Embryonic stem (ES) cells are isolated from the inner cell mass of developing blastocysts which have unlimited self-renewal capacity and the ability to differentiate into cells of all three primary germ layers in vitro and in vivo. Induced differentiation of embryonic stem cells into neural lineage had been achieved in 1981,1 since then various methods have been used to derive neural cells from ES cells. Two methods were widely used: suspension culture of embryoid bodies (EBs) followed by retinoic acid2,3 or growth factors treatment4,5 and selection; as well as coculture with stromal cells.6,7 Although it is possible to generate neural cells as neuron, astrocytes and oligodendrocyte, some problems exist. First, the final cultures are always a mixture of many different cell types because embryoid bodies are multicellular aggregations of cells of different lineages. Coculture with stromal cell circumvents EB formation step, but the contamination associated with the use of stromal cell lines and the difficulty of separation of the induced cells makes it unsuitable for therapeutic applications.
To overcome these problems, different systems have been developed, for example, Ying et al8 described a method for conversion of mouse ES cells to neural cells in monolayer culture by defined conditions. It is known that histone deacetylase inhibitors such as sodium butyrate, enhances histone acetylation, so that globularly activate gene expression. And it may serve as a mechanism for differentiation initiation. Here, we combined the differentiation induction effect of sodium butyrate with the induction and selection effects of N2B27 medium, derived homogenous and functional neural cells from mES cells in monolayer culture. The results suggested potential pathways for derivation of neural cells from ES cells, but also shed light on epigenetic mechanisms in neural differentiation.
Murine ES cells were cultured on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells in the Dulbecco's modified Eagle's medium (DMEM; Sigma, USA) supplemented with 15% fetal bovine serum (FBS, Hyclone, Rockville, USA), 2 mmol/L L-glutamine (Gibco-BRL, Grand Island, USA), 0.1 mmol/L 2-mercaptoethanol (MTG, Sigma), 1% non-essential amino acids (Invitrogen, USA), penicillin-streptomycin (Invitrogen), and 10 ng/ml mouse leukemia inhibitory factor (mLIF, Chemicon, Temecula, USA) to prevent spontaneous differentiation.
Based on the different adhesion rates between MEFs and ES cells to the gelatin-coated plate surface, a simple protocol was designed to separate the feeder MEFs from the mouse embryonic stem cells (mESCs). The resuspended mESCs were cultivated on gelatin-coated or poly-lysine coated plastic petri dishes (Falcon, Becton-Dickinson) at a density of (0.5-1.5)×104 /cm2 in differentiation medium supplemented with 2.5 mmol/L sodium butyrate (Sigma) for 2 days. The differentiation medium was DMEM supplemented with 15% new born calf serum, 2 mmol/L L-glutamine, 1 mmol/L MTG, 1% nonessential amino acids, penicillin-streptomycin. Then the cells were cultured in the N2B27 medium for 15 days. Medium was changed every 2 days. N2B27 is a 1:1 mixture of DMEM/F12 (Sigma) supplemented with N2 (Invitrogen), 50 ng/ml bovine serum albumin fraction V, 10 ng/ml basic fibroblast growth factor (FGF), 2 mmol/L L-glutamine, 1 mmol/L MTG, 1% nonessential amino acids and neurobasal medium supplemented with B27 (Invitrogen), 2 mmol/L L-glutamine, 1 mmol/L MTG and 1% nonessential amino acids. Immunocytochemistry analysis cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and washed with PBS and permeabilized with 0.3% Triton X-100 for 30 minutes. Then samples were incubated with primary antibodies for 1 hour at room temperature. After incubation with second antibodies for 30 minutes at room temperature, samples were examined under a confocal laser-scanning microscope (Carl Zeiss, Inc., USA). The following primary antibodies were used: mouse anti-Nestin (1:250, Chemicon), mouse anti-glial fibrillary acidic protein (GFAP, 1:250, Chemicon), and mouse anti-tubulin beta III (1:250, Chemicon). The secondary antibody was rhodamine-conjugated goat anti-mouse IgG secondary antibody (1:100, Chemicon).
Reverse transcriptional polymerase chain reaction (RT-PCR)
Total RNA was isolated from the differentiating cells at different stages using the TRIzol method (Invitrogen). Complementary DNA (cDNA) was synthesized using the SuperScript III first-strand cDNA synthesis system (Invitrogen) and PCR was followed. The target genes, primer sequences, annealing temperatures, and cycles were listed in the Table.
Cell cycle analysis
Flow cytometry was performed to measure the DNA content and cell cycle distribution of the ES-derived neural progenitor cells. The measurements were made with a Becton Dickinson FACS Calibur machine, adapted for excitation with a 488-nm argon laser, propidium iodide-stained nuclei emit fluorescent light at wavelengths between 564 nm and 606 nm, data analyses were performed using the CellQuest software (MD, USA).
Electrophysiological recordings were performed as discussed previously.9 Electrophysiological studies were initiated by replacing the culture medium with a defined recording solution and in the absence of Mg2+ for 3 hours, the potential of induced neural cells were monitored continuously by using Light-Addressable Potentiometric Senser (LAPS) array system (Biosensor National Special Laboratory, Department of Biomedical Engineering, Zhejiang University, China).
