Os autores agradecem Janet Rossant para o protocolo original. Este estudo foi financiado pelo Instituto Nacional de Saúde para conceder CA106308 WH.

Neste protocolo, descrevemos a preparação de blastocistos do mouse e do estabelecimento de linhas de células TS. Manipulações do mouse geral antes da coleta de blastocistos, incluindo a criação de acasalamento natural e na indução da ovulação super, basicamente, seguir o protocolo padrão ilustrado por Nagy et al. 2003 (pp146-150). Derivação e manutenção das células TS <ol><li> Cruz configuração cruzamento entre ratos de interesse. </li><li> Prepare fibroblastos embrionárias de camundongos (MEFs) como células alimentadoras. Dois dias antes da coleta de blastocistos, MEFs (2 x 10 6 células) são banhados em 100 pratos mm. No dia seguinte, essas células são tratadas com 10 ml de meio de TS contendo mitomicina C (20 mg / ml) para um 2 horas de incubação a 37 ° C CO, 5% 2. Isso é seguido por lavagem de células duas vezes com PBS. As células tratadas mitomicina C são então semeadas em placas de 12 poços (1 x 10 5 células / poço). </li><li> No dia da coleta de blastocisto (3,5 dpc, referido como um dia), MEF meio de cultura é substituído por TS médio mais FGF4/Heparin 1x (0,5 ml / poço). </li><li> Preparação para a coleta do útero: euthanize o mouse e localizar o útero (Figura 1A). </li><li> Remover o útero, segurando-o com uma pinça no colo do útero (localizado atrás da bexiga) e atravessam a junção do útero e colo do útero (Figura 1B). Puxar o útero, a guarnição da membrana e tecidos de gordura fora e cortar o útero abaixo da junção com o oviduto (Figura 1C). Coloque o útero (Figura 1D) em um pequeno volume (0,2 ml) de meio de TS em uma placa de 60 mm. </li><li> Lave cada corno uterino com 1 ml de meio de TS utilizando uma seringa de 1 ml com uma agulha de 26 gauge. Inserir a agulha na base do útero e lavar lentamente em uma placa milímetros novo 60 com uma média TS ml. O útero deve ser inflado e totalmente estendido durante a lavagem. </li><li> Coletar e transferir os blastocistos cuidadosamente em um prato limpo e bem 12 contendo meio de TS utilizando uma pipeta boca controlada. Lavar blastocistos três vezes por transferência serial através de médio TS em 12-lamelas. Blastocisto um lugar por poço (Figura 2A) em uma placa de 12 poços contendo os mitomicina C tratados com alimentadores MEF e cultura a 37 ° C, 5% de CO 2. </li><li> Os blastocistos chocará da zona pelúcida e anexar aos poços em 24 a 36 horas. A conseqüência de pequeno porte será prontamente observado no dia 3. Alimentar as culturas com 500 mL de médio TS fresco, mais 1x FGF4/Heparin. </li><li> No dia 4 ou 5, o resultado natural de blastocisto deve estar pronto para desagregação dependendo do seu tamanho. O tamanho ideal para TS desagregação das células é ilustrada na Figura 3B. Lavar as culturas, uma vez com 0,5 ml de PBS. Adicione 100 ml 0,25% de tripsina / EDTA e incubar por 5 minutos a 37 ° C CO, 5% 2. Quebrar o desdobramento em pedaços pequenos, pipetando cima e para baixo vigorosamente com uma pipetteman P100. Parar o tripsinização adicionando TS-70CM-1.5F4H (TS médio de 30%, 70% MEF CM-plus FGF4/Heparin 1.5x) no poço. Mudança média 8 horas após a desagregação e continuar a alimentar as células a cada 2 dias. </li><li> Entre os dias 6 e 10 (muito variável) colônias de células TS podem ser observados. Eles têm folha plana epiteliais, como a morfologia com uma fronteira clara colônia (Figura 2C). Continuar a alimentar as culturas a cada 2 dias </li><li> Quando as células TS a confluência de 50% (geralmente entre os dias 15 e 20), lave uma vez com PBS e adicione 100 ml 0,25% de tripsina / EDTA. Pipeta para cima e para baixo durante tripsinização para garantir uma suspensão de uma única célula próximo. Parar o tripsinização adicionando TS-70CM-1.5F4H e transferência para novas placas de 6-bem ou 60 milímetros contendo o mitomicina C tratados com alimentadores MEF (2,5 x 10 5 cells/6-well ou 5 x10 5 cells/60mm placa). </li><li> Mudança média 8 horas após a passagem e continuar a alimentar as culturas a cada 2 dias. </li><li> Depois de mais uma ou duas passagens (3-5 dias entre as passagens) nos