Differentiation Of Murine Stem Cells Research Articles

RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.

Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid-induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx(+/+) and Syx(-/-) embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx(-/-) embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx(+/+) embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx(-/-) embryoid bodies were thinner than those in Syx(+/+) cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx(-/-) embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

Development of biomimetic polycaprolactone films to promote the osteoblastic differentiation of murine stem cells

Event Abstract Back to Event Development of biomimetic polycaprolactone films to promote the osteoblastic differentiation of murine stem cells Jessica Jann1, Olivier Drevelle1, 2, Alex Daviau1 and Nathalie Faucheux1 1 Université de Sherbrooke, Chemical and Biotechnological Engineering Department, Canada 2 Ecole Polytechnique de Montréal, Chemical Engineering Department, Canada Introduction: These past years, marked by the increasing age of the global population, the frequency of bone loss and fractures has considerably increased. The technique most frequently used to overcome bone loss is autograft, but it has multiple post operational complications that arise in 8.5-20% of graft procedures[1]. As an alternative, biomimetic materials have been developed to favour mesenchymal stem cells (MSCs) adhesion[2]. For this purpose, the biomaterial is usually functionalized by peptides containing the sequence Arg-Gly-Asp (RGD) found in several proteins of the extracellular matrix[3]. RGD is recognized by cellular receptor integrins. However, osteoblastic differentiation of MSCs is also influenced by the bone morphogenetic proteins (BMPs). The co-localization of integrin and BMP receptors[4] highlights the potential of functionalizing biomaterials not only with adhesive peptides, but also with peptides derived from BMPs[5]. Saito et al have developed a peptide derived from the knuckle epitope of BMP-2 that promotes MSC differentiation into osteoblasts[6]. Since BMP-9 has a higher osteoinductive potential than BMP-2[7],[8], our research group has developed a peptide derived from BMP-9 that induces osteoblast differentiation in vitro and bone formation in vivo[9]-[11]. We also recently found that a peptide containing the RGD and synergy sequences of fibronectin (pFibro) promotes a higher MSC response to peptides derived from BMP-9 (pBMP-9 and SpBMP-9) in comparison to a peptide derived from the bone sialoprotein. The present study has therefore analysed the effect of polycaprolactone (PCL) films co-functionalized by pFibro and SpBMP-9 on MSC behaviours. Material and Methods: We first determined the number of murine C3H10T1/2 MSC attached to functionalized PCL after incubation for 0.5 to 4h. The cytoskeletal organization of these attached cells was also studied by labelling F-actin. The activation state of the canonical BMP pathway was determined using Western blots. Finally, by carrying out qPCR, we also analysed the activation of genes encoding osteogenic marker Dlx5 and Runx2 at days 3 and 6. Results and Discussion: At 4h, all functionalized PCL films promoted the attachment of C3H10 T1/2 cells. These cells organized their actin cytoskeleton. MSCs attached to PCL films functionalized with SpBMP-9 alone or a combination of pFibro with SpBMP-9 also contained a higher level of phosphorylated Smad 1/5/8, demonstrating that grafted SpBMP-9 was able to activate the canonical BMP pathway. At 6 days, the cells attached to PCL functionalized by SpBMP-9 alone or a combination of SpBMP-9 and pFibro contained higher level of Dlx5 and Runx2 transcripts, suggesting the osteoblastic commitment of the MSCs. Conclusion: A better understanding of cellular response to co-functionalized PCL is required. However, the co-functionalization of PCL by pFibro and SpBMP9 seems a promising approach to develop scaffold for tissue engineering application. NSERC

Effects of basic fibroblast growth factor, epidermal growth factor and platelet derived growth factor on osteogenetic differentiation of murine mesenchymal stem cells

Objective To investigate the effects of cytokine factors basic fibroblast growth factor (bFGF),epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) on prolification and osteogenesis of murine mesenchymal stem cells obtained from bone marrow (BMSCs).Methods Cell adhesion was used to purify the BMSCs in the presense or absence of bFGF,EGF and PDGF-BB (10 ag/L)in culture medium composed of DMEM/F12,10％ FBS,and penicillin/streptomycin (100 U/ml and 100 mg/L,respectively).Cells at the third passages were used for subsequent experiments.Flow cytometry was applied to detect the expression of cell-specific surface antigens CD45 and CD90.Osteogenic differentiation of BMSCs was measured by osteoblast-specific genes expression of collagen Ⅰ and osteocalcin,alkaline phosphatase assays and deposition of calcium mineralization.Results Cells in the absence of cytokines showed much faster adhesion rate than in the presence of cytokines after initial culture,but slower proliferative ability after passage,and displayed more senescense form.The expression of CD45 and CD90 in cells in the presence or absence of cytokines was [(0.48 ± 0.12)％,(82.78 ± 0.25)％] and [(27.34 ± 0.69)％,(41.93 ± 0.34)％],respectively.BMSCs in the presence of cytokines exhibited higher expression of osteoblast-specific genes,and better formation of calcium mineralization than control groups,indicating cells in the presence of cytokines possessed high potency for osteogenic differentiation.Conclusion Combination of three cytokines could improve the proliferation of mesenchymal stem cells in vitro and facilitate osteogenic differentiation. Key words: Mesenchymal stem cells; Cytokine; Osteogenic differentiation

