Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles

  • Authors:
    • Xuesong Chen
    • Qi Gu
    • Xiang Wang
    • Qingwen Ma
    • Huixiang Tang
    • Xiaoshuang Yan
    • Xinbing Guo
    • Hao Yan
    • Jie Hao
    • Fanyi Zeng
  • View Affiliations

  • Published online on: May 8, 2013
  • Pages: 25-34
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Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.


Neurons and glial cells constitute most of the central and peripheral nervous systems in the vertebrate that regulate and control a wide range of thinking and behaviors; their malfunction can cause serious nervous system diseases. Embryonic stem cells (ESCs) provide significant therapeutic promise as they can be induced to differentiate in vitro into various tissues and organs originated from all three germ layers, including neural cell lineages. Significant issues such as immunorejection, ethical concerns and safety have inhibited the advancement of ESCs toward clinical treatments, while induced pluripotent stem (iPS) cells have become an attractive option for regenerative medicine (1) and personalized medicine. Therapeutic use of iPS cells will require greater understanding and control of the reprogramming process, and demonstrations of safety such as lack of tumorigenesis, through studies that cannot be risked in humans. Mouse iPS cells are therefore a suitable model for studying basic mechanisms of development and differentiation and for evaluating the similarities between ES and iPS cells.

Several directed neuronal differentiation methods have been developed, including via embryoid bodies (EBs) (2,3), monolayer cultures (4), and stromal cell-derived inducing activity (SDIA) (1,5). In our study, we utilized a modified neuronal differentiation method (2) to induce a tetraploid complementation competent iPS cell line and two different ES cell lines to differentiate into neurons. Neurons derived from all three sources exhibited nearly the same differentiation patterns during approximately 20 days of in vitro culture. Derived cell populations are mixtures, and most cells are positive for the neuron-specific marker protein MAP2. Several neural differentiation stage marker genes were expressed by these cell mixtures as well, including Blbp (Fabp7), Nestin and Tuj1. Microarray profiling and single-cell PCR were employed to further analyze the neural lineage differentiation process. Following statistical comparisons and gene ontology analysis, 1,324 differentially expressed genes were identified, some of which are involved in cell morphology, synaptic transmission, neurogenesis and neuron recognition. The genes identified may be useful for investigating important signaling factors and pathways regulating neuronal differentiation.

Materials and methods

Cell culture

The mouse ES cell line CGR8.8 was a gift from Dr Yanru Chen (Stanford University), and the mouse iPSC line IP14D-1 was derived from B6/DBA2F1 fetal fibroblasts and was confirmed to be capable of developing into a complete embryo using the tetraploid complementation assay (6,7). Both R1 and IP14D-1 were cultured on mouse embryonic fibroblasts (MEFs) with mitotic inactivation, while CGR8.8 was cultured under feeder-free conditions using only 0.1% gelatin-coated culture dishes. The complete culture medium utilized for mouse pluripotent stem cells contained high glucose Dulbecco’s modified Eagle’s medium with 15% fetal bovine serum tested for ES, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, 1 mM MEM non-essential amino acids, 0.1 mM β-mercaptoethanol and 103 U/ml leukemia inhibitory factor (LIF) (ESG1107; Millipore). Culture medium was changed daily and cells were split every 2–3 days. E13.5 ICR mice were sterilely dissected and harvested cortices were made into cell suspension. They were cultured using Neurobasal culture medium containing 1% B27 supplement in a 37°C, 5% CO2 incubator ~7–10 days for further usage.

Induction of neural-specific embryoid body formation in conditioned culture medium

R1 and IP14D-1 were passaged on 0.1% gelatin-coated culture dishes prior to EB formation to avoid any effect of MEF cells. EBs of all three pluripotent stem cell clones were digested into single cells and cultured in suspension for the first four days in pluripotent stem cell culture medium without LIF. For further neural-specific induction, EBs were then transferred into neural induction culture medium (NIM) for the following 3 or 4 days. EBs were then transferred into culture plates coated with poly-D-lysine (PDL) and laminin for further adhesion culture with EB culture medium using the following method: coating with a 100 μg/ml PDL solution on clean coverslips in 6-well plates overnight at 37°C, then washing twice with water to remove PDL and adding laminin solution (5 μg/ml in Hank’s media) for a few hours to overnight, and rinsing once with Hank’s media prior to use.