alimentadores MEF, as células podem ser cultivadas TS sem eles. Porque MEFs atribuem à placa de cultura muito mais rápido do que as células TS após tripsinização, essa diferença na taxa de aderentes podem ser utilizados para separar células TS do alimentadores MEF. Depois de passar as células cultivadas, incube-los a 37 ° CO C, 5% 2 para 30 min. Mais MEFs irá anexar a placa e da população TS permanece em suspensão. Transfira o sobrenadante para um novo prato. Repita este processo para várias passagens para obter uma população TS pura. </li><li> Manter as células em TS TS-70CM-1.5F4H e passá-las a cada 4 dias (01:10 - 01:20 split) ou quando a cultura atinge 70% confluência. Passando as células com alta densidade (maior do que dividir 1:5) ou cultivo los quando excessivamente confluentes podem causar a sua diferenciação. </li><li> A identidade das células TS pode ser confirmado por imunocoloração de um marcador celular, Cdx2 (Figura 3). </li></ol> Congelamento de células TS <ol><li> Prepare 2x meio de congelamento (50% FBS, 30 de médio TS% e DMSO 20%). </li><li> Cocélulas TS llect por tripsinização, seguido por centrifugação a 450g por 6 min. Ressuspender-los em meio TS com igual volume de meio de congelamento 2x. </li><li> Congelar as células lentamente utilizando uma câmara de álcool isopropílico a -70 ° C e transferir para o nitrogênio líquido após 24-48 horas. </li><li> Uma placa de 60 mm (70% confluentes) é normalmente mantida em 2 frascos. </li></ol> Descongelamento células TS <ol><li> Descongelar rapidamente o frasco em 37 ° C, recolher em 10 ml de mídia TS por centrifugação (450g, 6 min) e ressuspender em 30% TS media, 70cm-1.5F4H. </li><li> Um frasco de células é semeado em uma placa de 60 mm. </li></ol> TS diferenciação celular em TGC Diferenciação das células TS nas células trofoblásticas gigantes (CTGs) pode ser induzida pela retirada do MEF-CM, FGF4 e heparina. O fenótipo TGC (núcleos grandes) podem ser detectados quatro dias após a indução (Tanaka et al., 1998 e Chiu et al. 2008). Imunomarcação de p450scc detectar a expressão deste marcador TGC na cultura diferenciada (Figura 4A). Citometria de fluxo PI (iodeto de propídio) células coradas mostra ainda a presença de células diferenciadas que contêm alto teor de DNA (Figura 4B). Estes resultados sugerem que as células sofrem endorreduplicação TS para se tornar CTGs poliplóides. Resultados representante <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Figura 1" /> Figura 1. Dissecção da úteros mouse. (A) Localização do útero é indicado por setas. (B) Corte através da junção do útero e colo do útero, como mostrado. (C) Corte do útero, logo abaixo do oviduto (OD) debaixo do ovário (OV). (D) Imagem do útero. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Figura 2" /> Fotos Representante figura 2. Ilustrar blastocisto mouse, conseqüência de blastocisto e colônia trofoblasto. (A) A blastocisto de rato coletados em E3.5 é mostrado. ICM, a massa celular interna; MT, trofectoderma mural; PT, trofectoderma polar. (B) A conseqüência trofoblasto em alimentadores MEF é mostrado. TS, células-tronco trofoblasto. (C) A trofoblasto colônia de células-tronco mostra uma clara vantagem em alimentadores MEF. Setas indicam o limite da célula. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Figura 3" /> Figura 3. Identificação de células TS por imunocoloração análise. TS (AD) e ES (controle, EH), as células estão sujeitos à identificação do marcador. Células estão com os anticorpos para reconhecer Oct4 (A, E) ou Cdx2 (B, F), e pela contra-DAPI (C, G) apresentado em imagens fundidas (D, H). Barra de escala, 50 mm. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Figura 4" /> Figura 4. Diferenciação de células in vitro TS. (A) Indiferenciado (undiff) ou diferenciados (Diff) células em cultura foram examinados para a expressão de um marcador TGC, p450scc (verde), contrastado com DAPI (vermelho). (B) análise por citometria de fluxo as células diferenciadas, marcadas com PI, mede o conteúdo de DNA (M1, 03:58 cópias; M2, mais de quatro cópias). A população M2 representa as células do trofoblasto poliplóides.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

