Differential Gene Expression in Oligodendrocyte Progenitor Cells, Oligodendrocytes and Type II Astrocytes

Tohoku J. Exp. Med., 2011,Differential 223, 161-176 Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 161

Jian-Guo Hu,1,2,* Yan-Xia Wang,3,* Jian-Sheng Zhou,2 Chang-Jie Chen,4 Feng-Chao Wang,1 Xing-Wu Li1 and He-Zuo Lü1,2

1Department of Clinical Laboratory Science, The First Affiliated Hospital of Bengbu Medical College, Bengbu, P.R. China 2Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu, P.R. China 3Department of Neurobiology, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China 4Department of Laboratory Medicine, Bengbu Medical College, Bengbu, P.R. China

Oligodendrocyte precursor cells (OPCs) are bipotential progenitor cells that can differentiate into myelin-forming oligodendrocytes or functionally undetermined type II astrocytes. Transplantation of OPCs is an attractive therapy for demyelinating diseases. However, due to their bipotential differentiation potential, the majority of OPCs differentiate into astrocytes at transplanted sites. It is therefore important to understand the molecular mechanisms that regulate the transition from OPCs to oligodendrocytes or astrocytes. In this study, we isolated OPCs from the spinal cords of rat embryos (16 days old) and induced them to differentiate into oligodendrocytes or type II astrocytes in the absence or presence of 10% fetal bovine serum, respectively. RNAs were extracted from each cell population and hybridized to GeneChip with 28,700 rat genes. Using the criterion of fold change > 4 in the expression level, we identified 83 genes that were up-regulated and 89 genes that were down-regulated in oligodendrocytes, and 92 genes that were up-regulated and 86 that were down-regulated in type II astrocytes compared with OPCs. The up-regulated genes, such as activating transcription factor 3 and myelin basic protein in oligodendrocytes or claudin 11 in type II astrocytes, might contribute to OPC differentiation and represent constitutive components of oligodendrocytes or type II astrocytes. The down-regulated genes in both oligodendrocytes and type II astrocytes, such as transcription factor 19, might be involved in maintaining self-renewal and/or represent the property of OPCs. These results provide new insights into the elucidation of the molecular mechanisms, by which OPCs differentiate to oligodendrocytes or type II astrocytes.

Oligodendrocytes (OLs) produce myelin in the central taining medium when cultured in vitro (Raff et al. 1983; nervous system (CNS). They wrap axons of neurons to Louis et al. 1992). Type II astrocytes displayed a typical produce myelin sheaths, provide trophic support and pro- process-bearing morphology and express both A2B5 and tection for neurons and their axons. The persistent defi- glial fibrillary acidic protein (GFAP) and may contribute to ciency in myelin results in the disorders of the CNS, such the structure of nodes of Ranvier (Ffrench-Constant and as multiple sclerosis and spinal cord injury. Oligodendro Raff 1986). OPC transplantation into the area of demyelin- cytes (OLs) are generated from oligodendrocyte precursor ation may be a feasible approach to repairing CNS disor- cells (OPCs), which arise at multiple locations in the devel- ders (Chari and Blakemore 2002; Keirstead et al. 2005). oping brain and spinal cord and migrate throughout the However, due to their bipotential differentiation potential, CNS (Qi et al. 2002; Kessaris et al. 2006). OPCs express when transplanted into lesion site, the majority of OPCs specific markers, such as a cell-surface ganglioside (A2B5) differentiate into astrocytes (Lu et al. 2010). Therefore, it is that is recognized by A2B5 monoclonal antibody (Raff et an important issue to control the implanted OPCs to differ- al. 1983) and platelet-derived growth factor receptor α entiate into OLs, but not astrocytes. For this purpose, the (PDGFRα), and they are bipotential in vitro (Raff et al. molecular mechanisms that control OPC differentiation 1983; Louis et al. 1992). OPCs can differentiate into OLs should be well understood. in serum-free medium or type II astrocytes in serum-con- Microarray technology provides a powerful tool for

Received December 30, 2010; revision accepted for publication January 29, 2011. doi: 10.1620/tjem.223.161 *These authors contributed equally. Correspondence: He-Zuo Lü, Ph.D., Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, 287 Chang Huai Road, Bengbu 233004, P.R. China. e-mail: [email protected]

