Os autores reconhecem o programa de esterilização canina na UNESP-FCAV para gentilmente fornecendo os ovários. Este trabalho foi financiado por doações da FAPESP (processo n º 2013/14293-0) e a CAPES.

Esta experiência foi realizada com os ovários de quatro cães femininos rafeiros doados após a cirurgia eletiva em um programa de esterilização canina. Este experimento foi aprovado pelo Comitê de ética no uso de animais da UNESP-FCAV (protocolo n º 026991/13).
1. preparação experimental
<ol><li>Preparar ou comprar 500 mL de fosfato salino de estéril Dulbecco (DPBS) sem cálcio ou magnésio.</li>
	<li>Preparar uma colagenase armazeno solução através da mistura de 40 µ g da enzima em 1 mL de DPBS. Filtrar usando um filtro de seringa 0,2 µm e armazenar alíquotas de 2 mL a-20 ° C.</li>
	<li>Prepare os meios de cultura a partir médio (DMEM) baixos de glicose do Eagle modificado de Dulbecco com 10% de soro fetal e 1% de antibióticos.</li>
	<li>Coletar o material esterilizado: tesoura e pinça, um frasco de cultura de tecidos, pratos, tubos e pipetas de 10 mL e 5 mL.</li>
	<li>Usar equipamento de laboratório de cultura de célula padrão: uma câmara de segurança biológica (CSB), uma incubadora de cultura celular fixado em 5% de CO2 e 38 ° C e uma centrífuga.</li>
	<li>Limpar o BSC: com as mãos enluvadas, esterilize-a com 70% EtOH, usando luzes UV.</li>
</ol>2. isolamento de células-tronco mesenquimais do ovário
<ol><li>Após a cirurgia, manter os ovários em PBS no gelo em um tubo cônico, até sua chegada ao laboratório.</li>
	<li>Preencha dois pratos de Petri de 100 mm com 10 mL de PBS.</li>
	<li>Remover os ovários de tubo e transferi-los para o primeiro prato de Petri com PBS. Lave-os com os movimentos de mistura.</li>
	<li>Delicadamente, recuperar o ovário e coloque-o no outro prato. Com uma pinça estéril e tesoura, mince o tecido em pedaços muito pequenos, cerca de 1 mm de tamanho.</li>
	<li>Transferir o tecido para um prato de Petri de 35mm e adicionar 2 mL de colagenase. Picar o tecido um pouco mais.</li>
	<li>Coloque o prato de Petri na incubadora a 38 ° C por 3 h e agite suavemente a placa de Petri com movimentos circulares a cada 20 min.</li>
	<li>Após 3h, retire o prato de Petri da incubadora.</li>
	<li>Transfira o conteúdo do prato para um tubo de 15 mL. Adicione 5 mL de mídia de expansão.</li>
	<li>Inverta suavemente o tubo antes da centrifugação. Centrifugar o tubo a 2100 x g durante 7 min. remover o sobrenadante e ressuspender o precipitado com 3 mL de mídia de expansão.</li>
	<li>Transferi o sedimento para uma garrafa de T25. Coloque a garrafa na incubadora com 5% CO2 a 38 ° C por 3 h. Nota: A parte mais importante do procedimento é mudar a mídia após 3 h de incubação.</li>
	<li>Retire a garrafa da incubadora e remova a mídia com o tecido restante. Adicione 3 mL de mídia expansão fresco para a garrafa.</li>
	<li>Mudar a cada 48 h mídia e observar a garrafa para celular confluência. Nota: As principais etapas do processo podem ser observadas na Figura 1.</li>