Não há conflitos de interesse declarados.

Neste vídeo, demonstramos o processo de coletar E3.5 blastocistos de procedimentos uterino e experimental para estabelecer linhagens de células TS. Descrevemos também a condição para manter a stemness de células TS e induzir sua diferenciação em células diferenciadas. Dois passos fundamentais para obter linhagens de células pura TS são o tempo para desagregação conseqüência (passo 9) e processamento de primeira passagem (passo 11). Tem sido relatado que a primitiva endoderme células derivadas podem surgi...

Reagentes Médio TS: RPMI-1640, 20% FBS, piruvato de sódio 1 mM, 100 mM beta-mercaptoetanol e 100 mg / ml de penicilina-estreptomicina. Mitomicina C: Dissolver 2 mg mitomicina C (Sigma M-0503) em 2 ml de PBS e adicione essa mistura de 2 ml em 200 ml de médio TS (10 ng / ml). Fazer 20 alíquotas (10 ml) e armazenar a -20 ° C até o uso. Adicionar uma alíquota por 100 placa mm. Embrionárias de camundongos médio de fibroblastos-condição (MEF-CM): Placa MEFs em 100 placas mm (1 x 10 6 / placa). No dia seguinte, adicione 10 ml de meio de TS por prato e continuar a cultura por 48 horas. Recolher o meio de cultura, filtrá-los (0,45 mm) e armazenar a -20 ° C em 35 alíquotas ml. Descongelar cada alíquota, quando necessário, que pode ser armazenado a 4 ° C. O MEFs pode ser usado para se preparar para mais dois lotes da CM antes que eles se tornam confluentes. PBS / BSA: Dissolver BSA, fração V (Sigma A3311, de 0,1% (w / v)) em PBS (10 ml), através de um filtro de seringa de 0,45 milímetros de filtro e fazer um alíquotas ml em tubos de 1,5 ml. Armazenar a -70 ° C e descongelar um tubo quando necessário para preparar FGF4. FGF4 (1000x): Ressuspender liofilizado FGF4 (Sigma F8424, 25 mg) com 1 ml da solução de PBS / BSA. Misture bem e fazer 10 alíquotas (100 ml) em tubos de 1,5 ml e armazenar a -70 ° C. Descongelar cada tubo quando necessário e guardar o restante a 4 ° C, não re-congelar. Diluída 1000 vezes em meio TS para obter 1x FGF4 (25 ng / ml). Heparina (1000x): Ressuspender heparina (Sigma H3149, 10.000 unidades) em PBS para uma concentração final de 1 mg / ml (1000x). Fazer 100 ml alíquotas em tubos de 1,5 ml e armazenar a -70 ° C. Alíquotas descongelar quando necessário e guardar o restante a 4 ° C, não voltar a congelar. Diluir 1000 tempo em meio TS para obter 1x heparina (1 ng / ml).

derivation of mouse trophoblast stem cells from blastocysts

Especificação do trofectoderma é um dos eventos mais antigos diferenciação de desenvolvimento dos mamíferos. A linhagem trofoblasto derivados a partir da implantação medeia trofectoderma e gera a parte fetal da placenta. Como resultado, o desenvolvimento desta linhagem é essencial para a sobrevivência do embrião. Derivação de trofoblasto haste (TS) células de blastocistos mouse foi primeiramente descrita por Tanaka<em&gt; Et al.</em&gt; 1998. A capacidade das células TS para preservar a propriedade trofoblasto específicos e sua expressão de palco e de células de tipo específico marcadores após a estimulação adequada fornece um sistema modelo valioso para investigar o desenvolvimento da linhagem trofoblasto qual recapitulando eventos placentação cedo. Além disso, as células do trofoblasto são um dos poucos tipos de células somáticas submetidos a amplificação do genoma natural. Ainda que os percursos moleculares subjacentes poliploidização trofoblasto começaram a desvendar, o papel fisiológico e vantagem de amplificação do genoma do trofoblasto permanece em grande parte ilusória. O desenvolvimento de células-tronco em células diplóides trofoblasto poliplóide na cultura torna este<em&gt; Ex vivo</emSistema de&gt; uma excelente ferramenta para elucidar o mecanismo de regulação da replicação do genoma e da instabilidade na saúde e na doença. Aqui nós descrevemos um protocolo com base em relatórios anteriores com modificação publicado em Chiu<em&gt; Et al.</em&gt; 2008.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Neste vídeo, demonstramos o isolamento de blastocistos mouse ea derivação de células-tronco de blastocistos trofoblasto. Descrevemos também condições para a manutenção da propriedade de célula-tronco, bem como a indução de diferenciação em cultura.

Derivação de células-tronco do rato trofoblasto de blastocistos

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

derivation-of-mouse-trophoblast-stem-cells-from-blastocysts

Research

JoVE Journal

Biology

18.1K visualizações.  University of Rochester. Neste vídeo, demonstramos o isolamento de blastocistos mouse ea derivação de células-tronco de blastocistos trofoblasto. Descrevemos também condições para a manutenção da propriedade de célula-tronco, bem como a indução de diferenciação em cultura.

Vídeo: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento de Células-Tronco da retina do olho do rato

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolamento de células primário do mouse trofoblasto e Ensaio invasão do trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimento de série-tronco de esperma e células progenitoras (SSC) em Cultura para Derivação de longo prazo Adultos Linhas rato SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...