Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells have been tested for potential cell transplantation therapy for CNS disorders. Understanding their differentiation mechanism and identifying factors involved in driving excitatory and inhibitory neuron lineages should enhance the efficacy and efficiency of the cell transplantation therapy. We tested the hypothesis that selective expression of Src family tyrosine kinases is required for phenotype-specific differentiation and functional maturation of ES cell derived neurons. Cultured mouse pluripotent ES cells were treated with retinoic acid (RA) to induce neural differentiation. After RA induction, neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lck and an extension of Src kinase expression from cell body to neurite processes. ES cell derived neuron-like cells expressed neurofilament, synaptophysin, glutamate receptors, NMDA and kainate currents, became vulnerable to excitotoxicity and formed functional excitatory synapses. These developmental events were blocked or attenuated when cells were grown in the presence of Src family kinase inhibitor PP2. However, there was no change in the expression of GABAergic-specific protein GAD67 during PP2 treatment. Our data suggest that Src tyrosine kinases are involved in the terminal differentiation of excitatory neuronal phenotype during ES cell neural differentiation after RA induction.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.     

Effect of BMP4 preceded by retinoic acid and co‐culturing ovarian somatic cells on differentiation of mouse embryonic stem cells into oocyte‐like cells

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling are the key regulators for germ cell and meiosis induction, respectively. Gonadal tissue also provides an appropriate microenvironment for oocyte differentiation in vivo. The current study aimed to determine whether mimicking in vivo niche is more efficient for oocyte differentiation from embryonic stem (ES) cells. Here, differentiation of mouse ES cells toward oocyte‐like cells using embryoid body (EB) and monolayer protocols was induced in the presence (+BMP4) or absence (‐BMP4) of BMP4. On day 5, each group was co‐cultured with ovarian somatic cells in the presence or absence of RA (+RA or –RA) for an additional 14 days. Our results showed a significant increase in expression of meiotic markers in the +BMP4 condition in EB differentiation protocol. Further differentiation with ovarian somatic cells led to a subpopulation of oocyte‐like cell formation. Compared to the controls, the +RA condition resulted in a significant elevation of the meiotic gene expression in contrast to Oct4 that significantly decreased in both protocols. In the cells pre‐treated with BMP4 and then exposed to RA in the monolayer differentiation protocol, the gene expression levels of germ cell, Mvh, and maturation markers, Cx37, Zp2, and Gdf9, were also upregulated significantly. Therefore, it can be concluded that +BMP4 and +RA along with ovarian somatic cell co‐culture improved the rate of in vitro oocyte differentiation.     

Embryonic stem cell-derived neurogenesis

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.     

Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells represent an ideal source for cell engraftment in the damaged central nervous system (CNS). Understanding key signals that control ES cell differentiation may improve cell type-specific differentiation that is suitable for transplantation therapy. We tested the hypothesis that extracellular signal-regulated kinase (ERK) 1/2 phosphorylation is an early signaling event required for the neuronal differentiation of ES cells. Cultured mouse ES cells were treated with an all-trans-retinoic-acid (RA) protocol to generate neurally induced progenitor cells. Western blot analysis showed a dramatic increase in ERK 1/2 phosphorylation (p-ERK 1/2) 1-5 days after RA induction, which was attenuated in the presence of the p-ERK 1/2-specific inhibitor UO126. Phospho-ERK 1/2 inhibition significantly reduced the number of NeuN-positive cells and the expression of associated cytoskeletal proteins. In differentiating ES cells, there was increased nuclear translocation of STAT3 and decreased protein expression levels of GDNF, BDNF and NGF. STAT3 translocation was attenuated by UO126. Finally, caspase-3 activation was observed in the presence of UO126, suggesting that the ERK pathway also contributes to the survival of differentiating ES cells. These data indicate that ERK 1/2 phosphorylation is a key event required for early neuronal differentiation and survival of ES cells.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

PBX1 is dispensable for neural commitment of RA-treated murine ES cells

Experimentation with PBX1 knockout mice has shown that PBX1 is necessary for early embryogenesis. Despite broad insight into PBX1 function, little is known about the underlying target gene regulation. Utilizing the Cre–loxP system, we targeted a functionally important part of the homeodomain of PBX1 through homozygous deletion of exon-6 and flanking intronic regions leading to exon 7 skipping in embryonic stem (ES) cells. We induced in vitro differentiation of wild-type and PBX1 mutant ES cells by aggregation and retinoic acid (RA) treatment and compared their profiles of gene expression at the ninth day post-reattachment to adhesive media. Our results indicate that PBX1 interactions with HOX proteins and DNA are dispensable for RA-induced ability of ES to express neural genes and point to a possible involvement of PBX1 in the regulation of imprinted genes.     