161 162 J.G. Hu et al. large-scale gene expression studies (Hu et al. 2004). In the Chemicon), the markers for OLs, or both A2B5 and glial fibrillary present study, we cultured OPCs isolated from the spinal acidic protein (GFAP; 1:200; Sigma, St. Louis, MO, USA), the cords of embryonic rats at 16 days of gestation and induced marker for type II astrocytes overnight at 4°C. On the second day, them into OLs or type II astrocytes under different culture the cultures were incubated with rhodamine- or fluorescein isothiocy- conditions, respectively. We then employed a cDNA micro- anate (FITC)-conjugated secondary antibody (all from Jackson array to detect differential gene expression in three cell ImmunoResearch Lab, West Grove, PA, USA) for 1 hour at 37°C. After staining, the coverslips were rinsed and mounted with Gel/ types. Mount aqueous mounting media (Biomeda Corp., Foster City, CA, Materials and Methods USA) containing Hoechst 33342, a fluorescent nuclear dye (1 μg/ml; Sigma). The coverslips were examined using an Olympus BX60 Cell Culture microscope. For cell counts, at least five randomly selected fields OPCs were immunopanned from embryonic day 16 (E16) with a total of more than 500 cells were counted. In all experiments, Wistar rat spinal cords using an A2B5 antibody and a protocol modi- primary antibody omission controls were used to confirm the specific- fied from previous studies (Mayer-Proschel et al. 1997; Mujtaba et al. ity of the immunofluorescence labeling. 1999; Cao et al. 2005). Immunopanned OPCs were plated onto Poly- D-lysine (PDL)/fibronectin-coated 10 × 12 cm culture dishes and growth medium was added and changed every other day. The growth RNA Isolation and Hybridization to Affymetrix GeneChips medium contained DMEM/Ham’s F12 (Invitrogen, Carlsbad, CA, OPCs, differentiated OLs and type II astrocytes (5 days) derived USA), 1 × N2 and 1 × B27 supplements (Invitrogen), fibroblast from three rats were used in cDNA microarray detection. Purified growth factor 2 (FGF-2) (20 ng/ml; Sigma, St. Louis, MO), and cells were rinsed twice with PBS and total RNA was extracted using platelet-derived growth factor aa (PDGF-aa) (10 ng/ml; Chemicon, SV total RNA isolation system (Promega, Madison, USA) according Temecula, CA). After 5~7 days, the cells were passaged. In all cases, to the manufacturer’s instruction. RNA was treated with DNase I an aliquot of cells was analyzed the following day to determine the (Promega) to eliminate genomic DNA contamination. The integrity efficiency of immunopanning. Only those preparations in which and purification of RNA samples were monitored by formaldehyde/ >95% of the bound cells expressed A2B5 and at passage 2 (P2) were agarose gel electrophoresis. The concentration of RNA was deter- used. OLs and type II astrocytes were induced from the OPCs in the mined by repeated OD measurements of aliquots at wavelength of presence of thyroid hormone [tri-iodothyronine (T3)] or serum after 260 nm. withdrawal of bFGF and PDGF-AA according to the method The microarray hybridization analysis was performed by described previously (Fu et al. 2007; Lu et al. 2008). Briefly, for OL Biochip Co., Ltd. (Shanghai, China) according to the manufacturer’s differentiation, PDGF and bFGF were withdrawn from the OPC- protocol (Affymetrix, Santa Clara, CA, USA). Biotinylated cRNA TM medium, T3 was added (30 μM). The OPCs were allowed to differ- samples were prepared according to the standard Enzo Bioarray entiate for 5 days. Antibodies against O1 (a marker of pre-myelinat- protocol (Enzo Life Sciences, Farmingdale, NY, USA). In brief, total ing OLs), receptor interaction protein (Rip) and myelin basic protein RNA (1 μg) from each sample was converted to double-stranded TM (MBP) were used to identify OLs. For type 2-astrocytes differentia- cDNA with the Bioarray Single-Round RNA Amplification and tion, OPCs were cultured in basal-OPC-medium supplemented with Labeling Kit (Enzo Life Sciences). After second-strand synthesis, the 10% fetal bovine serum (FBS) for 5 days. Antibodies against A2B5 cDNA was purified with the cDNA Purification Kit (Enzo Life and GFAP were used to identify type 2 astrocytes. Sciences). The resulting double-stranded DNA was then used to gen- All embryonic rats were obtained from pregnant Wistar rats erate multiple copies of biotinylated cRNA by in vitro transcription bred in the Animal Care Facility at Bengbu Medical College, Bengbu, with the Bioarray HighYield RNA Transcript Labeling Kit (Enzo Life P.R. China. All animal care was carried out in accordance with the Sciences). For each sample, biotinylated cRNA (10 μg) spiked with National Institute of Health Guide for the Care and Use of Laboratory bioB, bioC, bioD, and cre (Hybridization Control) was hybridized to Animals (NIH Publications No. 80-23; revised 1996) and was an Affymetrix Rat 230 2.0 Microarray for 16 h at 45°C. Following approved by the Animal Care Committee of the Use of Laboratory hybridization, all arrays were washed and stained in an Affymetrix Animals, Bengbu Medical College. Gene-Chip Fluidics Station. Stained arrays were scanned with an Affymetrix GeneChip1 Scanner 3000. Quality checks and data analy- Immunocytochemistry ses were carried out using Affymetrix GeneChip Operating Software For identification of cell purity, in some experiment, OPCs were and Expression Console. All microarray data reported in the manu- plated onto PDL/fibronectin-coated coverslips in 35-mm dishes at a script are described in accordance with MIAME guidelines. density of 5 × 104 cells/coverslip (12 mm). OPCs were cultured, and The processed image file of the Affymetrix Rat 230 2.0 array OLs and type II astrocytes were induced from OPCs as described contains over 31,000 probe sets representing ~28,700 well-substanti- above. The OPCs, OLs and astrocytes cultured on coverslips were ated rat genes. The probe set level data were analyzed with either rinsed in phosphate-buffered saline (PBS) and fixed with 4% parafor- Rosetta Resolver Gene Expression Data Analysis System (Rosetta, maldehyde (PFA) in PBS for 20 min at room temperature (RT). After Seattle, WA, USA) or Partek Genomics Suite, version 6.4, build three rinses in PBS (10 min each), the cells were incubated with 10% 6.09.0129 (Partek, St. Louis, MO, USA). The fold change was pre- normal goat serum (NGS) in PBS in the presence (for intracellular sented as mean ± S.E.M of the ratio value (n = 3). We used a combi- antigens) or absence (for surface markers) of 0.3% Triton X-100 for 1 nation of 4-fold change in signal intensity and p < 0.05 as criteria to hour at RT and then with one of the monoclonal primary antibodies consider whether the differential expression of a gene was significant. against A2B5, NG2 and PDGFRα (1:100; all from Chemicon, Temecula, CA, USA), the markers for OPCs, O4 (a specific antigen Real-time RT-PCR on the surface of young OLs), O1, Rip and MBP (1:100; all from cDNA from 5 μg of total RNA was synthesized using Avian Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 163 myeloblastosis virus (AMV) reverse transcriptase (Takara, Japan). GFAP and βIII-tubulin. OPCs displayed typical bipolar or Each single-stranded cDNA was diluted for subsequent PCR amplifi- tripolar morphology (Fig. 1A) and expressed the markers cation by monitoring glyceraldehyde-3-phosphate dehydrogenase for OPCs including PDGFR (Fig. 1B, E; 95.36 ± 4.59%), (GAPDH) as a quantitative control. Real-time RT-PCR was carried NG2 (Fig. 1C, E; 94.63 ± 3.37%) and A2B5 (Fig. 1D, E; out using by SYBR Premix Ex Taq (Perfect real-time) kit (Takara) in 96.35 ± 3.95%). In addition, a small percentage (4.86 ± the Rotor Gene 3,000 system (Corbet Research, Sydney, Australia). 0.89%) of O4 expressing cells and a smaller percentage of Results were analyzed by Rotor Gene 5.0 software. The expression GFAP (only 2.81 ± 1.4%) expressing cells were detected levels of target genes were standardized against those of GAPDH (Fig.1E). These data suggest that OPCs obtained were gene detected in parallel in identical cDNA samples. All the assays were performed in triplicate. highly purified. To obtain the differentiated OLs and type II astrocytes, parallel OPC cultures were maintained in T3- or 10% FBS-containing basal-OPC-medium (without Results PDGF-AA and bFGF), repectively. In the presence of T3, Identification of OPCs, OLs and type II astrocytes OPCs readily differentiated into O1 (Fig. 2A, D; 78.36 ± OPCs isolated and cultured for this study were identi- 4.37%), Rip (Fig. 2B, D; 95.36 ± 2.37%) and MBP-positive fied by morphological and immunocytochemical analyses cells (Fig. 2C, D; 79.28 ± 5.48%). The majority of them using following antibodies: NG2, A2B5, PDGFRa, O4, Rip, displayed typical highly branched morphology of OLs. In

Fig. 1. Identification of OPCs. The isolated and cultured OPCs from the spinal cords of E16 rat embryos were cultured in the growth medium containing PDGF-AA and bFGF. A: The cells display bipolar or tri-polar morphology, the typical morphology of OPC. B~D: Almost all cells were immunopositive for PDGFRα (B), NG2 (C) and A2B5 (D), the specific markers of OPCs. E: Quantification for PDGFRα+, NG2+ and A2B5+ cells in these OPC cultures. Data are given as means ± s.d., n = 3. Cells in B~D were counterstained with Hoechst 33342 (blue), a nuclear dye. A: bar = 50 µm; B~D: bar = 25 µm. 164 J.G. Hu et al.