</ol>3. expansão de células-tronco mesenquimais do ovário
<ol><li>
Passagem de células
	<ol>
<li>Quando as células chegam a confluência, remova a mídia do frasco.</li>
		<li>Lave o frasco com 3 mL de PBS. Remova a PBS.</li>
		<li>Adicione 1,5 mL de tripsina e colocar a garrafa na incubadora a 38 ° C por 3 min. Bata levemente o frasco para ajudar as células a se separar. Nota: Células devem alcançar confluência aproximadamente 5 - 7 d após o chapeamento inicial.</li>
		<li>Adicionar 3 mL de mídia de expansão e transferir o conteúdo para um tubo cônico.</li>
		<li>Centrifugar o tubo a 2100 x g durante 7 min. remover o sobrenadante e adicionar 1 mL de mídia de expansão.</li>
		<li>Homogeneizar cuidadosamente o conteúdo do tubo.</li>
		<li>Tome uma alíquota de 10 µ l da amostra para realizar a célula contando e colocar o tubo com as células de volta na incubadora.</li>
		<li>Lugar de 10 µ l da mistura para o hemocytometer.</li>
	</ol>
</li>
	<li>
Contar as células
	<ol>
<li>Use o objectivo de X 10 do microscópio e concentrar as linhas de grade do hemocytometer.</li>
		<li>Conte as células em cinco pequenos quadrados (Figura 2).</li>
		<li>Multiplique o número contado por 50.000 para estimar o número de células por mililitro.</li>
	</ol>
</li>
</ol>4. diferenciação de células-tronco mesenquimais do ovário
Nota: Diferenciação ensaios foram realizados de acordo com as diretrizes estabelecidas em Hill et al . 9.
<ol><li>Semente de 1,0 x 104 células por poço, em triplicado, usando um prato de cultura 4-bem.</li>
	<li>Adicione 1 mL de mídia de expansão.</li>
	<li>Agite suavemente o prato com movimentos circulares.</li>
	<li>Após 24 h, substitua os meios de cultura com os meios de diferenciação desejável.</li>
	<li>Para osteogênica, adipogenic e chondrogenic de diferenciação, incubam as células com os meios de indução para 30 d, com substituição de mídia cada 3 kits de diferenciação específica d. foram utilizados para cada um destes ensaios.</li>
	<li>Para a diferenciação da linhagem neurogênica, Incube as celulas para 10 d na mídia da indução, com a substituição de mídia cada 3 d. A mídia utilizada aqui contidos DMEM baixa glicose, ácido valproico de 2 mM, 1 hidrocortisona µM, 10 µM forskolin, cloreto de potássio de 5 mM, 5 µ g/mL insulina e 200 µM butil hidroxianisolo.</li>
	<li>Para os precursores de endoderme, incube 5D em mídia endoderme conforme as instruções do fabricante reagente.</li>
	<li>Para a diferenciação da linhagem primordial de células germinativas, Incube as celulas na mídia indução para 14 d, com substituição de mídia cada 3 d. A mídia utilizada aqui contidos DMEM, 10% FBS, piruvato de sódio 1 mM, 10 ng/mL LIF, aminoácidos não essenciais de 1 mM, 2 mM L-glutamina, 5 µ g/mL insulina, 0,1 mM β-Mercaptoetanol, 60 putrescina µM, 20 transferrina µ g/mL, 10 ng/mL do mouse fator de crescimento epidérmico, humano de 1 ng/mL fator de crescimento fibroblástico básico, 40 ng/mL humanos gliais celular fator neurotrófico derivado de linha e penicilina de 15 mg/L.</li>
</ol>