Twelve hours later, EB culture medium was replaced with NIM and cultured for one week. NIM was a condition culture medium which included Neurobasal Medium (21103-049) with L-glutamine, NEAA, N2 (17502-048), B27 (17504-044) (Gibco), bFGF, EGF and all-trans retinoic acid (R2625; Sigma) for another week. bFGF and EGF were removed from NIM for subsequent culturing.

Samples of the original R1, CGR8.8, IP14D-1 lines, derived neuronal cells (R1_Ne, CGR8.8_Ne and IP14D-1_Ne), and primary cultured neurons as positive control were harvested in TRIzol for RNA isolation. Reverse transcription of 0.5 μg total RNA produced cDNA for PCR of Pax6, Sox1, Blbp, Nestin, Tuj1, Ncam, Map2, which test whether the pluripotent stem cells differentiate into neurons. All PCR primers used are listed in Table I.

Table I.

Primers for pluripotent stem cell directed neuronal differentiation.

Table I.

Primers for pluripotent stem cell directed neuronal differentiation.

Forward primerReverse primerSize (bp)CyclesParameters
Pax6 5′-GAAATCCGAGACAGATTATTATCCGAG-3′ 5′-CCATTTGGCCCTTCGATTAGA-3′4953594°C-40 sec; 58°C-30 sec; 72°C-40 sec
Sox1 5′-CCAAGAGACTGCGCGCGCTG-3′ 5′-GGGTGCGCCGGGTGTGCGTG-3′3813594°C-40 sec; 62°C-30 sec; 72°C-40 sec
Blbp 5′-TGAGTACATGAAAGCTCTGGGCGT-3′ 5′-TGAGCTTGTCTCCATCCAACCGAA-3′2243594°C-40 sec; 58°C-30 sec; 72°C-30 sec
Nestin 5′-CTGGAACAGAGATTGGAAGGCCGCT-3′ 5′-GGATCCTGTGTCTTCAGAAAGGCTGTCAC-3′4033594°C-40 sec; 59°C-30 sec; 72°C-40 sec
Tuj1 5′-ATCCACCTTCATTGGCAACAGCAC-3′ 5′-ACTCGGACACCAGGTCATTCATGT-3′1733594°C-40 sec; 58°C-30 sec; 72°C-30 sec
Ncam 5′-TTCCTGTGTCAAGTGGCAGGAGAT-3′ 5′-AGATCTTCACGTTGACAGTGGCCT-3′2293594°C-40 sec; 60°C-30 sec; 72°C-30 sec
Map2 5′-AGCCGCAACGCCAATGGATT-3′ 5′-TTTGTTCCGAGGCTGGCGAT-3′3133594°C-40 sec; 58°C-30 sec; 72°C-40 sec
Gapdh 5′-GCAAATTCAACGGCACAGTC-3′ 5′-TCTTCTGGGTGGCAGTGATG-3′3993594°C-40 sec; 59°C-30 sec; 72°C-40 sec
Immunocytochemistry confirmation

Mouse pluripotent stem cells grown on coverslips were washed with PBS twice and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were washed again with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15 min. The cells were then blocked with 1% BSA, 0.5% Triton X-100 in PBS for 1 h at room temperature. Mouse anti-mouse MAP2 primary antibodies were diluted 1:200 in 1% BSA, 0.5% Triton X-100 in PBS and incubated with fixed cells at 4°C overnight. After triple washing with PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:1,000) secondary antibody for 1 h at room temperature. DAPI counter-staining was used for cell nuclei.

Microarray assays of gene expression

Total RNA was extracted from three replicates of each cell population, including primary cultured neurons, three pluripotent stem cells and corresponding neuron populations. cDNA was hybridized to Affymetrix Mouse Gene 1.0 ST Arrays following reverse transcription, in vitro transcription amplification and quality control using the manufacturer’s standard protocols.

