Este trabalho foi financiado pela Veterans Affairs e subvenções NIH 5U01HL100398 e 1R01HL104040.

1. Corpo formação embrionária (EB), utilizando fundo 96-redonda bem placas de microtitulação <ol><li> Cultivar células TE de camundongos em placas de 10 centímetros de cultura de células com o mouse médio de células ES suplementado com LIF. </li><li> Quando as células ES estão prontos para uso (geralmente na confluência 50-70%), remova a mídia ES e lavar as células uma vez com 5ml de PBS estéril. </li><li> Adicionar 2 ml de tripsina 0,05% / EDTA para cada placa e incubar as placas a 37 ° C por 3 ~ 5 minutos, Quench tripsina EDTA com 3 ml de mídia ES. </li><li> Transferência de células para tubos falcon de 50ml e giram a 1000 rpm por 3 minutos. </li><li> Remover o sobrenadante e ressuspender pellet celular com uma quantidade desejada de EB mídia. </li><li> Conte o número de células e diluir as células a 5 x10 3 células / ml no EB mídia. </li><li> Use pipeta multicanal para adicionar 100μl de EB mídia contendo células em cada poço de 96 - round placas de microtitulação. </li><li> Lugar 96 - rodada placas de microtitulação bem em uma incubadora. </li></ol> 2. Identificação de janelas de tempo crítico da via de sinalização chave (s) para cardiogenesis <ol><li> Adicionar moduladores de vias de sinalização específica e de controle do veículo em cada poço de placas de microtitulação de 96 poços contendo células-tronco embrionárias em momentos diversos, tais como o dia 0, dia 1, dia 2, dia 3 e dia 4, etc Metade dos poços em cada 96 bem-placa (48 poços) são usados ​​para cada ponto de hora de início do tratamento. </li><li> Lavar a moduladores mudando media EB para cada 48 poços em diferentes pontos de tempo de parada. Por exemplo, para obter dois dias de tratamento para as células ES a partir de dia 0, lavar a moduladores no dia 2. Para qualquer tratamento mais de 48 horas, a mudança de mídia EB suplementado com moduladores fresco a cada dois dias até que os pontos de tempo desejado parar. </li><li> Examine contração EB sob um microscópio após o dia 7. Pontuação dos poços com a contratação EBs como positivos para obter porcentagens de contratação para cada EBs cursos tempo diferente de tratamento. </li><li> Os pontos momento crítico para cardiogenesis ES são os prazos em que continha o maior percentual de contratação EBs tratados por moduladores de sinalização quando comparado com o controle do veículo. </li></ol> 3. Verificação de janelas de tempo de sinalização para cardiogenesis em EBs feita a partir de gotas de suspensão <ol><li> Prepare placas de Petri, adicionando com 3-4 ml de PBS para evitar a evaporação. </li><li> Siga os passos 1,1 ~ 1,5 para preparar células-tronco embrionárias em 2.5x10 4 células / ml densidade celular final. </li><li> Despeje a mistura de células bacterianas em prato de fácil acesso. Usando pipeta multicanal, adicione 20 gotas mL (contendo cerca de 500 células) para tampas invertidas. Não permita que as gotas individuais ao toque. Geralmente uma tampa pode caber até 80 gotas. Vire a tampa para trás sobre o prato contendo PBS. Incubar por 24 horas ou 48 horas para permitir a formação EB em uma incubadora. </li><li> Lavar EB formado a partir de gotas de suspensão de cada tampa com 3 ml de mídia e piscina EB 2 tampas de EBs a nova placa de Petri. Adicionar mídia EB para um volume total de 10ml. </li><li> Transferência EBs em suspensão para 0,2% de gelatina revestido de 6 placas bem no dia 4. Em geral 30 EBs são transferidos para cada poço. Adicionar mídia EB para obter o volume de 2 ml final para cada poço. </li><li> Incubar moduladores de sinalização no período de tempo identificados a partir de 96 poços experimentos prato. </li><li> Observe a contração EB sob microscópio após o dia 7 e cardiogenesis pode ser ainda caracterizada por RT-PCR de positividade, e outros, se necessário. </li></ol>

<ol>
	<li>Fukuda, K., Yuasa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+a+source+of+regenerative+cardiomyocytes.">Stem cells as a source of regenerative cardiomyocytes.</a> Circulation research. 98, 1002-1013 (2006).</li><li>Foley, A. C., Mercola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+induction+by+Wnt+antagonists+depends+on+the+homeodomain+transcription+factor+Hex.">Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex.</a> Genes Dev. 19, 387-396 (2005).</li><li>Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., Komuro, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stage-specific+biphasic+roles+of+Wnt/beta-catenin+signaling+in+cardiomyogenesis+and+hematopoiesis.">Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis.</a> Proc Natl Acad Sci U S A. 103, 19812-19817 (2006).</li><li>Hao, J., Daleo, M. A., Murphy, C. K., Yu, P. B., Ho, J. N., Hu, J., Peterson, R. T., Hatzopoulos, A. K., Hong, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin,+a+selective+small+molecule+inhibitor+of+BMP+signaling,+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Dorsomorphin, a selective small molecule inhibitor of BMP signaling, promotes cardiomyogenesis in embryonic stem cells.</a> PloS one. 3, e2904-e2904 (2008).</li></ol>

Não há conflitos de interesse declarados.