<ol>
	<li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D., Atala, A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li><li>Ahmed, N., Thompson, E. W., Quinn, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li><li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17 (2005).</li><li>Gong, S. P., 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=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li><li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145 (2004).</li><li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li><li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391 (2017).</li><li>Dominici, M. L. B. 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li><li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li><li>Kuznetsov, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li></ol>

Os autores não têm nada para divulgar.

Aqui nós fornecemos evidências que MSCs podem ser isolados de tecido ovariano canino, que é considerado lixo biológico após ovariectomia. Devido ao fato de que muitos tipos de células podem ser encontrados no ovário, propusemos um protocolo para selecionar MSCs baseadas sua rápida aderência ao plástico, que com sucesso selecionado células que cresceram em uma monocamada com uma morfologia de fibroblasto tipo.
O primeiro relatório da derivação do MSCs da medula óssea baseou-se na...

O número de estudos publicados que foco sobre células-tronco tem aumentado substancialmente na última década, um esforço de investigação que tem sido alimentado pelo objetivo coletivo de descobrir terapias da medicina regenerativa poderosa. Células-tronco têm dois marcadores definição primárias: autorenovação e diferenciação. Células-tronco mesenquimais são responsáveis por volume de negócios de tecido normal e têm uma capacidade mais restrita de diferenciação em comparação com células-tronco embrionárias1. Recentemente, muitos estudos têm mostrado uma vasta gama de diferenciação do MSCs, e um tema em discussão é se existem diferenças entre as células-tronco embrionárias e adultas em todos os2.
O epitélio de superfície ovarium é uma camada não confirmada de células, relativamente menos diferenciada, que expressa ambos os marcadores epiteliais e mesenquimais3, mantendo a capacidade de se diferenciarem em diferentes tipos de células em resposta a sinais ambientais4. A localização exata de células-tronco no ovário não é bem conhecida; no entanto, tem sido proposto que bipotential progenitores na túnica albugínea originar células germinativas5. Estudos imunológicos têm a hipótese de que estas células têm uma origem do estroma6 ou estão localizados em ou proximal para a superfície do ovário7. Uma vez que as células-tronco mesenquimais express numerosos receptores que desempenham um papel importante na aderência de célula8, um experimento foi concebido para testar a hipótese de que a seleção de uma população de células com a rápida adesão iria isolar uma população de células claramente characterizable como mesenquimais na natureza. Recentemente, nosso grupo relatou a derivação de MSCs de tecido ovariano com base na sua capacidade de aderência à superfície plástica do prato cultura nas primeiras 3 horas de cultura, a fim de obter uma população purificada de células exibindo rápida adesão9. Aqui, descrevemos o método desenvolvido por isolamento de células-tronco mesenquimais do tecido do ovário.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DPBS&#160;</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">14190144</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Collagenase I</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">17100017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tissue flask</td>
			<td style="width:151px;">Corning</td>
			<td style="width:123px;">CLS3056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DMEM low glucose</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">11054020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FBS</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12484-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">TrypLE express</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12604021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Adipogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Chondrogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Osteogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">STEMdiff Definitive Endoderm Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:123px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Penicillin-Streptomycin</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD45</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2035S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD34</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2411GA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD90</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1036G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD44</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Nestin</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">&#946;-Tubulin&#160;</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB1637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DDX4</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:123px;">PA5 -23378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR80F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR120F</td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation and differentiation of canine ovarian mesenchymal stem cells

Interesse em células-tronco mesenquimais (MSCs) aumentou na última década devido a sua facilidade de expansão de isolamento e cultura. Recentemente, estudos têm demonstrado a capacidade de diferenciação de largura que estas células possuem. O ovário representa um candidato promissor para terapias baseadas em células, devido ao fato de que é rica em MSCs e que ela frequentemente é descartada após cirurgias de ovariectomia como resíduos biológicos. Este artigo descreve os procedimentos para o isolamento, a expansão, e diferenciação de MSCs derivado do ovário canino, sem a necessidade de ordenação de células técnicas. Este protocolo representa uma importante ferramenta para a medicina regenerativa devido a ampla aplicabilidade destas células altamente diferenciável em ensaios clínicos e terapêuticos, usa.

Isolamento de células-tronco mesenquimais de ovário canino:
O procedimento de isolamento de MSC ovário está resumido na Figura 1. Após a cirurgia, tecido de picagem, digestão de colagenase e uma mudança de mídia 3 h após o início da cultura, uma população de MSC putativa com propriedades de plástico adesivo rápidas foi com êxito isolada de tecido ovar...

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Aqui, descrevemos um método para o isolamento, expansão e diferenciação de células-tronco mesenquimais de tecido ovariano canina.

Derivação e diferenciação de células-tronco mesenquimais ovário canina

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

O objetivo geral deste procedimento é isolar, expandir e diferenciar as hastes mesenquimais derivadas do tecido ovariano canino. Este experimento foi realizado com os ovários de quatro cães do sexo feminino doados após cirurgia eletiva em um programa de esterilização canina. Este experimento foi aprovado pelo Comitê de Ética sobre o uso de animais da Universidade Estadual de São Paulo, FVAC.Resultados representativos. Discussão.conclusão.

derivation-differentiation-canine-ovarian-mesenchymal-stem

Research

JoVE Journal

Developmental Biology

5.6K visualizações.  São Paulo State University (UNESP). O objetivo geral deste procedimento é isolar, expandir e diferenciar as hastes mesenquimais derivadas do tecido ovariano canino. Este experimento foi realizado com os ovários de quatro cães do sexo feminino doados após cirurgia eletiva em um programa de esterilização canina. Este experimento foi aprovado pelo Comitê de Ética sobre o uso de animais da Universidade Estadual de São Paulo, FVAC.Resultados representativos. Discussão.conclusão.

Vídeo: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem 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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Derivação eficiente do epitélio pigmentar da retina células de células-tronco

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Derivação de Cardiomyocytes altamente purificada de induzidas pelo homem pluripotentes células estaminais usando pequenos Diferenciação modulada-Molecule e Subsequente Glucose Fome

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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

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

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

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Regulação epigenética da Cardiac diferenciação de células estaminais embrionárias e Tecidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolamento e expansão de CTM / células estromais derivadas de placenta humana Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Diferenciação de células-tronco mesenquimais humanas em células casos funcionais

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Derivação livre de integração de células-tronco pluripotentes induzidas por seres humanos com Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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

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

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

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

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

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 ...