Single cell PCR
Single cell isolation and cDNA synthesis

A micromanipulator was employed to transfer single cells into individual RNase-free EP tubes with 12 μl reverse transcriptase mixture on ice. Reverse transcription reactions were immediately performed to synthesize cDNA. Glycogen, ammonium acetate and cold ethanol were added and stored at −80°C overnight for precipitation.

In vitro transcription and cDNA synthesis

Single-cell cDNA was amplified by in vitro transcription (8,9), and the resulting aRNA was isolated using high-speed low-temperature centrifugal sedimentation. The amplified RNA was converted to single-stranded cDNA for PCR assays of gene expression. PCR conditions were as described above.

Microarray data analysis

We applied the RMA algorithm in Affymetrix Expression Console with default parameters to normalize and summarize probe signals. We utilized the online NIA Array Analysis Tool ( for hierarchical clustering and statistical testing (6,10,11). For identification of significantly different gene expression levels between pluripotent cell populations and differentiated cells, we set cutoff thresholds at 5% false discovery rate (FDR) and 2-fold magnitude of difference and conducted pairwise comparisons using ANOVA with multiple testing correction (6,10,11). Finally, the DAVID online database and tools ( were employed to annotate the differentially expressed genes by Gene Ontology categories, and affected pathways were examined using Ingenuity Pathway Analysis (


Pluripotent stem cell lines remain viable with shared culture conditions

The ES cell line R1 was established in 1991 from a blastocyst produced by crossing two 129 substrains (129S1/SvImJ and 129X1/SvJ) (12). R1 cells were cultured on inactivated MEF cells as recommended by the ATCC. The CGR8.8 mouse ES cell line was derived from the 129/Ola mouse strain. The induced pluripotent stem cell line IP14D-1 was induced from C57/DBA2F1 MEFs and can produce embryos by tetraploid complementation as previously reported (6). All three pluripotent stem cell lines were cultured using the same conditions before testing the differentiation procedure. Each showed classic stem cell characteristics such as colony growth with rapid proliferation, smooth edges and strong refraction. There were no obvious cell boundaries within stem cell colonies (Fig. 1A–C). Passages 20, 21 and 28 of R1, CGR8.8 and IP14D-1, respectively, were employed for neuronal differentiation.

Conditioned NIM is beneficial for neural induction from EBs

EB formation is the first stage of differentiation and allows pluripotent stem cells to be primed for further lineage specific development. We adapted a standard neural differentiation method with slight modifications to produce neurons via EB formation. EBs formed after 4 days of suspension culture and then 0.5 μM ATRA was added into NIM for another 4 days of suspension culture (Fig. 1D–F). We used a modified ‘4+4’ induction method developed by Bain et al (2). In the second 4 days of EB suspension culture, we replaced EB medium with Neurobasal containing some supplements, growth factors and RA without serum, which was more suitable for promoting neuronal differentiation as serum may have a negative effect on neural induction via RA (13).

Pluripotent stem cells are induced into MAP2 positive neurons

Following suspension culture induction, EBs were transferred into PDL and laminin co-coated plates and cultured with EB culture medium. After 12 h of adhesion, numerous EB cells proliferated and extended from EBs. A week later, cells were cultured in serum-free culture conditioned medium (NIM) for neuronal differentiation. Total EB extension occurred after 2 weeks, and cells showed neuron-like morphology (Fig. 2A).

Neurons derived from each of the three pluripotent stem cell lines were harvested to test for neuronal gene expression patterns. Several genes specifically expressed in neurons and during neural development processes were also expressed by the cell populations differentiated from all three pluripotent cell lines, such as Blbp (Fabp7), Nestin, Tuj1 and Map2 (Fig. 2B). MAP2 protein is a marker for mature neurons, and the majority of cells (~75%) from all three populations are positive for MAP2 antigen expression (Fig. 2C).

Global gene expression profiles of cells derived from mouse ESCs and iPSCs are similar

Microarrays were employed to analyze directed neural differentiation of ESCs and iPSCs at a global RNA expression level (Fig. 3A). Gene expression profiles of induced neurons from ES and iPS cells are nearly identical, particularly between derivatives of the two ES cell populations. However, there are some differences between induced neurons from ESCs and iPSCs, despite the confirmation that the iPS cell line IP14D-1 has a developmental capacity equivalent to ES cells. Moreover, the expression profiles of all these induced neurons are to some extent not consistent with primary cultured neurons. Hierarchical clustering of expression patterns grouped the three induced neuron populations as more related to each other than to primary cultured neurons, but all neurons were distinct from the precursor pluripotent stem cells (Fig. 3B).

