-o através do orifício central do inserto. </li><li> Dilui-se a amostra de sangue com um volume igual de DPBS estéril contendo 2% de soro humano (v / v). Por exemplo, dilui-se 5 ml da amostra com 5 ml de DPBS + 2% de soro humano. </li><li> Adicionar a amostra diluída pipetando-o para o lado do tubo. Tome cuidado para não derramar a amostra diretamente através do orifício central. </li><li> Centrifugar a 1.200 xg durante 10 min à temperatura ambiente. </li><li> Remova cuidadosamente o sobrenadante acima da camada de PMBC. </li><li> Recolher a camada de PBMC (turva) para um novo tubo. </li><li> Adicionar 15 ml de fosfato de Dulbecco tamponado com solução salina (DPBS) contendo 2% de soro humano no tubo e centrifugar a 150 xg durante 10 min à temperatura ambiente com o freio. </li><li> Descartar o sobrenadante e ressuspender o sedimento em 15 ml de DPBS contendo 2% de soro humano. Contar o número de células. </li><li> Centrifugar a 690 xg durante 10 min à temperatura ambiente. Remover todo o sobrenadante do tubo. </li><li> Ressuspender as células em 1 x 10 7 células por 15 ml de Dulbecco modificado por Iscove, meio (IMDM) contendo 1% antibióticos (v / v) e 10% de soro humano (v / v). </li><li> Incubar as células S / N a 37 ° C em 5% de CO2, antes do isolamento de células CD34 +. </li></ol></li><li> Selecção positiva de células CD34 + a partir de PBMC utilizando CD34 Multisort Kit: NOTA: Trabalhe rápido e usar soluções pré-arrefecido a tornar as células frio e evitar tamparde anticorpos na superfície da célula e rotulagem de células não-específica por o de instruções do kit. <ol><li> Recolhe todos os PMBC em um tubo de 50 ml e contar o número de células totais no meio. </li><li> Centrifuga-se as células a 300 xg durante 10 min. Remova todo o sobrenadante completamente. </li><li> Ressuspender 8 até 10 células por 300 uL de tampão de talão (DPBS contendo 0,5% de soro humano (v / v) e EDTA 2 mM). </li><li> Adicionar 100 ul de reagente de bloqueio FcR e adicionar 100 ul de MultiSortMicroBeads CD34. </li><li> Misturar bem e incubar durante 30 min no frigorífico (2 - 8 ° C). </li><li> Lavar as células por adição de 2 ml de tampão de talão 10 por 8 células e centrifugar a 300 xg durante 10 min à temperatura ambiente. Remover o sobrenadante completamente. </li><li> Ressuspender 8 até 10 células em 500 ul de tampão. </li><li> Coloque coluna MACS LS no campo magnético de um separador MACS e preparar a coluna por lavagem com 3 ml de tampão de grânulo. </li><li> Aplicar a suspensão de células para center da coluna. Células não marcadas fluirá através do qual podem ser recolhidos, se preferir. </li><li> Lavar a coluna três vezes com 3 ml de tampão de grânulo. </li><li> Remova a coluna do separador e colocá-lo em um tubo de 15 ml. </li><li> Adicionar 5 ml de tampão de talão para a coluna e lave imediatamente as células magneticamente marcadas, empurrando o êmbolo para dentro da coluna. </li><li> Adicionar 20 ul de Reagente de lançamento Multisort por 1 ml de suspensão de células. Misturar bem e incubar durante 10 min no frigorífico (2 - 8 ° C). </li><li> Adicionar 1 - 2 ml de tampão de talão por 10 7 células e centrifugar as células a 300 xg durante 10 min à temperatura ambiente. Remover o sobrenadante completamente. </li><li> Ressuspender as células em 50 ul de tampão de talão por 10 7 células. </li><li> Adicionar 30 ul de reagente de parada Multisort por 10 7 células e incubar durante 15 min no microondas. </li><li> Adicionar 5 ml de tampão de grânulo e centrifugar as células a 300 xg durante 10 min à temperatura ambiente. Descartar o sobrenadante. </li></ol></li></ol> 2. Determinação dos Induced Neurais células-tronco de células CD34 + <ol><li> As células CD34 + cultura: NOTA: medium recentemente preparado deve ser utilizado no prazo de 7 dias, quando armazenados na geladeira. Perda significativa de células CD34 pode ser observada após 24 h, mas a proliferação significativa de células deve ser perceptível, especialmente após o dia 5. Se o número de células diminui continuamente ou não existe proliferação, implica qualquer má qualidade de células médias ou CD34 e as células não deve ser utilizado no passo seguinte. <ol><li> Ressuspender as células e