Embryonic stem cells as a model for studying regulation of cellular differentiation

Mouse embryonic stem (ES) cells can be differentiated in vitro into near homogeneous populations of both neurons and skeletal muscle as well as other cell types. We previously showed that treatment of pluripotent ES cells with retinoic acid (RA) induced differentiation into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons. The reasons for generation of only GABA neurons as opposed to other neuronal cell types were not known. We have extended our previous work and now show that with RA induction of ES cells we not only obtain GABA neurons, but also dopaminergic neurons. Critical for the production of dopaminergic neurons after RA induction was the post-induction plating conditions used. No dopaminergic neurons were detected if cells were plated in serum-free media optimized for neuronal survival. However, significant numbers of dopamine neurons could be detected when cells were plated in media containing fetal calf serum. These observations support the conclusion that RA acts as a general neural inducing agent and that conditions post-induction either selectively support survival of a particular class of neuronal cells or that the conditions post-induction actually further instruct cells to differentiate into different types of neurons.     

Mouse embryonic stem cells are not susceptible to cytomegalovirus but acquire susceptibility during differentiation

BACKGROUND: Cytomegalovirus (CMV) is the most significant infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection in humans. The timing of infection and the susceptibility of cells in early gestational stages are not well understood. In this study we investigated the susceptibility of embryonic stem (ES) cells to CMV infection during differentiation. METHODS: ES cell lines were established from transgenic mice integrated with the murine CMV (MCMV) immediate-early (IE) promoter connected with a reporter lacZ gene. The susceptibility of the ES cells was analyzed in terms of viral gene expression and viral replication after induction of differentiation. RESULTS: ES cells were nonpermissive to MCMV infection in the undifferentiated state. Upon differentiation, permissive cells appeared approximately 2 weeks after the leukemia inhibitory factor was removed. Upon neural differentiation by retinoic acid (RA), glial cells showed specific susceptibility in terms of expression of the viral antigen. The MCMV IE promoter was not activated in ES cells from the transgenic mice. Activation of the IE promoter was detected approximately 2 weeks after induction of differentiation and observed predominantly in glial cells. Upon MCMV infection of the ES cells, viral infection was correlated with the activation of the IE promoter. CONCLUSIONS: ES cells are nonpermissive to MCMV infection and acquire permissiveness about 2 weeks after induction of differentiation, especially in glial cells. Acquisition of permissiveness in differentiated ES cells may be associated with activation of the IE promoter.     

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.     

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 neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol 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 patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. 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 Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). 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.     

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.     

Cdk6在维持ES细胞自我更新中的作用

细胞周期蛋白依赖性激酶6（cyclin dependent kinase 6,Cdk6）对胚胎早期发育有着重要的作用.然而,它在胚胎干（embryonic stem,ES）细胞中的生物学功能仍不清楚.在该研究中,我们运用RNA干扰技术和基因表达分析方法探索了Cdk6在小鼠胚胎干细胞中的功能及分子机制.结果表明：Cdk6的表达水平与小鼠ES细胞的自我更新密切相关.首先,维甲酸（RA）处理和白血病抑制因子（LIF）去除实验显示 ,随着ES细胞的分化,Cdk6的表达水平明显降低.更为重要的是,RNA干扰介导的Cdk6表达抑制导致ES细胞自我更新相关基因的显著下调,同时伴随细胞分化基因的表达激活,提示Cdk6对维持ES细胞自我更新至关重要.     

STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells.     

Involvement of Crosstalk between Oct4 and Meis1a in Neural Cell Fate Decision

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6–12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.