Validation of microarray data

We selected differentially expressed candidates from the microarray data for genes related to neurogenesis and pluripotency. Ncam (neural cell adhesion molecule), En1 and Lix1l were selected as neuron marker genes, and Nestin, Bcl2, Nav3, Ntf3 and Zfp9 were selected as genes related to neural differentiation. Each of the neurogenesis-related genes was upregulated during direct neural differentiation as assayed by microarrays and quantitative PCR. RNA expression differences for the neuron marker genes were also confirmed. Additionally, the three well-known pluripotency genes Oct4, Nanog and Klf4 were downregulated during the differentiation process (Fig. 4A).

We also used single cell PCR to validate gene expression levels in individual neurons derived both from R1 and IP14D-1. We randomly chose five genes from the microarray candidate lists for each cell population, as well as selected genes with known relevance in pluripotency or neural differentiation. Expression of genes involved in pluripotency maintenance, such as Fgf4 and Lin28, decreased during neural differentiation. Genes involved in neural lineage development, including Ndn and Nr6a1, were confirmed to be upregulated (Fig. 4B).

Differential expression between ES/iPS and derived neurons

At an FDR <5% and a difference threshold greater than 2-fold, almost 5,000 differentially expressed genes were identified by pairwise comparisons between pluripotent stem cells and their corresponding induced neurons. There are 1,126 differentially expressed genes common to all three pairwise comparisons (Table II), including 824 upregulated genes and 302 downregulated genes during neural lineage development.

Table II.

Number of differentially expressed genes detected after induced neural differentiation.

Table II.

Number of differentially expressed genes detected after induced neural differentiation.

Pairwise comparisonsUpregulated genes (pluripotent > neurons)Downregulated genes (pluripotent < neurons)
IP14D-1 vs. IP14D-1_neuron2,2782,103
IP14D-1 vs. primary neuron4,5743,684
Shared set1,634613
R1 vs. R1_neuron2,6842,707
R1 vs. primary neuron4,0333,778
Shared set1,829962
CGR8.8 vs. CGR8.8_neuron2,5992,378
CGR8.8 vs. primary neuron4,0993,680
Shared set1,791934
Common among 3 inductions824302
Fold-change >2, FDR <0.05

[i] FDR, false discovery rate.

Annotation analysis of regulated genes in ES/iPS cell differentiation

We investigated functional categories within sets of differentially expressed genes using Ingenuity Pathway Analysis tools. Among the 1,126 differentially expressed genes, nearly 700 can be classified into annotated categories that include transcription regulators, growth factors and ion channel formation (Table III). We also tested for statistically significant overrepresentation of Gene Ontology (GO) categories, and classified 302 upregulated genes into biological processes involved in synaptic transmission, regulation of membrane potential, axonogenesis, and neuron recognition, as well as basic functions such as transcription, metabolism and biosynthesis (Table IV). We ranked the GO categories according to overrepresentation P-value, and with the exception of some basic biological processes, the most important categories involved in neural development are focused on neuron projection morphogenesis and axonogenesis.

Table III.

Molecular function categories for differentially expressed genes.

Table III.

Molecular function categories for differentially expressed genes.

CategoryNo. of mapped genes overexpressed in pluripotent cell vs. neuronsNo. of mapped genes underexpressed in pluripotent cell vs. neuronsSum
Transcription regulator672693
Growth factor8311
Transmembrane receptor549
Ion channel178
Ligand-dependent nuclear receptor347
Translation regulator505
G-protein coupled receptor134

Table IV.

Gene ontology analysis of upregulated genes.

Table IV.

Gene ontology analysis of upregulated genes.