cultura de CD34 + em células CD34 mantendo médio (meio StemSpan SFEM contendo trombopoietina humana (TPO, 100 ng / ml), semelhante a fms tirosina quinase 3 (Flt-3) ligando (100 ng / mL) e factor de células estaminais (SCF, 100 ng / ml), interleucina-6 (IL-6, 20 ng / ml) e interleucina-7 (IL-7, 20 ng / ml)) em uma placa de 24 poços (de até 3 x 10 5 / po em 1 ml de meio por poço), a 37 ° C em 5% de CO2. </li><li> After 24 horas, recolher as células flutuantes e centrifugar as células a 110 xg à temperatura ambiente para se livrar de todas as células unidas e os resíduos celulares. Descartar o sobrenadante e ressuspender as células em 1 ml de meio fresco mantendo CD34 e sementes de uma nova placa de 24 poços (1 x 10 5 / po) a 37 ° C em 5% de CO2 durante 5 dias adicionais. </li><li> Substituir metade do meio todos os outros dias por aspiração cuidadosamente a metade superior de sobrenadante enquanto as células CD34 + são flutuante sobre o fundo das cavidades (Figura 1A). </li></ol></li><li> Transfecção Sendai virus: <ol><li> No dia 5, as células CD34 recolher e centrifugar as células a 170 xg durante 10 min à temperatura ambiente. Descartar o sobrenadante. </li><li> Ressuspender as células em meio fresco a 1 x 10 5 / poço numa placa de 24 poços em 1 ml de meio. </li><li> Descongelar um kit de reprogramação cytotune-iPS Sendai por primeira imersão das partes inferiores dos tubos num banho de água a 37 ° C durante 10 segundos e, em seguida, descongelar completamente umt RT. Resumidamente centrifugar os tubos e colocá-los no gelo. Prepare a mistura de vírus Sendai, misturando o volume recomendado de cada vírus listados no Certificado de Análise (COA) para o lote correspondente. MOI 15 é recomendado. </li><li> Adicionar mistura de vírus Sendai a cada poço de células CD34. Misture os poços agitando suavemente. Incubar as células a 37 ° C em 5% de CO2. </li><li> Observe a eficiência de transfecção após 24 horas. Note-se que a formação de agregados de células e esferas são indicadores para a transfecção bem sucedida (Figura 1B). </li><li> Mudar metade do meio por transferência cuidadosamente a metade superior do meio de outro poço. Se existem esferas de células da nova cavidade, adicionar a mesma quantidade de meio fresco. </li><li> Alterar meio cada outro dia repetindo o passo 2.2.6. </li></ol></li><li> Indução de células-tronco neurais: NOTA: Dependendo das condições de celulares, cerca de 5 ou 7 dias após a transfecção, as células aderentes monocamada chegará a 30 -50% de confluência (Figura 1C). <ol><li> Remove-se os sobrenadantes contendo células flutuantes e esferas e, em seguida, adicionar 1 ml de meio de células progenitoras neurais (Dulbecco Modified Eagle Médium: Mistura Nutriente F-12 (DMEM / F12), contendo suplemento 1x N2, 0,1% (w / v) de albumina de soro bovino ( BSA), 1% (v / v) antibióticos, 20 ng / ml de factor de crescimento de fibroblastos básico (bFGF), e 20 ng / ml de factor de crescimento epidérmico (EGF)) para as células aderentes. </li><li> Opcionalmente, dissociar as esferas de células por pipetagem suave e centrifugar as células a 170 xg durante 10 min. Remover o sobrenadante e ressuspender as células em 1 ml de meio de células progenitoras neurais numa nova placa de 24 poços revestida com matrigel por instrução como back-up. </li><li> Mudar a forma todos os outros dias, até as células chegar a 60 - 80% confluentes, geralmente após 1 semana. </li><li> Desprezar o sobrenadante e adicionar em 1 ml de meio-tronco neurais de células (meio sem soro, NSC SFM). Dissociar as células usando um raspador de células seguidopor pipetagem muito gentil. </li><li> Remover as células e semear todos eles para um poço de uma placa de 6 poços. Adicionar mais 1 ml de meio de células estaminais neurais para fazer 2 ml de meio para cada poço. Incubar as células a 37 ° C em 5% de CO2. </li><li> Alterar meio cada outro dia. </li><li> Quando as células atingiram 60% de confluência, dissociar e replate as células na proporção de 1: 3 seguindo os passos em 2.3.4 e 2.3.6. </li></ol></li><li> Congelamento de células-tronco neurais: <ol><li> Prepare célula estaminal neural meio de congelação pela adição de 20% de dimetilsulfóxido (DMSO) para o meio de