O método de gota em suspensão tem sido o método convencional usado para a formação de EB e na diferenciação in vitro. No entanto, é trabalhoso e limita a flexibilidade experimental, devido a preocupações logísticas. Da mesma forma, os resultados são também mais difíceis de validar como a habilidade do pesquisador é crucial para a formação de EB bem sucedido e manipulação como gotas de suspensão. Um método mais simples é formar EBs em uma placa de fundo redondo de 96 poços como um processo de etapa única. Este formato permite a diferenciação in vitro a ser padronizada e pode acomodar mais condições experimentais, devido à facilidade de formação de EB e manipulação downstream (por exemplo, adição ou remoção modulador). Além disso, o formato de 96 poços da placa pode ser otimizado para ser uma ferramenta de triagem eficiente nas células ES mouse.

<ul><li> Rato CGR8 células são usados ​​principalmente. </li><li> ES médio: media GMEM suplementado com 10% inativado pelo calor FBS, 2 mM L-glutamina, 0,05 mM 2-mercaptoetanol, e 200 U / ml murino LIF. </li><li> EB médio: Prepare media IMDM com 20% inativado pelo calor FBS, 1,6 mM L-glutamina, 0,08 mM 2-mercaptoetanol, e 1X MEM não-solução de aminoácidos essenciais. </li></ul>

modified mouse embryonic stem cell based assay for quantifying  cardiogenic induction efficiency

Diferenciação das células-tronco pluripotentes é rigidamente controlado por regulação temporal e espacial de várias vias de sinalização chave. Um dos obstáculos à sua compreensão tem sido os métodos variados em correlacionar as alterações dos principais eventos de sinalização para a eficiência diferenciação. Descrevemos aqui o uso de um tronco embrionárias de rato ensaio de células (ES), com base para identificar janelas de tempo crítico para Wnt / β-catenina e ativação do sinal BMP durante a indução cardiogênico. Marcando para a contratação de corpos embrionárias (EBs) em um formato de placa de 96 poços, podemos rapidamente quantificar a eficiência cardiogênico e identificar janelas de tempo crucial para Wnt / β-catenina e ativação do sinal BMP em um curso de tempo seguintes tratamentos modulador específico. O principal descrito aqui não se limita a indução cardíaca por si só, e pode ser aplicado para o estudo de muitas outras linhagens celulares. Além disso, o formato de 96 poços tem o potencial de ser desenvolvido como um alto throughput, ensaio automatizado para permitir o teste de hipóteses mais sofisticado experimental.

Watch this Scientific Journal Video about Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency at JoVE.com

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Nós descrevemos o uso de um ensaio de células de rato ES base para identificar janelas de tempo crítico para Wnt / β-catenina e ativação do sinal BMP durante a indução cardiogênico. O método fornece uma plataforma padronizada, que quantifica a eficiência confiável cardiogênico, e é aplicável ao estudo de linhagens de célula.

Modificado embrionárias de camundongos com células-tronco Ensaio baseado para quantificar a eficiência de indução cardiogênico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

modified-mouse-embryonic-stem-cell-based-assay-for-quantifying

Research

JoVE Journal

Biology

9.6K visualizações.  Vanderbilt University School of Medicine. Nós descrevemos o uso de um ensaio de células de rato ES base para identificar janelas de tempo crítico para Wnt / β-catenina e ativação do sinal BMP durante a indução cardiogênico. O método fornece uma plataforma padronizada, que quantifica a eficiência confiável cardiogênico, e é aplicável ao estudo de linhagens de célula.

Vídeo: Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

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 ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Entrevista: biorreatores e apareceu-Modified 3D-andaimes para Investigação em Células Estaminais

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análise de Crescimento Humano embrionárias Colony Stem Cell

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Diferenciação eficiente de células-tronco embrionárias em neurônios motores

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparação de células embrionárias de camundongos Fibroblastos adequados para a cultura de células-tronco embrionárias humanas e pluripotentes induzidas

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP Ensaio Reporter para o Estudo de genes que regulam o mouse Manutenção células-tronco embrionárias e auto-renovação

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...