CategoryBiological processCountP-valueFold enrichment
Transcription relatedPositive regulation of transcription, DNA-dependent192.8E-053.2
Positive regulation of transcription from RNA polymerase II promoter175.6E-053.3
Regulation of transcription from RNA polymerase II promoter221.9E-042.5
Regulation of transcription, DNA-dependent351.8E-031.7
Regulation of transcription482.0E-031.5
Metabolism relatedPositive regulation of macromolecule metabolic process279.8E-073.0
Positive regulation of RNA metabolic process193.1E-053.2
Phosphorus metabolic process251.3E-032.0
Regulation of RNA metabolic process352.3E-031.7
Regulation of cellular protein metabolic process94.4E-022.3
Biosynthesis relatedPositive regulation of macromolecule biosynthetic process222.1E-052.9
Positive regulation of cellular biosynthetic process223.8E-052.8
Positive regulation of biosynthetic process224.2E-052.8
Cell adhesion relatedCell adhesion224.9E-052.7
Homophilic cell adhesion81.5E-034.7
Cell-cell adhesion112.0E-033.3
Regulation of cell-matrix adhesion31.7E-0214.9
Phosphorylation relatedRegulation of phosphorylation142.5E-043.4
Protein amino acid phosphorylation223.1E-042.4
Regulation of protein amino acid phosphorylation77.0E-034.1
Ion homeostasis relatedIon homeostasis139.2E-043.1
Cellular calcium ion homeostasis69.9E-034.6
Homeostatic process178.4E-032.1
Cellular ion homeostasis113.9E-033.0
Cellular chemical homeostasis114.7E-032.9
Cation homeostasis81.5E-023.1
Di-, tri-valent inorganic cation homeostasis71.6E-023.4
Regulation of metal ion transport43.1E-025.8
Regulation of sodium ion transport31.9E-0213.9
Cell morphogenesis relatedNeuron projection morphogenesis137.7E-065.2
Cell morphogenesis163.5E-053.6
Cell morphogenesis involved in neuron differentiation125.7E-054.6
Regulation of neuron projection development43.0E-025.9
Cell motion relatedCell motion201.1E-063.8
Cell migration144.0E-054.1
Axon guidance61.3E-024.2
Neural crest cell migration34.2E-029.1
Synaptic transmission relatedRegulation of membrane potential77.3E-034.1
Synaptic transmission81.4E-023.2
Transmission of nerve impulse91.6E-022.8
Regulation of postsynaptic membrane potential34.9E-028.3
Neural development relatedAxonogenesis122.0E-055.2
Neuron recognition45.8E-0423.1
Regulation of neurogenesis95.9E-044.8
Neuron development139.2E-043.1
Regulation of nervous system development91.3E-034.2
Axonal fasciculation34.1E-0329.7
Neural crest cell development41.2E-028.4
Negative regulation of neurogenesis41.6E-027.5
Regulation of axonogenesis41.8E-027.1
Negative regulation of axonogenesis32.1E-0213.0


During the last decade, mouse and human ES cells have been induced to differentiate into several cell types to study developmental potential in vitro and to develop valuable therapeutics (1,14,15). Diseases of the nervous system have a serious impact on human health, therefore, a number of methods have been adapted to induce ESCs into neurons with the aim of curing disease (1,16). We used a classic induction method with slight modifications to cause mouse ES and iPS cells to differentiate into neurons. RA, an efficient induction factor for neural development, was added to the EB culture medium to promote EBs to differentiate into the neural lineage as suggested by previous in vitro research (17,18). With a low RA concentration in culture, pluripotent cells, both ES and EC cells, could differentiate into neurons (19). RA is particularly critical for GABAergic neuron differentiation of ESCs (3,20). To maintain cellular proliferation and promote expansion from EBs, we pretreated cell culture dishes with PDL and laminin at least 2 h (or, optimally, overnight) at 37°C. Both substances promote efficient cell adhesion and stretching (21). We added growth factors into the neural induction medium to increase cell proliferation and directed differentiation (22,23). These factors were previously shown to protect induced neurons by promoting resistance to apoptosis and necrosis (24), perhaps via regulation of genes such as Bcl-2 expressed during the neural directed differentiation process (25).

By comparing RNA expression levels among induced neurons from pluripotent stem cells and to primary cultured neurons isolated from ICR E13.5 mice, we found ES and iPS cells follow almost the same neural differentiation process, and the derived neurons have marker gene expression patterns identical to primary cultured neurons.

