Generation of neural progenitor cells by chemical cocktails and hypoxia

Abstract

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors.

Figure 1

Induction of ciNPCs from MEFs by a chemical cocktail VCR under physiological hypoxic condition. (A) Induction of AP-positive compact cell colonies by VCR under hypoxia. MEFs were cultured under 21% (normoxia) or 5% (hypoxia) O₂ in the presence of VCR (0.5 mM VPA, 3 μM CHIR99021 and 1 μM Repsox). Representative pictures of compact cell colonies and colonies stained with alkaline phosphatase (AP) were shown. Colonies were counted on day 15 following VCR treatment. Bars represent the number of colonies per 200 000 cells initially plated. (B) Relative expression levels of pluripotency-related genes were measured by quantitative RT-PCR and shown. All sample data are normalized to that of day 0, which is considered as 1. (C) Induction of NPC-like cells from VCR-treated MEFs. VCR-treated MEFs were cultured in neural expansion medium. Representative images of MEFs and ciNPCs at passages 1, 5 and 13 were shown as indicated. (D) Relative expression levels of NPC-specific genes were monitored by qRT-PCR and shown. All sample data are normalized to that of MEFs, which is considered as 1. (E) Expression of Nestin, Pax6 and Sox2 was visualized by immunostaining. Nuclei were counterstained with DAPI. Representative images were shown. Nestin/Pax6 or Nestin/Sox2 double positive cells were counted and shown in right panel. (F) Scheme of the experimental strategy for induction of ciNPCs from mouse fibroblasts. Generally, initial fibroblasts were plated in DMEM and this day is termed as “Day −2”. 2 days later, cells were transferred into KSR medium with chemical compounds and cultured under 5% O₂. Compact cell colonies formed in about 10 days. Cell mixtures containing colonies were then further cultured in neural expansion medium with growth factors. ciNPCs formed around day 20 were further enriched by rounds of neurosphere suspension culture. Scale bars represent 50 μm. Data are represented as the mean ± SEM. At least three independent experiments were performed. ^***P < 0.001, ^**P < 0.01.

Figure 2

Proliferation and self-renewal of ciNPCs. (A) Representative images of ciNPCs at passage 13 stained for Ki67 and Nestin. (B) Neurospheres of ciNPCs at passage 13 versus those of NPCs at passage 5 cultured in suspension. (C) Nestin, Pax6 and Sox2 expression of ciNPCs at passage 23 in monolayer. (D) Nestin, Pax6 and Sox2 expression of ciNPC neurospheres at passage 25. Nuclei were counterstained with DAPI. Scale bars represent 50 μm. Representative images from at least three independent experiments were shown.

Figure 3

Genome-wide transcriptional profiling of ciNPCs. (A) Heatmap and hierarchical clustering of genes with significance from microarray analysis data. Samples of MEFs, NPCs, ciNPCs at passages 5 and 13 were compared. In heatmap, red indicates increased expression whereas green stands for decreased expression as compared to that in the MEF control sample. (B) Pairwise scatter plot analysis of 251 genes selected by the term “neuro” in the microarray data of ciNPCs p5 and p13 versus that of MEFs and NPCs. Red dots represent upregulated gene expression, whereas green dots represent downregulated ones, and the grey dots stand for no significant difference. (C) A Venn diagram illustrating the overlaps among the expression changes (≥ 10-fold, P < 0.05) identified in ciNPC p5, p13-1, p13-2 and control NPCs compared with that of MEFs. (D) Gene ontology (GO) analysis of the 774 overlapping genes in C. The P values represent the EASE scores.