Fig. 2. Identification of OLs and type II astrocytes derived from OPCs. The isolated and cultured OPCs from the spinal cords of E16 rat embryos were cultured in different media for 5 days. A~C: In the medium containing T3 and without PDGF-AA and bFGF, the cells induced from OPCs were immunopositive for O1 (A), MBP (B) and Rip (C), the specific markers of OLs. D: Quantification for O1+, Rip+ and MBP+ cells in OLs derived from OPCs. Data are given as means ± s.d., n = 3. E~G: In the medium containing 10%FBS without PDGF and bFGF, the cells induced from OPCs were immunopositive for GFAP (E) and A2B5 (F), the specific markers of type II astrocytes. H: Quantification for + + GFAP A2B5 (GFAP/A2B5) and Rip+ cells in type II astrocytes derived from OPCs. Data are given as means ± s.d., n = 3. Cells in B~F were counterstained with Hoechst 33342 (blue), a nuclear dye. Bar = 25 µm. Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 165

Fig. 3. Relative level of some gene transcripts confirmed by real time RT-PCR assay. Differentially expressed genes in the microarray data were confirmed with real time RT-PCR (n = 3). The levels of target genes were standardized against the level of the GAPDH gene. The results of real-time RT-PCR showed that 14 of 15 genes were consistent with the cDNA microarray data. The only exception was GAP43 in the last graph. the presence of 10% FBS, over 95% of the cells were flat- regulated or down-regulated genes. Of these, 192 genes tened with multiple processes and positive for both A2B5 had a significant change in expression in OLs compared and GFAP (Fig. 2E-H). These cells were morphologically with OPCs (83 up-regulated and 89 down-regulated), and and immunocytochemically type II astrocytes. No neurons 178 in type II astrocytes compared with OPCs (92 up-regu- were observed under these differentiating conditions. These lated and 86 down-regulated). results suggest that OPCs, OLs and type II astrocytes pre- To examine the reliability of our microarray data, we pared here were three distinct cell populations with high selected randomly 15 up-regulated genes or down-regulated purity and unique phenotypic characteristics, and thus could genes, and investigated their expression levels by real-time be readily used to detect differential gene expression among RT-PCR in the same RNA samples that had been used for the three cell populations. microarray analysis. The results of real-time RT-PCR were consistent with those from the cDNA microarray data in 14 Generation of a comprehensive database of gene expression of the 15 genes (Fig. 3). and verification of the results To identify the detailed program of OPC differentia- Differential gene expression between OPCs and OLs tion towards OLs or type II astrocytes and biological char- To learn more about differentiation and property of acteristics of three cell populations, we analyzed the global OLs, we analysis gene expression profiles of OPCs and gene expression changes between them using Affymetrix OLs which were differentiated from OPCs in the presence Rat 230 2.0 array. Four-fold change in signal intensity and of T3. Both up-regulated and down-regulated genes p < 0.05 were used as the criteria of the significant differen- exceeding 4 folds in OLs were listed in Tables 1 and 2, tial expression of a given gene. Genes with changed respectively. Based on the potential function of these expression in every spot were defined as the commonly up- genes, we categorized them into several classes which 166 J.G. Hu et al.

Table 1. Upregulated gene expression in OLs compared with OPCs (n = 3). Gene Gene Title Gene Symbol Fold change ID Receptor activity angiotensin II receptor, type 1 Agtr1a BI290306 42.22 ± 5.14 bradykinin receptor B1 Bdkrb1 NM_030851 7.46 ± 1.56 Carcinoembryonic antigen-related cell adhesion molecule 3 Ceacam3 AI145313 19.70 ± 2.38 CD44 antigen Cd44 AF065147 14.93 ± 1.68 endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, Edg2 NM_053936 24.25 ± 2.51 2 purinergic receptor P2Y, G-protein coupled 1 P2ry1 U22830 6.06 ± 1.37 retinoic acid receptor responder 2 Rarres2 BI282993 4.05 ± 1.21 transferrin Tf AA945178 6.96 ± 1.52 glutamate receptor, ionotropic, NMDA2C Grin2c U08259 5.65 ± 1.01 integrin beta 4 Itgb4 NM_013180 45.25 ± 4.22 interleukin 6 receptor, alpha Il6ra NM_017020 34.30 ± 4.12 met proto-oncogene Met NM_031517 29.86 ± 3.56 Neurexin 3 Nrxn3 BE109405 21.11 ± 2.51 neurotensin receptor 2 Ntsr2 NM_022695 14.93 ± 1.82 tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a BI303379 4.287 ± 1.09 asialoglycoprotein receptor 1 Asgr1 NM_012503 119.50 ± 15.32 interleukin 13 receptor, alpha 1 Il13ra1 AY0442518 4.40 ± 0.92 G protein-coupled receptor 37-like 1 Gpr37l1 AF087947 5.65 ± 1.31 thyroid hormone receptor beta Thrb J03819 22.63 ± 3.57 C1q and tumor necrosis factor related protein 5 C1qtnf5 BI278078 6.06 ± 1.52 Growth factor activity heparin-binding EGF-like growth factor Hbegf NM_012945 4.28 ± 0.78 neurturin Nrtn AI598581 6.49 ± 1.34 placental growth factor Pgf BF281271 16.00 ± 2.32 bone morphogenetic protein 1 Bmp1 AB012139 4.25 ± 0.99 bone morphogenetic protein 2 Bmp2 AA944827 4.28 ± 1.05 bone morphogenetic protein 4 Bmp4 NM_012827 10.56 ± 1.69 neuregulin 1 Nrg1 U02315 27.86 ± 4.21 Protein binding CD59 antigen Cd59 NM_012925 7.46 ± 1.38 CD9 antigen Cd9 AI227627 11.31 ± 2.14 heat shock 70kDa protein 2 Hspa2 NM_021863 4.00 ± 0.68 lectin, galactose binding, soluble 3 Lgals3 NM_031832 36.76 ± 5.62 nidogen 2 Nid2 BM389302 6.96 ± 1.55 Ras association (RalGDS/AF-6) domain family 4 Rassf4 AI227769 6.06 ± 1.14 SH3 and multiple ankyrin repeat domains 1 Shank1 BG381587 10.56 ± 1.88 sphingomyelin phosphodiesterase, acid-like 3A Smpdl3a BM389498 8.57 ± 1.53 growth arrest and DNA-damage-inducible 45 beta Gadd45b BI287978 25.99 ± 4.28 heat shock 22kDa protein 8 Hspb8 NM_053612 8.57 ± 2.01 Transcription cofactor activity and transcription regulator pirin Pir AI176041 5.27 ± 1.01 activating transcription factor 3 Atf NM_012912 104.10 ± 15.36 activating transcription factor 5 Atf5 BM391471 4.92 ± 0.97 inhibitor of DNA binding 3 Id3 AF000942 5.27 ± 1.21 inhibitor of DNA binding 1 Id1 M86708 5.65 ± 1.22 Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 167