This level of similarity extended to genomic expression patterns from microarray assays. Neurons derived from all three types of pluripotent cells had mostly overlapping gene expression by cluster analysis, and the differences between these patterns and that of primary cultured neurons is likely due to a heterogeneous mixture of cell types in the induced populations. The number of differentiated cells never exceeded 75%, so samples taken from induced cell populations contained a mixture of neuron and non-neuron RNA.

As an independent validation of the microarray results, we used quantitative RT-PCR of several well-known pluripotency and neuron-relevant genes. As expected, pluripotency genes were downregulated and neural genes were upregulated during differentiation. We also employed single cell PCR in the validation tests, which has become a relatively accurate technology for assessing gene expression (2628). Primary cultured neurons served as positive control. Genes selected for the PCR assays showed expression patterns consistent with the microarray results. After confirming the concordance of selected microarray and qPCR assays, we conducted a statistical analysis on the full microarray data set to test for gene expression differences between ES/iPS cells and their derived neurons. We identified 824 genes highly expressed in pluripotent cells and 302 genes highly expressed in both induced neurons and primary cultured neurons. The resulting lists of candidate genes were used for pathway and gene ontology analyses that highlighted several biological processes apparently operating during induced neural differentiation. The cadherin super-family contains calcium-dependent cell adhesion molecules including protocadherins (Pcdh) that are important regulators of mouse and human nervous system development (2932). The Pcdh family is involved in maintenance of spinal interneurons, axon convergence and synaptic development, particularly connectivity between neuronal cells (29). In the present study, we detected increased expression of several members of this family including Cdh2, Cdh10, Pcdh7, Pcdh16, Pcdh18 and Pcdh22. The solute carrier gene family (Slc) encodes another group of key proteins in neural development for maintaining neuron functions such as ion transport, glutamate transport, and neurotransmitter symporters. Genetic variants of Slc9a9 affect the development of attention-deficit/hyperactivity disorder, a common behavioral disorder with over-activity and inattentiveness (3335). This gene, therefore, may play a part in nervous system development, and we observed more than 2-fold upregulation during neuron differentiation.

We also detected differential expression of Sema family genes including 3a, 3d, 4c and 6d which have roles in nervous system development (36). The Sema3a and Npn1 genes were upregulated during the directed neuronal differentiation process. Both genes are involved in a neural development regulatory pathway for motor and sensory axon outgrowth (37). microRNA let-7 is known to promote neural lineage differentiation (38,39) and to suppress expression of several pluripotency genes (40). The precursor transcript for let-7 was upregulated 20-fold on average during induced neural differentiation.

Our analysis of in vitro directed neuronal differentiation indicates that iPSCs follow almost the same differentiation process as mouse ESCs. Neurons induced from iPSCs and ESCs have similar morphology, neuron marker expression, and global gene expression patterns. Several genes related to known neuronal differentiation processes showed statistically significant changes in expression, and these patterns may be useful for optimizing induction methods, improving the efficiency of neural differentiation from pluripotent stem cells, and understanding neuronal differentiation mechanisms underlying nervous system development.


This study was supported by grants from the National Natural Science Foundation of China (81125003, 30900259), the Hi-Tech Research and Development Program of China (2011AA020116), the China National Basic Research Program (2010CB945200), the Science and Technology Committee of Shanghai Municipality (10140900200), and the Shanghai Leading Academic Discipline Project (S30201) to FZ.



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July 2013
Volume 32 Issue 1

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Chen, X., Gu, Q., Wang, X., Ma, Q., Tang, H., Yan, X. ... Zeng, F. (2013). Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles. International Journal of Molecular Medicine, 32, 25-34.
Chen, X., Gu, Q., Wang, X., Ma, Q., Tang, H., Yan, X., Guo, X., Yan, H., Hao, J., Zeng, F."Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles". International Journal of Molecular Medicine 32.1 (2013): 25-34.
Chen, X., Gu, Q., Wang, X., Ma, Q., Tang, H., Yan, X., Guo, X., Yan, H., Hao, J., Zeng, F."Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles". International Journal of Molecular Medicine 32, no. 1 (2013): 25-34.