Development and Characterisation of Cardiomyocytes Derived from Murine Embryonic Stem Cells of a DCM Caused by a SCN5A Mutation

Introduction: Mutations on Nav1.5 sodium channels have been reported recently in patients with complex cardiac arrhythmias associated with Dilated cardiomyopathy (DCM). Nav1.5 channels are encoded by SCN5A gene and allow the action potential generation in the heart. Its involvement in structural heart disease such as DCM is not well elucidated. We recently identified a novel mutation (R219H) on this channel protein to cause DCM associated with a complex arrhythmic phenotype Gosselin-Badaroudine P., et al., (2012): PlosONE, 7(5):e38331. The biophysical characterisation in Xenopus oocytes expression system revealed a proton (H+) leak at hyperpolarised voltages through the mutant protein. The aim of this study is to develop a cellular model based on the differentiation of murine stem cells carrying the R219H mutation to characterise the H+ leakage in physiological conditions.Methods: Murine stem cells differentiated to cardiomyocytes were dissociated and the H+ leak current is measured by the patch clamp method. The morphology of the derived cardiomyocytes was investigated by immunocytochemistry. Intracellular pH and acidification is also observed through fluorescence techniques (BCECF).Results and conclusion: The patch clamp experiments confirmed the presence of a H+ leak at hyperpolarized voltages due to the R219H mutation. After maturation, the use of typical sarcomere markers (myosin light chain 2v and troponin T) indicates that mutant cardiomyocytes like cells also exhibited a progressive disorganization of their contractile apparatus. This cellular model describes for the first time a complex morphological pathology caused by the dysfunction of an ion channel. Finally, the study of current proton leak in physiological conditions provides helpful understanding on its pathological impact.

Modeling of epigenome dynamics identifies transcription factors that mediate Polycomb targeting

Although changes in chromatin are integral to transcriptional reprogramming during cellular differentiation, it is currently unclear how chromatin modifications are targeted to specific loci. To systematically identify transcription factors (TFs) that can direct chromatin changes during cell fate decisions, we model the relationship between genome-wide dynamics of chromatin marks and the local occurrence of computationally predicted TF binding sites. By applying this computational approach to a time course of Polycomb-mediated H3K27me3 marks during neuronal differentiation of murine stem cells, we identify several motifs that likely regulate the dynamics of this chromatin mark. Among these, the sites bound by REST and by the SNAIL family of TFs are predicted to transiently recruit H3K27me3 in neuronal progenitors. We validate these predictions experimentally and show that absence of REST indeed causes loss of H3K27me3 at target promoters in trans, specifically at the neuronal progenitor state. Moreover, using targeted transgenic insertion, we show that promoter fragments containing REST or SNAIL binding sites are sufficient to recruit H3K27me3 in cis, while deletion of these sites results in loss of H3K27me3. These findings illustrate that the occurrence of TF binding sites can determine chromatin dynamics. Local determination of Polycomb activity by REST and SNAIL motifs exemplifies such TF based regulation of chromatin. Furthermore, our results show that key TFs can be identified ab initio through computational modeling of epigenome data sets using a modeling approach that we make readily accessible.

Heme Oxygenase 1 Promotes Murine Stem Cell Differentiation into Beating Cardiomyocytes

Heme Oxygenase 1 Promotes Murine Stem Cell Differentiation into Beating Cardiomyocytes

The embryonic stem cell test as tool to assess structure-dependent teratogenicity: the case of valproic acid.

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.

Effects of aFGF and HGF on differentiation of murine hepatic stem cells

目的 初步探讨了酸性成纤维细胞生长因子(aFGF)及肝细胞生长因子(HGF)对大鼠胎肝干细胞分化成熟的影响.方法采用胶原酶消化法分离胎龄13 d的SD大鼠胎肝细胞,分别接种于Ⅰ、Ⅱ、Ⅲ、Ⅳ组细胞培养瓶中进行体外培养.Ⅰ组:培养液中含酸性成纤维细胞生长因子(aFGF)和肝细胞生长因子(HGF);Ⅱ组:培养液中含aFGF;Ⅲ组:培养液中含HGF;Ⅳ组培养液中无细胞因子,此组作为对照组. 检测在相同培养时间(第3、5、7、9、11天)细胞培养上清液中甲胎蛋白(AFP)和白蛋白(ALB)的含量;并用免疫细胞化学法鉴定在无细胞因子干预培养3 d后,胎肝干细胞对细胞角质素-19(CK-19)的表达.结果Ⅰ组、Ⅱ组及Ⅲ组分别与Ⅳ组比较,在相同培养时间的培养上清液中AFP的含量均较少,而ALB的含量均较多,且差异均有显著性意义(AFP:t=6.264,3.221,2.591, P 0.05;ALB: t=0.616, P>0.05);而Ⅳ组分别与Ⅱ组和Ⅲ组比较,在相同培养时间的培养上清液中ALB的含量均较少,而AFP的含量均较多,差异也均有显著性意义(AFP:t=3.221,2.591, P 90%.结论采用胶原酶消化法可从胎龄13 d的大鼠胎肝中分离出肝干细胞;aFGF及HGF均能促进肝干细胞向肝细胞分化成熟,并且两者有一定的协同作用。