Table 1. Continued Gene Gene Title Gene Symbol Fold change ID Structural constituent of myelin sheath myelin basic protein Mbp NM_017026 55.72 ± 7.62 myelin oligodendrocyte glycoprotein Mog NM_022668 27.86 ± 3.99 myelin-associated glycoprotein Mag NM_017190 4.03 ± 0.58 myelin protein zero-like 1 Mpzl1 BM384017 4.73 ± 0.95 peripheral myelin protein 22 Pmp22 AA943163 4.73 ± 0.97 proteolipid protein Plp NM_030990 36.76 ± 4.56 tenascin R Tnr NM_013045 4.25 ± 0.86 tetraspanin 2 Tspan2 AI228231 9.84 ± 1.82 Extracellular matrix structural constituent chitinase 3-like 1 Chi3l1 AA945643 7.46 ± 1.55 matrix Gla protein Mgp NM_012862 42.22 ± 5.98 matrilin 1, cartilage matrix protein Matn1 BM384214 4.28 ± 0.89 Kinase activity ret proto-oncogene Ret AJ299017 48.50 ± 6.21 phosphorylase kinase gamma 1 Phkg1 NM_031573 6.49 ± 1.23 protein kinase inhibitor beta, cAMP dependent, catalytic Pkib NM_012627 14.93 ± 2.11 receptor interacting protein kinase 5 Ripk5 BG664535 4.92 ± 0.94 mitogen-activated protein kinase 4 Mapk4 BF395788 5.65 ± 1.11 Rho family GTPase 3 Rnd3 BF548080 8.57 ± 1.35 PCTAIRE-motif protein kinase 3 Pctk3 AA964856 29.86 ± 3.99 Phospholipase inhibitor activity annexin A1 Anxa1 NM_012904 36.76 ± 4.87 annexin A4 Anxa4 BM385237 24.25 ± 2.96 annexin A5 Anxa5 NM_013132 7.46 ± 1.42 Growth factor binding insulin-like growth factor binding protein 5 Igfbp5 NM_012817 73.52 ± 9.21 insulin-like growth factor binding protein 7 Igfbp7 AI233246 11.31 ± 1.76 Others UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl- Galnt14 AI763990 29.86 ± 3.57 transferase 14 (transferase activity) heat shock 27kDa protein 1 (protein bindin) Hspb1 NM_031970 19.70 ± 2.57 chemokine (C-X-C motif) ligand 14 (chemokine activity) Cxcl14 BG380414 5.27 ± 0.87 interleukin 15 (cytokine activity) Il15 AF015718 9.84 ± 1.58 syndecan 4 (cytoskeletal protein binding) Sdc4 NM_012649 6.49 ± 1.24 adrenomedullin (hormone activity) Adm NM_012715 36.76 ± 4.88 neuropeptide Y (hormone activity) Npy NM_012614 73.52 ± 7.52 matrix metallopeptidase 14 (metalloendopeptidase activity) Mmp14 X83537 4.59 ± 0.76 jagged 2 (Notch binding) Jag2 AI715578 16.00 ± 2.01 protein phosphatase 1, regulatory (inhibitor) subunit 1B (protein phosphatase Ppp1r1b AA942959 274.40 ± 21.50 inhibitor activity) apelin, AGTRL1 ligand (receptor binding) Apln AI177057 6.06 ± 1.02 tumor necrosis factor (ligand) superfamily, member 13 (receptor binding) Tnfsf13 BF286267 6.96 ± 1.20 renin 1 (signal transducer activity) Ren1 J02941 11.31 ± 1.46 aldo-keto reductase family 1, member B7 (aldehyde reductase activity) Akr1b7 NM_053781 9.85 ± 1.65 apolipoprotein D (lipid transporter activity) Apod NM_012777 4.25 ± 0.67 plectin 1 ( structural constituent of cytoskeleton) Plec1 NM_022401 7.46 ± 1.08 168 J.G. Hu et al.

Table 2. Downregulated gene expression in OLs compared with OPCs (n = 3). Gene Gene Title Gene Symbol Fold change ID Receptor activity neuropeptide Y receptor Y1 Npy1r BI395810 0.15 ± 0.021 hyaluronan mediated motility receptor (RHAMM) Hmmr AF336825 0.06 ± 0.008 gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 3 Gabra3 NM_017069 0.09 ± 0.008 Insulin-like growth factor 2 receptor Igf2r BI299621 0.13 ± 0.022 gamma-aminobutyric acid (GABA-A) receptor, subunit beta 2 Gabrb2 NM_012957 0.18 ± 0.025 glutamate receptor, ionotropic, AMPA1 (alpha 1) Gria1 BG376217 0.05 ± 0.007 growth hormone receptor Ghr AI170771 0.15 ± 0.018 platelet derived growth factor receptor, alpha polypeptide Pdgfra AI232379 0.16 ± 0.019 Fc receptor, IgG, low affinity III Fcgr3 BI281955 0.22 ± 0.017 latrophilin 2 Lphn2 NM_134408 0.22 ± 0.015 Neurexophilin 1 Nxph1 AW523488 0.04 ± 0.003 Max dimerization protein 3 Mxd3 U45986 0.20 ± 0.016 Protein binding CDK5 regulatory subunit associated protein 2 Cdk5rap2 BF564964 0.20 ± 0.014 kinesin family member 2C Kif2c NM_134472 0.01 ± 0.001 cell division cycle 20 homolog Cdc20 U05341 0.01 ± 0.002 cyclin B2 Ccnb2 AW253821 0.03 ± 0.002 cyclin B1 Ccnb1 L11995 0.04 ± 0.003 ERBB receptor feedback inhibitor 1 Errfi1 AI169756 0.16 ± 0.013 stathmin 1 Stmn1 NM_017166 0.18 ± 0.014 leucine rich repeat and fibronectin type III domain containing 2 Lrfn2 BG380772 0.20 ± 0.021 Down syndrome cell adhesion molecule Dscam NM_133587 0.22 ± 0.024 transforming, acidic coiled-coil containing protein 3 Tacc3 AI556917 0.01 ± 0.002 Dihydropyrimidinase-like 5 Dpysl5 AA851257 0.04 ± 0.003 kinesin family member 15 Kif15 BF396282 0.08 ± 0.006 cadherin 22 Cdh22 NM_019161 0.09 ± 0.010 erythrocyte protein band 4.1-like 3 Epb4.1l3 AB032828 0.13 ± 0.018 stathmin-like 2 Stmn2 NM_053440 0.13 ± 0.014 myosin, heavy polypeptide 10, non-muscle Myh10 AA946388 0.23 ± 0.021 BMP/retinoic acid-inducible neural-specific protein 2 Brinp2 BF393519 0.25 ± 0.022 Transcription factor activity and transcription regulator CREB binding protein Crebbp BF391144 0.25 ± 0.020 RNA binding motif protein 14 Rbm14 BG670091 0.19 ± 0.022 B-cell CLL/lymphoma 11A Bcl11a AA964818 0.06 ± 0.001 Calsenilin Csen BF411924 0.16 ± 0.015 Sin3-associated polypeptide, 18kDa Sap18 BF406814 0.23 ± 0.021 POU domain, class 3, transcription factor 1 Pou3f1 M72711 0.01 ± 0.001 Transcription factor 4 Tcf4 AI639517 0.10 ± 0.012 E2F transcription factor 8 E2f8 AI231053 0.20 ± 0.021 transcription factor 19 Tcf19 BM387190 0.05 ± 0.004 pituitary tumor-transforming 1 Pttg1 NM_022391 0.07 ± 0.005 E2F transcription factor 1 E2f1 AI713319 0.16 ± 0.017 BCL2-associated transcription factor 1 Bclaf1 BI295614 0.09 ± 0.008 Structural molecule activity lamin B1 Lmnb1 NM_053905 0.03 ± 0.005 nestin Nes NM_012987 0.14 ± 0.024 procollagen, type V, alpha 3 Col5a3 NM_021760 0.05 ± 0.006 thrombospondin 4 Thbs4 X89963 0.06 ± 0.007 Extracellular matrix structural constituent proline arginine-rich end leucine-rich repeat protein Prelp AI011747 0.15 ± 0.020 fibrillin 2 Fbn2 NM_031826 0.06 ± 0.007 Nidogen 1 Nid1 AI235948 0.15 ± 0.021 Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 169