células estaminais neurais. Mantenha o meio de congelamento no gelo. </li><li> Dissociar as células em uma placa de 6 poços, usando o raspador de células, seguido por pipetagem suave. Contar as células. Centrifuga-se as células a 170 xg durante 10 min à temperatura ambiente. Descartar o sobrenadante. </li><li> Ressuspender as células em meio de células estaminais neurais a 1 x 10 6 / ml. Adicionar o mesmo volume de meio de congelação de células. </li><li> Adicionar 1 ml (5 x 15 0 número total de células) de células num frasco de congelação. Congelar as células utilizando um recipiente de congelamento Sr. Froster seguindo as instruções. </li></ol></li></ol> 3. Neural Diferenciação Celular <ol><li> Células neuronais diferenciação: <ol><li> Quando as células-tronco neurais chegar a 80% de confluência, retire as células-tronco neurais usando um policial de borracha e depois dissociar pipetando suavemente. </li><li> Contar as células e ajustar a concentração de 1 x 10 5 / ml em meio de células estaminais neurais. </li><li> Para a diferenciação neuronal, as células na placa com poli-D-lisina / laminina lamelas revestidas em placas de 24 poços a 1 x 10 5 / poço. Incubar as células a 37 ° C em 5% de CO2. </li><li> Substituir o meio com meio de diferenciação neuronal (DMEM / F12 contendo 1x suplemento N2, 1x suplemento de B27, 300 ng / ml de cAMP, 0,2 mM de vitamina C, 10 ng ml cérebro / factor neurotrófico derivado (BDNF) e 10 ng / ml de derivado de células gliais factor neurotrófico (GDNF)) after 24 h. Incubar as células a 37 ° C em 5% de CO2. </li><li> Mudar meio de diferenciação neuronal, duas vezes por semana durante 2 semanas. </li></ol></li><li> Diferenciação astrogliais: <ol><li> Dissociar as células-tronco neurais por pipetagem. </li><li> Contar as células e ajustar a concentração de células a 5 x 10 4 células / ml. Semente as células em placas de 24 poços. Incubar as células a 37 ° C em 5% de CO2. </li><li> Substituir o meio com DMEM / F12 com 10% de soro fetal bovino (FBS) e incuba-se as células a 37 ° C em 5% de CO 2 incubadora. </li><li> Mudar meio duas vezes por semana durante 2 semanas. Se as células atingem confluência antes de 2 semanas, replate as células seguintes passos 3.2.1 e 3.2.2. </li></ol></li><li> Oligodendrocyte diferenciação: <ol><li> Dissociar as células-tronco neurais por pipetagem. </li><li> Contar as células e sementes de 1 x 10 5 células em revestidas placas de 24 poços poli-L-Ornitina em 1 ml de oligodendrócitos diferenciaramção que consiste de meio DMEM / F12 contendo suplemento N2, 10 ng / ml de factor de crescimento derivado das plaquetas-AA (FCDP-AA), 2 ng / ml de neurotrofina-3 (NT-3), 2 ng / ml de sonic hedgehog ( Shh), e 3 nM de tri-iodotironina (T3). Incubar as células a 37 ° C em 5% de CO2. </li><li> Mudar meio duas vezes por semana durante 2 semanas. </li><li> Substituir o meio com DMEM / F12 com suplemento 1xN2 e 3 nM de T3 e da cultura das células durante uma semana adicional para a produção de proteína básica de mielina (MBP). </li></ol></li></ol> 4. Imunofluorescência Coloração <ol><li> Substituir mídia com paraformaldeído 4% (PFA). Incubar durante 10 minutos à temperatura ambiente. </li><li> Descartar PFA e lavar com PBS contendo 0,1% de Triton X-100 (PBS-T), 3 vezes, 5 min cada vez. </li><li> Permeabilizar as células com PBS contendo 0,5% de Triton X-100 durante 10 min à TA. </li><li> Bloquear as células com PBS-T contendo 4% de soro de cabra durante 20 min à TA. </li><li> Incubar as células com Antibo primária correspondentemorre diluído em PBS-T contendo 0,1% de BSA durante 2 horas à temperatura ambiente ou O / N na sala fria. </li><li> Lavam-se as células por três vezes com PBS-T, a 5 minutos de cada vez. Incubar as células com os correspondentes anticorpos secundários acoplados com um fluorocromo utilizando 1: 400 em PBS-T contendo 0,1% de BSA durante 1 hora num agitador no escuro. </li><li> Lavam-se as células com PBS-T contendo 4 &#39;, 6-diamidino-2-fenilindole (DAPI) durante 10 min à TA. </li><li> Lavam-se as células mais duas vezes com PBS-T, durante 5 minutos cada à temperatura ambiente. </li><li> Observar as células marcadas utilizando um microscópio e tirar fotos (Figura 3). </li></ol>

