Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation

Abstract

Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

Fig. 1

Human neural progenitor cells (NPC) in culture. a Human NPC maintained under the serum-free culture conditions formed free floating growing spheres. b Human NPC spheres exposed to 10% FBS rapidly attached on the plastic surface, followed by vigorous outgrowth of a sheet of adherent cells from the attachment face. a, b Phase-contrast photomicrographs. c RT-PCR amplified for 32 cycles of nestin (NES, lanes 1 and 2), musashi homolog 1 (MSI1, lanes 3 and 4), neurofilament heavy polypeptide (NFH, lanes 4 and 6), myelin basic protein (MBP, lanes 7 and 8), and glial fibrillary acidic protein (GFAP, lanes 9 and 10) expressed in human NPC under the serum-free (S−) and the 10% FBS-containing (S+) culture conditions

Fig. 2

Nestin, GFAP, and ID1 expression in human NPC in culture. Human NPC spheres attached on poly-L-lysine-coated cover glasses were incubated for 72 h in the NPC medium with (S+) or without (S−) inclusion of 10% FBS, and processed for double-labeling immunocytochemistry for nestin, GFAP, or ID1. a S−, NPC sphere, merge of nestin (green) and GFAP (red), b S+, vigorous outgrowth of adherent cells from the attachment face of the sphere, merge of nestin (green) and GFAP (red), c S+, outgrowth of adherent cells, merge of ID1 (green) and GFAP (red), d S+, outgrowth of adherent cells, nestin (green), e the same field as d, GFAP (red), and f merge of d and e

Fig. 3

Validation of microarray data by real-time RT-PCR and western blot analysis. Human NPC spheres were incubated for 72 h in the NPC medium with (S+) or without (S−) inclusion of 10% FBS, and then total cellular RNA or protein extract was processed for real-time RT-PCR and western blot analysis. a–f Real-time RT-PCR. The levels of target genes were standardized against the levels of the G3PDH gene. a ID1, b ID3, c GFAP, d NPTX1, e DLL1, and f FOS. g, h Western blot. The blots were reprobed with anti-HSP60 antibody to serve HSP60 for an internal control. g GFAP and h ID1

Fig. 4

ID1 and DLL1 expression in various human cell lines. Total RNA of human cell lines, such as NTera2 teratocarcinoma, Y79 retinoblastoma, SK-N-SH neuroblastoma, IMR-32 neuroblastoma, U-373MG astrocytoma, HMO6 microglia, HeLa cervical carcinoma, and HepG2 hepatoblastoma was processed for real-time RT-PCR analysis. The levels of target genes were standardized against the levels of the G3PDH gene. a ID1 and b DLL1

Fig. 5

Molecular network analysis of ID1, ID2, ID3, DLL1, and DLL3. KeyMolnet, a bioinformatics tool for analyzing molecular interaction on the curated knowledge database, identified the shortest route connecting the start point molecules of ID1, ID2, and ID3 (red) and the end point molecules DLL1 and DLL3 (blue). The pathway based on the molecules showed a significant relationship with transcriptional regulation by SMAD or CREB and Notch signaling pathway. The molecular network indicated HASH1 (MASH1), neurogenin 3 (NGN3), and TBX18 as principal regulators of DLL1 expression. The molecular relation is shown by solid line with arrow (direct binding or activation), solid line without arrow (complex formation), and dash line with arrow (transcriptional activation), and dash line with arrow and stop (transcriptional repression). Thick lines indicate the core contents, while thin lines indicate the secondary contents of KeyMolnet

Fig. 6

Activation of the DLL1 promoter by MASH1 was counteracted by ID1. a, b Coimmunoprecipitation analysis. Recombinant MASH1 protein tagged with Flag and ID1 protein tagged with Myc were coexpressed in HEK293 cells. Immunoprecipitation (IP) followed by Western blotting (WB) was performed by using the antibodies against Flag and Myc. The lanes (1–3) represent (1) input control of cell lysate, (2) IP with anti-Flag or anti-Myc antibody, and (3) IP with normal mouse or rabbit IgG. c, d Dual luciferase assay. Two non-overlapping regions of the human DLL1 promoter, consisting of the region #1 spanning −1,253 and −254 or the region #2 spanning −2,946 and −1,786, were cloned into the Firefly luciferase reporter vector. It was co-transfected with the Renilla luciferase reporter vector (an internal control) in HEK293 cells, which were introduced with none (CNT), MASH1, or both MASH1 and ID1 expression vectors at 36 h before transfection of the luciferase reporter vectors. At 16 h after transfection of the luciferase reporter vectors, cell lysate was processed for dual luciferase assay. The ratio of Firefly (F)/Renilla (R) luminescence (RLU) is indicated

Fig. 7

BMP4 upregulates ID1 and GFAP expression in human NPC. Human NPC were incubated for 72 h in the NPC medium with (+) or without (−) inclusion of 50 ng/ml recombinant human BMP4, and then total cellular RNA was processed for real-time RT-PCR analysis. The levels of target genes were standardized against the levels of the G3PDH gene. a ID1 and b GFAP

Fig. 8

The serum-induced astrocyte differentiation of human NPC is characterized by a counteraction between ID and Delta family genes. The present observations raise the possible scenario that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein, probably MASH1-mediated Delta expression in human NPC