Table 2. Continued Gene Gene Title Gene Symbol Fold change ID Kinase activity JNK/SAPK-inhibitory kinase JIK AI101685 0.18 ± 0.022 phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 Pik3r1 D64048 0.08 ± 0.009 serine/threonine kinase 6 Stk6 AA996882 0.13 ± 0.012 checkpoint kinase 1 homolog Chek1 NM_080400 0.13 ± 0.014 cell division cycle 2 homolog A Cdc2a NM_019296 0.04 ± 0.005 cyclin D1 Ccnd1 X75207 0.06 ± 0.007 calcium/calmodulin-dependent protein kinase I gamma Camk1g AW251224 0.08 ± 0.008 SH3-binding kinase 1 Sbk1 AB010154 0.09 ± 0.008 microtubule associated serine/threonine kinase 1 MAST1 AI072348 0.10 ± 0.011 Rous sarcoma oncogene Src BI282912 0.18 ± 0.013 mitogen activated protein kinase kinase kinase 1 Map3k1 NM_053887 0.25 ± 0.020 cyclin-dependent kinase 5, regulatory subunit 1 (p35) Cdk5r1 NM_053891 0.25 ± 0.025 Growth factor activity bone morphogenetic protein 7 Bmp7 AI013715 0.07 ± 0.008 fibroblast growth factor 14 Fgf14 NM_022223 0.04 ± 0.005 fibroblast growth factor 13 Fgf13 NM_053428 0.18 ± 0.023 DNA binding high mobility group box 2 Hmgb2 NM_017187 0.08 ± 0.009 Breast cancer 1 Brca1 BF404972 0.19 ± 0.023 DEK oncogene (DNA binding) Dek AI073244 0.23 ± 0.025 Chemokine activity chemokine (C-X-C motif) ligand 12 Cxcl12 BF283398 0.03 ± 0.005 chemokine (C-C motif) ligand 20 Ccl20 AF053312 0.08 ± 0.009 Calcium ion binding visinin-like 1 Vsnl1 AI227991 0.04 ± 0.005 matrix metalloproteinase 16 Mmp16 NM_080776 0.16 ± 0.015 Protocadherin 8 Pcdh8 AW523567 0.16 ± 0.016 Notch binding jagged 1 Jag1 NM_019147 0.10 ± 0.008 delta-like 3 (Drosophila) Dll3 BE107343 0.14 ± 0.015 delta-like 1 (Drosophila) Dll1 NM_032063 0.03 ± 0.004 Signal transducer activity regulator of G-protein signaling 8 Rgs8 NM_019344 0.15 ± 0.020 chondroitin sulfate proteoglycan 4 Cspg4 NM_031022 0.16 ± 0.018 Nucleic acid binding insulin-like growth factor 2, binding protein 3 Igf2bp3 BF414160 0.13 ± 0.015 replication factor C (activator 1) 3 Rfc3 AI103093 0.15 ± 0.016 Transferase activity heparan sulfate (glucosamine) 3-O-sulfotransferase 2 Hs3st2 BG376092 0.23 ± 0.021 Beta galactoside alpha 2,6 sialyltransferase 2 St6gal2 AW524749 0.10 ± 0.011 Others growth associated protein 43 (calmodulin binding) Gap43 NM_017195 0.20 ± 0.024 cell division cycle associated 7 (copper ion binding) Cdca7 AA859235 0.02 ± 0.003 insulin-like growth factor binding protein 3 (growth factor binding) Igfbp3 NM_012588 0.13 ± 0.012 follistatin-like 1 (heparin binding) Fstl1 BI290885 0.25 ± 0.022 Thymopoietin (hormone activity) Tmpo AI044110 0.11 ± 0.014 epsin 2 (lipid binding) Epn2 AW521110 0.22 ± 0.026 matrix metallopeptidase 11 (metallopeptidase activity) Mmp11 NM_012980 0.08 ± 0.008 kinesin family member 11 (motor activity) Kif11 BF562797 0.03 ± 0.005 cell division cycle 25 homolog B (protein tyrosine phosphatase activity) Cdc25b NM_133572 0.18 ± 0.025 170 J.G. Hu et al.

Table 3. Upregulated gene expression in AS compared with OPCs (n = 3). Gene Gene Title Gene Symbol Fold change ID Receptor activity asialoglycoprotein receptor 1 Asgr1 NM_012503 147.03 ± 20.45 CD44 antigen Cd44 NM_012924 19.69 ± 20.14 erythropoietin receptor Epor NM_017002 18.37 ± 2.65 interleukin 6 receptor, alpha Il6ra NM_017020 45.25 ± 6.20 transferrin Tf AA945178 9.18 ± 1.58 angiotensin II receptor, type 1 (AT1A) Agtr1a BI290306 42.25 ± 5.23 bradykinin receptor B1 Bdkrb1 NM_030851 14.92 ± 1.87 Collagen-binding factor Endo180 Endo180 AI102514 51.98 ± 6.21 galanin receptor 2 Galr2 NM_019172 5.65 ± 1.02 interleukin 13 receptor, alpha 1 Il13ra1 AY044251 4.28 ± 0.87 Neurexin 3 Nrxn3 BE109405 21.11 ± 3.24 Neuropilin 2 Nrp2 AA859669 6.49 ± 1.11 neurotensin receptor 2 Ntsr2 NM_022695 22.62 ± 3.01 platelet-derived growth factor receptor-like Pdgfrl BM384311 59.71 ± 7.21 Prostaglandin F receptor Ptgfr BF548045 4.59 ± 0.78 purinergic receptor P2Y, G-protein coupled 1 P2ry1 U22830 17.14 ± 2.10 ret proto-oncogene Ret AJ299017 97.00 ± 10.87 thyroid hormone receptor beta Thrb J03819 24.25 ± 3.10 Tumor necrosis factor receptor superfamily, member 6 Tnfrsf6 AI231531 6.96 ± 1.15 Growth factor activity bone morphogenetic protein 2 Bmp2 NM_017178 7.46 ± 1.52 bone morphogenetic protein 6 Bmp6 AI230985 39.39 ± 5.34 fibroblast growth factor 2 Fgf2 NM_019305 5.65 ± 1.25 heparin-binding EGF-like growth factor Hbegf NM_012945 7.46 ± 1.34 neurturin Nrtn AI598581 17.15 ± 2.45 inhibin beta-A Inhba NM_017128 13.93 ± 1.54 neuregulin 1 Nrg1 U02319 5.28 ± 0.84 VGF nerve growth factor inducible Vgf NM_030997 6.49 ± 1.08 Hormone activity galanin Gal NM_033237 4.92 ± 0.68 neuropeptide Y Npy NM_012614 119.42 ± 15.68 thyroglobulin Tg AF221622 9.19 ± 1.46 adrenomedullin Adm NM_012715 39.39 ± 4.98 natriuretic peptide precursor type C Nppc NM_053750 9.84 ± 1.62 Protein binding annexin A2 Anxa2 NM_019905 103.96 ± 17.25 annexin A5 Anxa5 NM_013132 12.12 ± 1.87 contactin 3 Cntn3 NM_019329 8.00 ± 1.35 fibulin 5 Fbln5 NM_019153 5.27 ± 1.01 nidogen 2 Nid2 BM389302 6.96 ± 1.16 annexin A1 Anxa1 NM_012904 73.51 ± 9.36 CD59 antigen Cd59 NM_012925 12.99 ± 1.85 CD9 antigen Cd9 AI227627 5.65 ± 1.08 desmuslin Dmn BG373779 32.00 ± 4.56 EGF-containing fibulin-like extracellular matrix protein 1 Efemp1 BF284634 55.71 ± 6.98 growth arrest and DNA-damage-inducible 45 beta Gadd45b BI287978 21.11 ± 2.85 lectin, galactose binding, soluble 3 Lgals3 NM_031832 24.25 ± 2.98 p300/CBP-associated factor Pcaf Z83046 4.00 ± 0.56 reelin Reln NM_080394 17.14 ± 2.11 retinal pigment epithelium 65 Rpe65 NM_053562 42.22 ± 5.12 heat shock 27kDa protein 1 Hspb1 NM_031970 73.51 ± 8.95 transgelin Tagln NM_031549 103.96 ± 12.45 Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 171