<ol>
	<li>Azari, 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=Purification+of+immature+neuronal+cells+from+neural+stem+cell+progeny.">Purification of immature neuronal cells from neural stem cell progeny.</a> PLoS ONE. 6, e20941 (2011).</li><li>Azari, H., Sharififar, S., Fortin, J. M., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroblast+assay:+an+assay+for+the+generation+and+enrichment+of+neuronal+progenitor+cells+from+differentiating+neural+stem+cell+progeny+using+flow+cytometry.">The neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry.</a> J Vis Exp. (62), (2012).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Zhang, N., An, M. C., Montoro, D., Ellerby, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Human+Huntington's+Disease+Cell+Model+from+Induced+Pluripotent+Stem+Cells.">Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells.</a> PLoSCurr. 2, RRN1193 (2010).</li><li>Park, I. 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=Disease-Specific+Induced+Pluripotent+Stem+Cells.">Disease-Specific Induced Pluripotent Stem Cells.</a> Cell. 134 (5), 877-886 (2008).</li><li>Song, B., 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=Neural+differentiation+of+patient+specific+iPS+cells+as+a+novel+approach+to+study+the+pathophysiology+of+multiple+sclerosis.">Neural differentiation of patient specific iPS cells as a novel approach to study the pathophysiology of multiple sclerosis.</a> Stem Cell Research. 8 (2), 259-273 (2012).</li><li>Vierbuchen, T., 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=Direct+conversion+of+fibroblasts+to+functional+neurons+by+defined+factors.">Direct conversion of fibroblasts to functional neurons by defined factors.</a> Nature. 463 (7284), 1035-1041 (2010).</li><li>Pfisterer, U., 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=Direct+conversion+of+human+fibroblasts+to+dopaminergic+neurons.">Direct conversion of human fibroblasts to dopaminergic neurons.</a> Proceedings of the National Academy of Sciences. 108, 10343-10348 (2011).</li><li>Lee, S. -. T., 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=Direct+Generation+of+Neurosphere-Like+Cells+from+Human+Dermal+Fibroblasts.">Direct Generation of Neurosphere-Like Cells from Human Dermal Fibroblasts.</a> PLoS ONE. 6, e21801 (2011).</li><li>Thier, M., 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=Direct+Conversion+of+Fibroblasts+into+Stably+Expandable+Neural+Stem+Cells.">Direct Conversion of Fibroblasts into Stably Expandable Neural Stem Cells.</a> Cell Stem Cell. 10 (4), 473-479 (2012).</li><li>Lujan, E., Chanda, S., Ahlenius, H., S&#252;dhof, T. C., Wernig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+mouse+fibroblasts+to+self-renewing,+tripotent+neural+precursor+cells.">Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells.</a> Proceedings of the National Academy of Sciences. 109, 2527-2532 (2012).</li><li>Wang, T., 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=Derivation+of+neural+stem+cells+from+human+adult+peripheral+CD34++cells+for+an+autologous+model+of+neuroinflammation.">Derivation of neural stem cells from human adult peripheral CD34+ cells for an autologous model of neuroinflammation.</a> PLoS ONE. 8, e81720 (2013).</li><li>Jin, C. 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=Recombinant+Sendai+virus+provides+a+highly+efficient+gene+transfer+into+human+cord+blood-derived+hematopoietic+stem+cells.">Recombinant Sendai virus provides a highly efficient gene transfer into human cord blood-derived hematopoietic stem cells.</a> Gene Ther. 10 (3), 272-277 (2003).</li><li>Ban, 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=Efficient+generation+of+transgene-free+human+induced+pluripotent+stem+cells+(iPSCs)+by+temperature-sensitive+Sendai+virus+vectors.">Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.</a> Proceedings of the National Academy of Sciences. 108, 14234-14239 (2011).</li><li>Goolsby, J., 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=Hematopoietic+progenitors+express+neural+genes.">Hematopoietic progenitors express neural genes.</a> Proceedings of the National Academy of Sciences. 100, 14926-14931 (2003).</li></ol>