Protection of peritoneal macrophages by granulocyte/macrophage colony-stimulating factor (GM-CSF) against dexamethasone suppression of killing of Aspergillus, and the effect of human GM-CSF

Murine peritoneal macrophages in vitro could kill Aspergillus fumigatus conidia, and this activity could be suppressed with dexamethasone. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) alone did not boost killing, but GM-CSF treatment concurrently with dexamethasone reversed the dexamethasone suppression. Both recombinant human and recombinant murine GM-CSF were equivalent in this activity, even though the human reagent reportedly does not stimulate differentiation of murine stem cells. Recombinant human GM-CSF could also reverse dexamethasone suppression of bronchoalveolar macrophage conidiacidal activity. Sequential studies with peritoneal macrophages indicated that recombinant human GM-CSF pretreatment also blocked dexamethasone suppression, but the GM-CSF treatment given after dexamethasone did not block the suppressive effect. Recombinant human GM-CSF did not boost spleen cell proliferation to a mitogenic stimulus, and did not reverse dexamethasone suppression of proliferation. These studies suggest GM-CSF treatment prior to and concurrent with steroid immunosuppression may ameliorate the steroid effect on tissue macrophage antifungal activity, but does not affect steroid suppression of T-cell immunity.

A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.

During human placental development, specialized cells allocated to the extraembryonic lineage (cytotrophoblasts) invade the uterus, anchoring the conceptus to the decidua and tapping a supply of maternal blood. This unusual behavior requires cytotrophoblasts to assume highly specialized characteristics; some are commonly associated with tumor cells, while others are typical of endothelia. Here we investigated the transcriptional mechanisms that control cytotrophoblast differentiation/invasion. Specifically, we examined the cells' expression of a number of transcription factors, at the RNA level, as they differentiated along the invasive pathway in vitro. Since basic helix-loop-helix (bHLH) proteins play important roles in murine trophoblast differentiation, we first examined their expression by cytotrophoblasts. As in murine placental development, expression of the human homologue of Mash-2 was confined to progenitor cells. But expression of Hand-1, which promotes differentiation of murine trophoblast giant cells, was not detected. We also found that cytotrophoblasts upregulated the expression of bHLH/PAS factors that function in adaptive responses to hypoxia, including hEPAS-1, which is expressed primarily in endothelial cells. Quite unexpectedly, we discovered that cytotrophoblasts express high levels of mRNA encoding the human homologue of the Drosophila neuronal fate gene, glial cells missing-1 (gcm-1). We also found evidence of crosstalk between the bHLH and GCM-1 regulatory networks. Together, these results offer insights into the transcriptional mechanisms that govern cytotrophoblast differentiation/invasion. Interestingly, these mechanisms suggest analogies with those that govern differentiation of murine stem cells allocated to both the intra- and extraembryonic lineages.

Temporospatial Relationships between Macroglia and Microglia during in vitro Differentiation of Murine Stem Cells

Embryonic stem (ES) cells of the permanent line BLC6 derived from a 129/Sv Gat mouse blastocyst were differentiated as spheroid aggregates (embryoid bodies, EBs) in the presence of retionic acid. After 2 days in suspension, EBs were plated on gelatine-coated glass coverslips and cultivated for 5, 9, and 16 days post plating (DPP) in normal medium. In this study we investigated whether the well-known retinoic acid-induced differentiation of ES cells into neurons (identified by immunostaining for neuron-specific enolase and synaptophysin) was accompanied by cells expressing astroglial (GFAP), oligodendroglial (O4), and microglial (5C6, galectin-3) markers. Whereas differentiation of neurons was closely related to their centrifugal migration towards the periphery of the EBs, the maturation of neuroglia followed a strict time-dependent manner. At 5 DPP, only neurons but no cells expressing glia-specific markers, were observed. At 9 DPP, GFAP-positive and O4-positive macroglial cells appeared. At 16 DPP, microglial cells (5C6-positive and galectin-3-positive) occurred. The established dynamic of relationships between neuronal and nonneuronal cells shows that the model of EBs is similar to the sequence differentiation of the nervous tissue. Thus, enabling in vivo observation of neurons, astrocytes, oligodendrocytes, and microglia, the model of EBs provides a basis for further investigations on the relationships between neurons and neuroglia under various experimental conditions.