Table 3. Continued Gene Gene Title Gene Symbol Fold change ID Transcription factor activity and transcription regulator Microphthalmia-associated transcription factor Mitf AI071509 6.96 ± 1.32 pleiomorphic adenoma gene 1 Plag1 BI290063 4.284 ± 0.56 pirin Pir AI176041 6.49 ± 1.02 inhibitor of DNA binding 1 Id1 M86708 11.31 ± 1.68 inhibitor of DNA binding 3 Id3 AF000942 13.92 ± 1.88 Inhibitor of DNA binding 4 Id4 AW916745 4.92 ± 0.87 activating transcription factor 3 Atf3 NM_012912 111.43 ± 15.32 Integrin binding laminin, alpha 5 Lama5 BI274917 4.92 ± 0.75 glycoprotein (transmembrane) nmb Gpnmb NM_133298 5.27 ± 0.74 Kinase activity mitogen-activated protein kinase 4 Mapk4 BF395788 9.84 ± 1.21 cyclin-dependent kinase inhibitor 1A Cdkn1a AI010427 21.11 ± 3.22 Rho family GTPase 3 Rnd3 AI598323 9.18 ± 1.54 Early growth response 3 Egr3 AA964492 45.25 ± 6.98 paired box gene 4 Pax4 AF053102 21.11 ± 2.35 CCAAT/enhancer binding protein (C/EBP), beta Cebpb NM_024125 137.18 ± 15.32 Heterochromatin protein 1, binding protein 3 Hp1bp3 BG372713 19.69 ± 2.31 Jun-B oncogene Junb NM_021836 17.14 ± 1.98 Kruppel-like factor 9 Klf9 BM390366 45.25 ± 5.97 Metadherin Mtdh BE110589 34.29 ± 4.21 GPI anchor binding CD24 antigen Cd24 BI285141 6.49 ± 1.14 CD14 antigen Cd14 NM_021744 6.49 ± 1.32 Metalloendopeptidase activity matrix metallopeptidase 14 (membrane-inserted) Mmp14 X83537 4.28 ± 0.79 tissue inhibitor of metalloproteinase 1 Timp1 NM_053819 8.57 ± 1.32 Structural molecule activity claudin 11 Cldn11 NM_053457 19.69 ± 2.58 glial fibrillary acidic protein Gfap NM_017009 6.49 ± 1.11 junction plakoglobin Jup NM_031047 6.49 ± 0.97 plectin 1 Plec1 NM_022401 17.14 ± 2.14 Procollagen, type XXIII, alpha 1 Col23a1 BE115264 36.75 ± 4.23 Thrombospondin 1 Thbs1 AI406660 17.14 ± 2.24 gelsolin Gsn BI285576 5.27 ± 0.96 tubulin, beta 6 Tubb6 BG153275 10.55 ± 1.65 myocilin Myoc NM_030865 5.27 ± 0.78 Others chemokine (C-X-C motif) ligand 10 (chemokine activity) Cxcl10 U22520 6.06 ± 1.02 interleukin 15 (cytokine activity) Il15 AF015718 19.69 ± 2.36 insulin-like growth factor binding protein 5 (growth factor binding) Igfbp5 NM_012817 111.43 ± 15.66 matrix Gla protein (extracellular matrix structural constituent) Mgp NM_012862 147.03 ± 18.32 clusterin Clu AF314657 12.99 ± 1.54 N-myc downstream regulated gene 1 Ndrg1 BM384099 16.00 ± 1.98 noggin Nog AA859752 8.57 ± 1.34 stomatin Stom BI295949 4.00 ± 0.56 thyroid hormone responsive protein Thrsp NM_012703 55.71 ± 6.32 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl- Galnt14 AI763990 55.87 ± 6.21 transferase 14 tumor necrosis factor (ligand) superfamily, member 13 (tumor necrosis factor Tnfsf13 AA800814 4.28 ± 0.58 receptor binding) 172 J.G. Hu et al.

Table 4. Downregulated gene expression in AS compared with OPCs (n = 3). Gene Gene Title Gene Symbol Fold change ID Receptor activity adenosine A2a receptor Adora2a NM_053294 0.20 ± 0.031 adenylate cyclase activating polypeptide 1 receptor 1 Adcyap1r1 D16465 0.13 ± 0.020 Insulin-like growth factor 2 receptor Igf2r BI299621 0.16 ± 0.018 opioid binding protein/cell adhesion molecule-like Opcml NM_053848 0.23 ± 0.024 hyaluronan mediated motility receptor (RHAMM) Hmmr AI171185 0.09 ± 0.010 glutamate receptor, metabotropic 3 Grm3 AW522430 0.08 ± 0.007 platelet derived growth factor receptor, alpha polypeptid Pdgfra AI232379 0.08 ± 0.009 growth hormone receptor Ghr AI170771 0.18 ± 0.021 gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 3 Gabra3 NM_017069 0.05 ± 0.006 Down syndrome cell adhesion molecule Dscam NM_133587 0.11 ± 0.012 Growth factor activity bone morphogenetic protein 3 Bmp3 NM_017105 0.15 ± 0.018 fibroblast growth factor 13 Fgf13 NM_053428 0.13 ± 0.012 transforming growth factor alpha Tgfa BG670310 0.19 ± 0.023 bone morphogenetic protein 7 Bmp7 AI013715 0.14 ± 0.012 Protein binding 0.14 ± 0.015 thymopoietin Tmpo NM_012887 0.12 ± 0.010 synapsin II Syn2 BI295795 0.16 ± 0.017 kinesin family member 2C Kif2c NM_134472 0.01 ± 0.001 chondroitin sulfate proteoglycan 5 Cspg5 AF292102 0.12 ± 0.014 platelet-activating factor acetylhydrolase, isoform 1b, alpha1 subunit Pafah1b3 NM_053654 0.09 ± 0.011 Discs, large (Drosophila) homolog-associated protein 2 Dlgap2 AW533496 0.08 ± 0.010 sprouty protein with EVH-1 domain 1, related sequence Spred1 BF388068 0.07 ± 0.006 Syntaxin 6 Stx6 BE097409 0.04 ± 0.005 cadherin 22 Cdh22 NM_019161 0.18 ± 0.021 leucine rich repeat and fibronectin type III domain containing 2 Lrfn2 BG380772 0.16 ± 0.015 prostaglandin F2 receptor negative regulator Ptgfrn NM_019243 0.25 ± 0.026 transforming, acidic coiled-coil containing protein 3 Tacc3 AI556917 0.02 ± 0.024 cyclin B2 Ccnb2 AW253821 0.02 ± 0.023 antigen identified by monoclonal antibody Ki-67 Mki67 AI714002 0.05 ± 0.006 cell division cycle 20 homolog Cdc20 U05341 0.08 ± 0.009 Cd200 antigen Cd200 NM_031518 0.20 ± 0.028 stathmin 1 Stmn1 NM_017166 0.16 ± 0.018 cyclin B1 Ccnb1 L11995 0.07 ± 0.008 erythrocyte protein band 4.1-like 3 Epb4.1l3 AB032828 0.05 ± 0.004 doublecortin Dcx BE109057 0.12 ± 0.014 karyopherin (importin) alpha 2 Kpna2 NM_053483 0.19 ± 0.023 Transcription factor activity and transcription regulator cyclin E Ccne1 AW913890 0.23 ± 0.025 B-cell CLL/lymphoma 11A (zinc finger protein) Bcl11 AW254062 0.06 ± 0.008 forkhead box M1 Foxm1 NM_031633 0.04 ± 0.006 myelin transcription factor 1 Myt1 AW531604 0.06 ± 0.007 transcription factor E2a Tcfe2a NM_133524 0.08 ± 0.009 transcription factor 19 Tcf19 BM387190 0.08 ± 0.009 E2F transcription factor 8 E2f8 AI231053 0.23 ± 0.026 pituitary tumor-transforming 1 Pttg1 NM_022391 0.07 ± 0.008 POU domain, class 3, transcription factor 1 Pou3f1 M72711 0.04 ± 0.005 Extracellular matrix structural constituent fibrillin 2 Fbn2 NM_031826 0.14 ± 0.012 Nidogen 1 Nid1 AI235948 0.15 ± 0.015 Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 173