The study is funded by NIH intramural funds.

Um protocolo detalhado para gerar directamente células estaminais neurais a partir de células progenitoras hematopoiéticas periféricas é fornecido. Em comparação com células de fibroblasto, de sangue periférico humano é mais acessível. Usando o protocolo apresentado, mais do que 1 x 10 5 células progenitoras hematopoiéticas ou células positivas CD34, podem ser enriquecidos a partir de 10 ml de sangue total. Embora a contaminação com plaquetas normalmente não é pr...

In vitro, as culturas neuronais têm sido utilizados como uma ferramenta fundamental para o estudo de doenças neurológicas. Animais primária (principalmente roedor) culturas neurais 1, 2 e linhas celulares neurais humanas derivadas de tumores ou outros gliomas são os mais vulgarmente utilizados em tais estudos. No entanto, tem sido reconhecido que existem diferenças significativas entre os roedores e células humanas. Muitos resultados baseados em roedores não pode ser traduzido para os seres humanos. Além disso, com a rápida evolução da análise de informação de massa genômica ea edição gene relativamente fácil e seqüenciamento do genoma inteiro, a tendência é cada vez mais orientada para a descoberta de genes de doenças propensas e delinear suas funções e papéis em doenças específicas, o que torna os poucos neuronal humana linhas celulares só têm limitado o uso. Teoricamente, as culturas neuronais primárias humanas derivadas a partir de amostras do sistema nervoso do paciente são a melhor escolha, mas eles são impossíveis de se obter; meth, portanto, alternativaods são necessárias. Nos últimos anos, algumas abordagens têm sido perseguidos, com dois sendo o mais distinguíveis. Seguindo o desenvolvimento da técnica de geração de células pluripotentes estaminais induzidas (IPSC) usando o rato e células somáticas humanas 3, 4, células neurais pode ser diferenciado a partir deles 5-7. No entanto, a geração e caracterização iPSC exige mão de obra intensiva, técnicas e tempo de entrada, às vezes até exageradamente. Pouco depois, foi desenvolvido outro método para transformar directamente as células neuronais a partir de células somáticas 8, 9. Como os neurónios resultantes são não-proliferativa, que limita a sua aplicação em estudos intensivos e rastreio de drogas, que requer uma grande quantidade de células. Para tirar vantagens de ambas as técnicas, derivação direta do tronco neurais / células progenitoras de células somáticas foi explorado por vários grupos de 10-12, que ignora o tedioso processo de geração iPSC e caracterização, mas ainda provides um número razoável de células-tronco neurais para diferenciação neural mais tarde. Mostrámos previamente que, após a introdução dos factores de transcrição Yamanaka em células progenitoras hematopoiéticas, células estaminais neurais podem ser gerados directamente utilizando uma célula progenitora neuronal seleccionando meio 13. Aqui, nós relatamos o método em detalhes.