Neural differentiation of mouse ES cells by sodium butyrate
Although we used both gelatin and poly-lysine as differentiation matrix, both of them showed similar results. Therefore, we presented results on gelatin matrix as representative in this paper. With the treatment of sodium butyrate, monolayer mouse ES cells underwent dramatic morphological changes (Figure 1A). After treated with 2.5 mmol/L sodium butyrate (NaB) for 2 days, nearly half of the cells detached and numerous dead cell observed, the number of dead cells was positively correlated with the concentration and treatment time of sodium butyrate, the residual cells changed into triangle or spindle morphologically. After withdrawal of sodium butyrate and continued culture in the N2B27 medium, the triangle and spindle cells stretched out processes of about the equal length as cell body from the ends (Figure 1B), the thin process was filamentous and the thick process was slightly narrower than the width of the cell body. At day 17, these cells displayed typical features of neuron on gelatin-coated plastic dishes: an overlapping network structure formed by a large number of processes.
If the time of treatment with sodium butyrate was less than 24 hours or the cell density was higher than 1.5 × 104/cm2, parts of the processes which extended from the triangle and spindle cells vanished gradually, finally even the triangle and spindle cell reduced gradually.
Identification and characterization of ES-derived neural cells
To confirm the neural identity of the differentiated cells, we demonstrated the expression to neural specific markers from several aspects. RT-PCR showed that a series of neuron marker genes were expressed during the differentiation process (Figure 2). These genes included the Nestin, TH and NeuroD. Nestin, a large intermediate filament protein (class Type VI), is a marker of early stage of neural differentiation during the development. TH and NeuroD are markers of maturation stage of neuron. From day 1 to day 15, expression of these genes continuously increased, while the ES cell marker gene Oct4 gradually faded, correlating well with the morphological transformation of the stem cells to the neuron-like cells.
To determine whether the ES-derived neural cells express typical markers of neural cells at the protein level, we evaluated nestin, tubulin beta III and GFAP by immunocytochemistry analysis. After treated with sodium butyrate for 2 days and N2B27 for 3 days, cells with extended processes were generated and most of them were nestin positive cells. After the 15-day culture in N2B27 after NaB treatment, these cells expressed tubulin beta III, but not GFAP. The result of immunocytochemistry analysis correlated well with the RT-PCR. Collectively, the results showed that the differentiated cells were neural cells.
Cell cycle analysis for ES-derived neural cells
During the 2-day treatment with sodium butyrate, the cells gradually stretched out processes with neural lineage molecular markers, and the cells underwent significant proliferation. As neural cells are mostly postmitotic cells, it is reasonable to postulate that exit from cell cycle is a critical step for neural differentiation. So we analyzed the cell cycle distribution after differentiation initiation by NaB. As shown in Figure 3, the FACS results demonstrated that approximately 38% of the differentiated cells and 43% of undifferentiated cells were in S phase, and the proportion of differentiated cells in G1/G0 phases was higher than undifferentiated cells.
Electrophysiological recordings of the ES-derived neural cells
In order to further confirm that the induced neural cells have the action potential, the induced cells were monitored continuously by LAPS array system. In the absence of any stimulate, the induced neural cells displayed occasional spontaneous action potentials, about 10 times per minute and low in voltage (Figure 4). Exposure to Mg2+ free medium for 3 hours, the cells displayed high frequency of action potential change with voltages ranging from 15 μV to 25 μV. Delorenzo et al9 reported an occurrence of spontaneous, recurrent epileptiform discharges in hippocampal neurons induced by low Mg2+ treatment. The potential changes we observed here coincided with Delorenzo's finding, suggesting that the induced cells have continuous epileptiform activity in low Mg2+ medium.
Our study showed that neural cells can be induced from ES cells by 2-day exposure to sodium butyrate treatment followed with culture in N2B27 medium. We speculated that sodium butyrate and N2B27 medium both promoted the neural differentiation.
Histone deacetylase (HDACs) catalyzes the removal of acetyl groups from histones. Histone deacetylase inhibitors, such as valproicacid, trichostatin A, sodium butyrate, thereby promote histone acetylation, relaxes the DNA, promote transcription of genes involving in important cellular processes.10-12 HDAC inhibitors have been shown to induce growth arrest, terminal differentiation and cell death of a broad variety of transformed cells in vitro and in vivo. Recently, HDAC inhibitors have been reported to induce the differentiation to hepatocyte-like cells from human embryonic stem cells,13 mediate cardiovascular differentiation in mouse embryonic stem cells exposed to laminar shear stress,14 mediate neuronal differentiation of multi-potent adult neural progenitor cells10 and activation genes of early pancreatic development in mouse embryonic stem cells.15 These studies suggested that HDAC inhibitors do not have special histone acetylation effect on particular areas of chromatin, so the type of cell lineage the ES cells adopt depend on the culture system that followed.
Datas from previous studies suggested that mouse ES cells neural differentiation depends on the FGF signaling.4,8 As N2B27 medium contain basic FGF, which had been regarded as capable of enhancing the survival and proliferation but not induction of neural progenitor cells, the minority of ES cells spontaneously differentiate into neural lineage sustain propagation.16 These findings indicate that HDAC inhibitors and basic FGF can actively drive the ES cells differentiate into neurons.
In summary, we have developed a simple but efficient method for direct differentiation of neurons from mouse ES cells without the formation of EBs or coculutre with other types of cells. Our findings suggested that HDAC inhibitors in combination with basic FGF can promote the ES-to-neural conversion.
We thank Dr. WU Rong-rong for her technical help and Mr. GU Bin for his helpful advice.
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