Table 4. Continued Gene Gene Title Gene Symbol Fold change ID Kinase activity mitogen activated protein kinase kinase kinase 1 Map3k1 NM_053887 0.25 ± 0.031 cyclin D1 Ccnd1 X75207 0.13 ± 0.015 calcium/calmodulin-dependent protein kinase I gamma Camk1g AW251224 0.19 ± 0.020 protein kinase C, beta 1 Prkcb1 M13706 0.12 ± 0.014 cyclin-dependent kinase 5, regulatory subunit 1 (p35) Cdk5r1 NM_053891 0.20 ± 0.023 phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 Pik3r1 D64048 0.14 ± 0.015 Transferase activity UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 2 B4galt2 AI177506 0.07 ± 0.008 acetyl-Coenzyme A acetyltransferase 2 Acat2 AI412322 0.18 ± 0.021 Ribophorin II Rpn2 BF407155 0.20 ± 0.025 Structural molecule activity collagen triple helix repeat containing 1 Cthrc1 AA958001 0.08 ± 0.009 nestin Nes NM_012987 0.18 ± 0.021 procollagen, type V, alpha 3 Col5a3 NM_021760 0.09 ± 0.010 internexin, alpha Inexa NM_019128 0.23 ± 0.028 Nucleic acid binding calsenilin, presenilin binding protein, EF hand transcription factor Csen NM_032462 0.01 ± 0.001 hairy and enhancer of split 5 (Drosophila) Hes5 NM_024383 0.11 ± 0.013 BCL2-associated transcription factor 1 Bclaf1 BI295614 0.15 ± 0.018 high mobility group box 2 Hmgb2 NM_017187 0.08 ± 0.007 Zinc finger protein (C2H2 type) 276 Zfp276 BE115520 0.05 ± 0.006 insulin-like growth factor 2, binding protein 3 Igf2bp3 AI230709 0.06 ± 0.006 checkpoint kinase 1 homolog (S. pombe) Chek1 NM_080400 0.16 ± 0.014 serine/threonine kinase 6 Stk6 AA996882 0.09 ± 0.008 cell division cycle 2 homolog A (S. pombe) Cdc2a NM_019296 0.04 ± 0.005 cyclin dependent kinase 2 Cdk2 AI012076 0.25 ± 0.032 Heterogeneous nuclear ribonucleoprotein A3 Hnrpa3 AI175966 0.1 ± 0.009 Chemokine activity chemokine (C-C motif) ligand 20 Ccl20 AF053312 0.18 ± 0.021 chemokine (C-X-C motif) ligand 12 Cxcl12 BF283398 0.13 ± 0.015 Notch binding delta-like 1 (Drosophila) Dll1 NM_032063 0.03 ± 0.004 jagged 1 Jag1 NM_019147 0.18 ± 0.021 Calcium ion binding Protocadherin 8 Pcdh8 AW523567 0.08 ± 0.007 S100 calcium binding protein A3 S100a3 NM_053681 0.14 ± 0.015 visinin-like 1 Vsnl1 NM_012686 0.10 ± 0.008 Others growth associated protein 43 (calmodulin binding) Gap43 NM_017195 0.19 ± 0.024 cell division cycle associated 7 (copper ion binding) Cdca7 AA859235 0.11 ± 0.012 RAP1, GTPase activating protein 1 (GTPase activator activity) Rap1ga1 BF284067 0.10 ± 0.008 dynamin 3 (GTPase activator activity) Dnm3 AF201839 0.10 ± 0.012 epsin 2 ( lipid binding) Epn2 AW521110 0.11 ± 0.014 dual specificity phosphatase 6 (protein tyrosine/serine/threonine phosphatase Dusp6 AI602811 0.11 ± 0.012 activity) cyclin A2 Ccna2 AA998516 0.02 ± 0.003 TRAF4 associated factor 1 Traf4af1 BM388478 0.05 ± 0.007 Ng23 protein Ng23 AI103527 0.09 ± 0.011 174 J.G. Hu et al. include receptor activity, growth factor activity, protein (Maxwell et al. 2005). It has been reported that hyaluronan, binding, transcription cofactor activity and transcription one of the ligands of HAMMR, accumulates in demyelin- regulator, extracellular matrix structural constituent, kinase ated lesions and inhibits OPC maturation through binding activity and so on. A detailed analysis demonstrated that to CD44, another receptor of hyaluronan (Back et al. 2005). some significantly regulated genes might be involved in OL The high expression of HAMMR on OPCs implicated that differentiation, including hyaluronan-mediated motility HAMMR might be another CD44-like receptor which can receptor (HAMMR), platelet derived growth factor receptor mediate hyaluronan to maintain self-renewal of OPCs. Our alpha polypeptide (PDGFRα), transferrin (Tf), ApoD, bone previous study demonstrated that platelet derived growth morphogenetic proteins (BMPs), etc. factor receptor alpha polypeptide (PDGFRα) is differentially expressed (12-fold) during the induction from NSCs Differential gene expression between OPCs and Astrocytes to OPCs (Hu et al. 2004). Further study showed that Similarly, gene expression profiles of OPCs and type PDGF-AA plays a dual role in mediating the number of OL II astrocytes which differentiated from OPCs in the pres- lineage cells: an early instruction of OPC differentiation ence of 10% serum were analyzed. Both up-regulated and from NSCs and later proliferation of committed OPCs (Hu down-regulated genes exceeding 4 folds in type II astro- et al. 2008). In the present study, we found that PDGFRα cytes were listed in Tables 3 and 4, respectively. There was strongly down-regulated in both OLs and type II astro- were also some significantly changed genes in serum- cytes, suggesting that PDGFRα is also a key factor in main- induced type II astrocyte differentiation, such as erythropoi- taining self-renewal and inhibiting differentiation of OPCs. etin receptor (EPOR), prostaglandin F receptor (PTGFR), Transcription factors (TFs) and binding proteins play prom- galanin receptor 2 (GALR2), platelet-derived growth factor inent roles in developmental processes and cell-lineage receptor-like (PDGFRL), metadherin (MTDH)/astrocyte specification (Ahmed et al. 2009; Burrows et al. 2010). elevated gene 1 (AEG-1), THBS1, myocilin, BMPs, and Here, we found that many OPC-expressed TFs and binding Noggin. proteins, such as E2F transcription factor 8, pituitary tumor- transforming 1 and cell division cycle 20 homolog, were Discussion down-regulated in both OLs and type II astrocytes, suggest- In the present report, the differential gene expression ing they may be involved in maintaining proliferation and profiling in OPCs, OLs and type II astrocytes were studied. self-renewal of OPCs. OPCs were obtained from E16 Wistar rats and were induced into OLs and type II astrocytes in the presence of T3 or Candidate genes that may intrinsically control OL-specific 10% serum in vitro, respectively. Immunocytochemistry differentiation and/or represent the property of OLs results demonstrated that OPCs, OLs and type II astrocytes As shown in Table 1, some genes were up-regulated in were characteristic and highly pure. Subsequently, using OLs compared with OPCs. Among them, several genes the Affymetrix Rat 230 2.0 array, the differential gene which encode structural constituent of myelin sheath were expression profiling in the three cell populations were iden- up-regulated markedly, including myelin basic protein tified. Among these genes, some may participate in OPC (MBP), myelin oligodendrocyte glycoprotein (MOgP), differentiation and others may constitute molecular identity myelin-associated glycoprotein (MAG), myelin protein of each cell population. zero-like 1 (Mpzl1), peripheral myelin protein 22 (PMP22), proteolipid protein (PLP), tenascin R (TNR), and tet- Gene profiles of OPCs raspanin 2 (Tspan2). These well-understood genes (David In the present study, we found that many genes were and Lacroix 2003) were confirmed again to highly express down-regulated in expression after OPCs differentiated into in OLs, but not type II astrocytes, suggesting that they OLs and/or type II astrocytes. Among these down-regu- indeed represent the unique characteristic of mature OLs. lated genes, some might be involved in maintaining self- It has been reported that