A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53^−/− ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.     

p53 in stem cells

p53 is well known as a "guardian of the genome" for differentiated cells,in which it induces cell cycle arrest and cell death after DNA damage and thus contributes to the maintenance of genomic stability.In addition to this tumor suppressor function for differentiated cells,p53 also plays an important role in stem cells.In this cell type,p53 not only ensures genomic integrity after genotoxic insults but also controls their proliferation and differentiation.Additionally,p53 provides an effective barrier for the generation of pluripotent stem celllike cells from terminally differentiated cells.In this review,we summarize our current knowledge about p53 activities in embryonic,adult and induced pluripotent stem cells.     

Progress in studies on the induction and differentiation of embryonic stem cells

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocyst stage embryo, have the potential to give rise to an entire organism and to generate every body cell type. Much improvement has been made in the field of induction and differentiation of ESCs during the last two years, such as the ESCs differentiation into germ cells (2003) and the cloning of human ESCs (2004), both of which were chosen respectively as one of the top ten achievements evaluated by academic journals. Great attention was also paid to the research of the new genes which could maintain ESCs in the undifferentiated state and the research of the induction and differentiation of ESCs.     

Progress in studies on the induction and differentiation of embryonic stem cells

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocysts, have the potential to give rise to an entire organism and to gen-erate every body cell type. During the last two years, much progress has been made in ESCs field, espe-cially in the induction and differentiation of ESCs. 1 ESCs differentiate into cells of different types 1.1 ESCs differentiate into germ cells In 2003, it was reported that mouse ESCs could differentiate into oocyte[1]. Oct-4 was …     

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.     

MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells

MicroRNAs （or miRNAs） are small non-coding RNAs （21-25 nucleotides） that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed （2-fold） after differentiation. MiR-124a was dramatically upregulated （more than 20-fold） while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos.     

Neural differentiation of human embryonic stem cells

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes.     

Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

p73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53

The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) that are able to self-renew or undergo differentiation depending on a complex interplay of extracellular signals and intracellular factors. However, the feedback regulation of differentiation-dependent ESC self-renewal is poorly understood. Retinoic acid (RA), a derivative of vitamin A, plays a critical role in ESC differentiation and embryogenesis. In the present study, we demonstrate that short-term treatment of murine (m) ESCs with RA during the early differentiation stage prevented spontaneous differentiation of mESCs. The RA-treated cells maintained self-renewal capacity and could differentiate into neuronal cells, cardiomyocytes, and visceral endoderm cells derived from three germ layers. The differentiation-inhibitory effect of RA was mimicked by conditioned medium from RA-treated ESCs and was accompanied with up-regulated expression of leukemia inhibitory factor (LIF), Wnt3a, Wnt5a, and Wnt6. Such RA-induced prevention of ESC differentiation was attenuated by a neutralizing antibody against LIF or by a specific Wnt antagonist Fz8-Fc and was totally reversed in the presence of both of them. Furthermore, knock-down of beta-catenin, a component of the Wnt signaling pathway, by small interfering RNA counteracted the effect of RA. In addition, RA treatment enhanced expression of endodermal markers GATA4 and AFP but inhibited expression of primitive ectodermal marker Fgf-5 and mesodermal marker Brachyury. These findings reveal a novel role of RA in ESC self-renewal and provide new insight into the regulatory mechanism of differentiation-dependent self-renewal of ESCs, in which Wnt proteins and LIF induced by RA have the synergistic action. The short-term treatment of ESCs with RA also offers a unique model system for study of the regulatory mechanism that controls self-renewal and specific germ-layer differentiation of ESCs.     

溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸（lysophosphatidic acid,LPA）对大鼠胚胎神经干细胞（neural stem cells,NSCs）分化为少突胶质细胞（galactocerebroside—positive,Gal-C阳性）和星形胶质细胞（grim fibrillary acidic protein-positive,GFAP阳性）的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析。结果显示：（1）加入不同浓度（0．010．0μmol／L）LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1．0μmol／LLPA组,少突胶质细胞所占百分比从对照组的8．5％增加到32．6％;（2）星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理组星形胶质细胞百分比与对照组相比均无显著性差异;（3）RT-PCR结果显示,大鼠胚胎NSCs的LPA1和LPA3受体表达明显,而LPA3受体表达很弱。以上结果表明,较低浓度的LPA可能作为细胞外信号,通过LPA1和LPA3受体促进大鼠胚胎NSCs向少突胶质细胞分化和生成,但对星形胶质细胞的分化过程无明显影响。     

The full spectrum of physiological oxygen tensions and step-changes in oxygen tension affects the neural differentiation of mouse embryonic stem cells

The beneficial impact of lowering oxygen tension to physiological levels has been demonstrated in a number of stem cell differentiation protocols. The majority of these studies compare normal laboratory oxygen tension with one physiological condition (typically 2-5% O(2) ). In this article, we investigated whether the full spectrum of physiological oxygen tensions (0-20% O(2) ) and step-changes in oxygen tension could enhance the production of neural populations from of embryonic stem cells (ESCs). We used a model system for the conversion of mouse ESCs into cells expressing one neuroectoderm stem cell marker (nestin) and two neural markers (βIII tubulin and microtubule-associated protein (MAP2)). 4-10% O(2) was associated with large increases in the total production of viable cells and the highest number of cells expressing Nestin, βIII tubulin, and MAP2. However, 4-10% O(2) also caused a reduction in the percentage of cells expressing all three markers. Step changes in oxygen tension at the mid-point of the differentiation process affected the total production of viable cells and the percentage of cells expressing all three markers. We found that the initial oxygen tension and the magnitude of the step-change were critical variables. A step increase from 0 to 2% O(2) mid-way through the protocol resulted in the highest percentage of cells expressing βIII tubulin (86.5%). In conclusion, we have demonstrated that the full spectrum of physiological oxygen tensions and step changes in oxygen tension represent a powerful tool for the optimisation of neural differentiation processes.     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.     

Murine embryonic stem cells as a model for human embryonic stem-cell research

Over the last several decades, murine embryonic stem cells (mESCs) have been used as a model for human embryonic stem cell (hESC) research. The relevance of this approach has not yet been proven. There is a great deal of evidence that is indicative of substantial differences between these two cell types. An analysis of the literature shows that the differences concern ESC proliferation, self-renewal, and differentiation. Consequently, mESC may be considered as a model object for hESC studies only for some aspects of their biology. The alternative model objects, such as primate ESC, are also discussed briefly in this review.