<table border="0" cellpadding="0" cellspacing="0" width="584">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Material List</td>
			<td style="width:171px;"></td>
			<td style="width:135px;"></td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:72px;">Comments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Lymphocyte Separation Medium</td>
			<td style="width:171px;">Lonza</td>
			<td style="width:135px;">17-829E</td>
			<td style="width:72px;">1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SepMate</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">15415</td>
			<td style="width:72px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Human Serum</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:135px;">34005-100</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Antibiotics</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:135px;">15240-062</td>
			<td align="right" style="width:72px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CD34 MultiSort Kit</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-056-701</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EDTA</td>
			<td style="width:171px;">Cellgro</td>
			<td style="width:135px;">46-034-Cl&#160;</td>
			<td style="width:72px;">2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">MACS LS Column</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-042-401</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">StemSpan SFEM Medium</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">9650</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IMDM</td>
			<td style="width:171px;">Quality Biologicals</td>
			<td style="width:135px;">112-035-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TPO</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-18</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Flt-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-19</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SCF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-07</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-6</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-06</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-7&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-07</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IPS Sendai Reprogramming Kit</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">A1378001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Cell scraper</td>
			<td style="width:171px;">Sarstedt</td>
			<td align="right" style="width:135px;">83.183</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMSO</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D2650</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CryoTube vials</td>
			<td style="width:171px;">Thermo</td>
			<td align="right" style="width:135px;">368632</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Mr. Frosty container</td>
			<td style="width:171px;">Thermo</td>
			<td style="width:135px;">5100-0001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMEM/F12</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">12400-024</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">N2 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A2934</td>
			<td style="width:72px;">0.1% (w/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">bFGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-18B</td>
			<td style="width:72px;">20 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">AF-100-15</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">B27 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17504-044</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NSC serum free medium</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:135px;">A1050901</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="40">
			<td height="43" rowspan="2" style="height:43px;width:208px;">Poly-D-lysine/laminin coated cover slips</td>
			<td rowspan="2" style="width:171px;">BD Bioscences</td>
			<td align="right" rowspan="2" style="width:135px;">354087</td>
			<td rowspan="2" style="width:72px;"></td>
		</tr>
		<tr height="3">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">cAMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A9501</td>
			<td style="width:72px;">300 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Vitamin C</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A0278</td>
			<td style="width:72px;">0.2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-02</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">GDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-10</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P4957</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PDGF-AA</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-13A</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NT-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-03</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Shh</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">1314-SH/CF</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">T3</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T6397</td>
			<td style="width:72px;">3 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PFA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:208px;">PBS</td>
			<td style="width:171px;">Quality Biological</td>
			<td style="width:135px;">119-069-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Goat serum</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G9023</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TritonX-100</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T9284</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse monoclonal anti-Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:135px;">AB5922</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-SOX2&#160; antibody</td>
			<td style="width:171px;">Applied Stemcell</td>
			<td style="width:135px;">ASA0120</td>
			<td style="width:72px;">Ready to use</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse anti-&#946;III-tubulin antibody</td>
			<td style="width:171px;">Promega</td>
			<td style="width:135px;">G712A</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:208px;">Rabbit anti-GFAP antibody</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G4546</td>
			<td style="width:72px;">1:100 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-O4 antibody</td>
			<td style="width:171px;">R&#38;D Systems</td>
			<td style="width:135px;">MAB1326</td>
			<td style="width:72px;">IgM; 1 ng/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 594 goat anti-rabbit antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11012</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 488 goat anti-mouse antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11001</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Alexa Fluor 488 goat anti-mouse IgM antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A21042</td>
			<td style="width:72px;">1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DAPI</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D9542</td>
			<td style="width:72px;">1 ug/ml</td>
		</tr>
	</tbody>
</table>

direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells

Culturas neuronais específicos doenças humanas são essenciais para a geração de modelos in vitro para doenças neurológicas humanos. No entanto, a falta de acesso para culturas neuronais adultas humanas primárias coloca desafios. Desenvolvimentos recentes em células-tronco pluripotentes induzidas (IPSC) fornece uma abordagem alternativa para derivar culturas neurais a partir de fibroblastos da pele através de paciente iPSC específico, mas este processo é trabalhoso, exige conhecimentos especiais e grandes quantidades de recursos, e pode levar vários meses. Isso impede que a ampla aplicação desta tecnologia para o estudo de doenças neurológicas. Para superar algumas destas questões, temos desenvolvido um método para obter células-tronco neurais diretamente do sangue periférico humano adulto, ignorando o processo de derivação da IPSC. As células progenitoras hematopoiéticas enriquecidos a partir do sangue periférico humano adulto foram cultivadas in vitro e transfectadas com vectores de vírus Sendai contendo factores de transcrição Sox2, Out3/4, KLF4, e c-Myc. Os resultados de transfecção em alterações morfológicas nas células, que são ainda seleccionadas usando meio progenitora neuronal humana contendo factor de crescimento de fibroblastos básico (bFGF) e factor de crescimento endotelial vascular (VEGF). As células resultantes são caracterizadas pela expressão de marcadores de células estaminais neuronais, tal como a nestina e SOX2. Essas células-tronco neurais pode ser ainda mais diferenciado para os neurônios, astroglia e oligodendrócitos em media diferenciação especificado. Utilizando amostras de sangue periférico humano facilmente acessíveis, este método pode ser utilizado para derivar células estaminais neurais para uma maior diferenciação para células neuronais in vitro de modelagem de desordens neurológicas e pode avançar estudos relacionados com a patogénese e tratamento dessas doenças.

<html> A qualidade das células CD34 é crítica para o sucesso da transdução de células estaminais neurais. As células CD34 alta qualidade proliferam durante o primeiro par de dias de cultura e aparecem como não aderentes, as células homogeneamente redondas, flutuando pouco acima do fundo de recipientes de cultura (Figura 1A). Os resultados de sucesso de infecção em agregados de células, que se expande ao longo do tempo (Figura 1B), enquanto os agregados de células não espec?...

Watch this Scientific Journal Video about Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells at JoVE.com

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Foi desenvolvido um método para derivar directamente células estaminais neurais humanos a partir de células progenitoras hematopoiéticas enriquecidas de células do sangue periférico.

Indução direta de Neurais Human células-tronco de sangue periférico células progenitoras hematopoéticas

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

direct-induction-human-neural-stem-cells-from-peripheral-blood

Research

JoVE Journal

Developmental Biology

12.4K visualizações.  National Institutes of Health.  Foi desenvolvido um método para derivar directamente células estaminais neurais humanos a partir de células progenitoras hematopoiéticas enriquecidas de células do sangue periférico. 

Vídeo: Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivação de Cardiac células progenitoras de células estaminais embrionárias

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Biologia do Desenvolvimento

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento de Neural Stem / células progenitoras da Região Periventricular do rato adulto e Medula Espinhal Humano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

A indução direta de hegemônicos endotélio e sangue por sobre-expressão de fatores de transcrição na pluripotentes humanas Células-Tronco

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Geração de Integração-livres induzidas células-tronco pluripotentes a partir de células mononucleares de sangue periférico humano Usando epissï¿½ica Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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

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

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

Dirigido a diferenciação de progenitores hematopoiéticos primitivo e definitiva de células pluripotentes humanas

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

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

Condrócitos diferenciadores de células-tronco pluripotentes induzidas por humanos derivadas de sangue periférico

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolamento de células progenitoras endoteliais de sangue humano do Cordão Umbilical

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion de célula única no Zebrafish intacta vida

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análise computacional do Germline Caenorhabditis elegans , para estudar a distribuição de núcleos, as proteínas e o citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Na geração de Vitro do Somite derivados de humanos induzida por células tronco pluripotentes

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...