transferrin (Tf), the iron trans- renewal of OPCs and/or represent the property of OPCs. port glycoprotein in biological fluids of vertebrates, can be Ligand-receptor interaction plays a determining role in synthesized by OLs. The Tf can selectively regulate MBP many cell biological processes. It is the first step for expression at the transcriptional level and increase the dif- growth factors, cytokines and other regulatory elements to ferentiation, maturation and myelination of OLs (Espinosa- mediate cell responses, such as proliferation, differentia- Jeffrey et al. 2002; Sow et al. 2006). Thus, our finding that tion, migration, angiogenesis, immune responses and cell high expression of Tf in OLs is consistent with previous death (Vermeer and Leuven 1990; Jones et al. 2008). Our report. In our previous study, we have shown that there was results showed that a number of receptor genes were down- a 3-fold increase in ApoD expression in OLs as compared regulated in expression after OPCs differentiated into OLs to NSCs (Hu et al. 2004). Here, ApoD expression in Ols or type II astrocytes. Hyaluronan-mediated motility recep- was found increasing more than 4-fold (but not type II tor (HAMMR) is first described by Turley (1982). It can astrocytes) as compared to OPCs. Further increased expres- bind hyaluronan, ERK kinase and microtubules and partici- sion of ApoD in OLs indicates that more lipid transport is pates in cell motility, signaling, and oncogenic events necessary in the progress of maturation of OLs. Bone mor- Differential Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 175 phogenetic proteins (BMPs) influence the lineage commit- 2009). In this study, we found a very high expression of ment of embryonic neural stem cell progeny by promoting MTDH in type II astrocytes (34-fold of OPCs). Thus, astroglial lineage determination (Gross et al. 1996) and MTDH/AEG-1 may play an important role in differentia- inhibiting oligodendroglial differentiation (Samanta and tion of type II astrocytes. THBS1 played diverse roles in Kessler 2004). Here, we showed that BMP4 had an the latter stage of tumorigenesis, which may function as an increased expression in oligodendrocytes, but didn’t change adhesive protein or a modulator of extracellular proteases to in type II astrocytes, which is consistent with Kondo’s promote tumor invasion (Kazerounian et al. 2008). In the report (Kondo and Raff 2004). Besides above-mentioned present study, the observation of THBS1 gene-expression in genes, there are still many other genes with increased- type II astrocytes indicates that THBS1 may transduce a expression in OLs. Among these genes, it is possible that growth-regulatory signal in these cells. Myocilin was the certain genes are involved in differentiation of OLs and/or first molecule discovered to be linked with primary open represent the property of OLs. Further functional research angle glaucoma (POAG), a blinding disease characterized of some candidate genes remains to be clarified. by progressive loss of retinal ganglion cells. It was reported that optic nerve head astrocytes constitutively express myo- Candidate genes that may contribute to type II astrocyte cilin in vivo (Noda et al. 2000; Ricard et al. 2001). In the fate and/or represent the property of type II astrocytes present study, a 5-fold increase in Myocilin gene-expression Our results showed that a number of receptor genes was found in type II astrocytes as compared to OPCs, con- were increased in expression when OPCs differentiated into firming the previous finding. Noggin is the antagonist of type II astrocytes. Among these up-regulated genes, some BMPs and inhibits astroglial differentiation induced by of them might contribute to OPC differentiation into type II BMPs. In the present study, our result showed over 8-fold astrocytes. Erythropoietin (EPO), a hematopoietic growth increase in Noggin expression in type II astrocytes. This is factor that stimulates proliferation and differentiation of similar to the previous report which found a strong expres- erythroid precursor cells, is also known to exert neuro- sion of Noggin in astrocytes in the developing optic nerve trophic activity in the CNS (Sugawa et al. 2002). EPO (Kondo and Raff 2004). mRNA expression was demonstrated only in human astro- In summary, using cDNA microarray technology, we cytes, while EPOR expression was found in human neu- have identified differentially expressed genes during the rons, astrocytes, and microglia. Neither EPO nor EPOR differentiation of OPCs into OLs or type II astrocytes. expression was found in OLs (Nagai et al. 2001; Sugawa et Among them, the up-regulated genes might contribute to al. 2002). These reports are consistent with our present OPC differentiation and represent constitutive components result which show an over 18-fold up-regulation of EPOR of OLs or type II astrocytes, whereas the down-regulated gene expression in type II astrocytes, but not OLs. There genes might be involved in maintaining self-renewal and/or fore, we suspect that EpoR maybe a mediator of prolifera- represent the property of OPCs. Results of this study con- tion of astrocytes as has been reported (Nagai et al. 2001; tribute to elucidation of molecular differences between Sugawa et al. 2002). Moreover, it is also possible that high OPCs and OLs or type II astrocytes and provide new expression of EpoR contributes to differentiation of type II insights into mechanism of OPC differentiation. astrocytes from OPCs. Another receptor, prostaglandin F receptor (PTGFR), was also found high expression in type Acknowledgments II astrocytes. It has been reported that PTGFR signalling This work was supported by the National Natural Science regulates endothelial cell network formation and prolifera- Foundation of China (NO. 30700439, 81071268), the Key tion (Keightley et al. 2010). Whether PTGFR also play a Project of Chinese Ministry of Education (NO. 210103), the role in type II astrocyte differentiation should be clarified in Science and Technological Fund of Anhui Province for Outstanding Youth (NO. 10040606Y13) and the Natural Science the future. Two other receptors, galanin receptor 2 Research Program of Education Bureau of Anhui Province (NO. (GALR2) and platelet-derived growth factor receptor-like 2006KJ098A, KJ2010B109). (PDGFRL), which have been shown increased gene-expression in type II astrocytes are also need to be further investi- Conflict of Interest gated. Metadherin (MTDH) was originally reported as a The authors declare no conflict of interest. novel late response gene, which can be induced to express in human fetal astrocytes after HIV-1 infection or treatment References with viral glycoprotein gp120 or TNF-α. Thus, MTDH is Ahmed, S., Gan, H.T., Lam, C.S., Poonepalli, A., Ramasamy, S., also named astrocyte elevated gene 1 (AEG-1) (Su et al. Tay, Y., Tham, M. & Yu, Y.H. (2009) Transcription factors and 2002). It has been reported that Metadherin acts in several neural stem cell self-renewal, growth and differentiation. Cell crucial aspects of tumor progression, including transforma- Adh. 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