Figure 4

Multipotency of ciNPCs in vitro and in vivo. (A) Expression of marker genes GFAP (astrocyte), Tuj1 (neuron), MAP2 (neuron), Olig2 (oligodendrocyte) and Mbp (oligodendrocyte) in ciNPC p13 cultured in differentiation medium. (B) ciNPC p13 differentiation using the neuronal differentiation medium for 4 weeks were stained with neuronal markers Tuj1, NeuN, synapsin, and glutamate, a marker for glutamatergic neuron. Nuclei were counterstained with DAPI. Representative images from at least three independent experiments were shown (A, B). (C) Current-clamp recordings of neurons derived from ciNPCs showing a representative train of action potentials with stepwise current injection. (D) Representative traces of spontaneous postsynaptic currents in ciNPC-derived neurons. (E) Voltage-clamp recordings of neurons derived from ciNPCs revealing functional voltage-dependent Na⁺ channels. Data are representatives of at least three independent experiments (C-E). (F) Olig2⁺ oligodendrocytes, GFAP⁺ astrocytes, and NeuN⁺ neurons derived from GFP-ciNPCs in vivo 1 month after transplantation. GFP-ciNPCs were transplanted to brains of E13.5 pups. One month after transplantation, the brain sections at the implantation sites were collected and subjected to immunostaining. 24 pups were transplanted with GFP-ciNPCs. Representative images were shown. Arrows indicate GFP⁺ cells expressing Olig2, GFAP or NeuN. Nuclei were counterstained with DAPI. Scale bars represent 50 μm in A and B, 15 μm in F.

Figure 5

Induction of ciNPCs by alternative chemical cocktails. (A) qRT-PCR analysis of Sox2 expression in MEFs treated with NLS (0.5 mM NaB, 1 mM LiCl and 1 μM SB431542) (left) or TLT (10 nM TSA, 0.3 mM Li₂CO₃ and 30 μM Tranilast) (right) under 5% O₂. All sample data are normalized to that of cells treated with DMSO at d0, which is considered as 1. (B) Morphology and expression of Nestin, Sox2 and Pax6 in ciNPC at passage 13 induced by NLS or TLT. Nuclei were counterstained with DAPI. Scale bars represent 50 μm. Representative images from at least three independent experiments were shown. (C) Relative expression levels of NPC-specific genes were monitored by qRT-PCR and shown. All sample data are normalized to that of MEFs, which is considered as 1. Data are represented as the mean ± SEM.

Figure 6

Generation and characterization of ciNPCs from mouse TTFs and human urinary cells. (A) Expression levels of pluripotent genes in TTFs treated with VCR under hypoxia were monitored by qRT-PCR analysis. All sample data are normalized to that of d0, which is considered as 1. (B) Morphology of TTFs and ciNPCs at passage 1 induced from TTFs. (C) Morphology and expression of Nestin, Sox2 and Pax6 ciNPC at passage 16 induced from TTFs. (D) qRT-PCR analysis of NPC-specific genes of TTF-derived ciNPCs at passage 16. All sample data are normalized to that of TTFs, which is considered as 1. (E) Expression of marker genes GFAP (astrocyte), Tuj1 (neuron), MAP2 (neuron), Olig2 (oligodendrocyte) and Mbp (oligodendrocyte) in TTF-derived ciNPCs p16 cultured in differentiation medium. (F) Phase-contrast images of human urinary cells (hUCs) before and after VCR treatment under 5% O₂. (G) Expession of pluripotent genes in hUCs treated with VCR under 5% O₂ for different days (d) was monitored by qRT-PCR analysis. All sample data are normalized to that of d0, which is considered as 1. (H) Morphology of hUC-derived ciNPCs at passage 5 cultured in monolayer or in suspension and control iNPCs cultured in monolayer. iNPCs were induced from human urinary cells by defined factors. (I) Expression of NPC-specific genes quantified by qRT-PCR analysis. All sample data are normalized to that of hUCs, which is considered as 1. (J) Expression of marker genes GFAP (astrocyte), Tuj1 (neuron) and MAP2 (neuron) in hUC-derived ciNPCs p5 cultured in differentiation medium. Nuclei were counterstained with DAPI. Scale bars represent 50 μm. Data are represented as the mean ± SEM. Representative images from at least three independent experiments were shown.