Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation

Abstract

Multipotent neural stem/progenitor cells (NSPCs) can be isolated from many regions of the adult central nervous system (CNS), yet neurogenesis is restricted to the hippocampus and subventricular zone in vivo. Identification of the molecular cues that modulate NSPC fate choice is a prerequisite for their therapeutic applications. Previously, we demonstrated that primary astrocytes isolated from regions with higher neuroplasticity, such as newborn and adult hippocampus and newborn spinal cord, promoted neuronal differentiation of adult NSPCs, whereas astrocytes isolated from the nonneurogenic region of the adult spinal cord inhibited neural differentiation. To identify the factors expressed by these astrocytes that could modulate NSPC differentiation, we performed gene expression profiling analysis using Affymetrix rat genome arrays. Our results demonstrated that these astrocytes had distinct gene expression profiles. We further tested the functional effects of candidate factors that were differentially expressed in neurogenesis-promoting and -inhibiting astrocytes using in vitro NSPC differentiation assays. Our results indicated that two interleukins, IL-1beta and IL-6, and a combination of factors that included these two interleukins could promote NSPC neuronal differentiation, whereas insulin-like growth factor binding protein 6 (IGFBP6) and decorin inhibited neuronal differentiation of adult NSPCs. Our results have provided further evidence to support the ongoing hypothesis that, in adult mammalian brains, astrocytes play critical roles in modulating NSPC differentiation. The finding that cytokines and chemokines expressed by astrocytes could promote NSPC neuronal differentiation may help us to understand how injuries induce neurogenesis in adult brains.

Fig. 1. Astrocytes derived from adult and newborn hippocampus and spinal cord expressed different sets of genes

(a) Sample correlation coefficient (cc) plots of signal intensity (representing gene expression levels) obtained by using MAS4.1 software (Affymetrix); Note that replicates (NBH1 vs. NBH2, ADS1 vs. ADS2) had similar gene expression profiles (high cc values), whereas samples from different tissues (NBH vs. ADS, NBH vs. ASF) had distinct gene expression profiles (low cc values); (b) A modified Venn Diagram showing numbers of genes that are unique or common among these astrocytes derived from different tissues; Among 5,190 genes that are “Present” in at least one astrocyte sample, 3,394 (65.4%) genes were shared by all four types of astrocytes, 914 (17.6%) genes were shared by 3 types of astrocytes, 537 (10.3%) genes were shared by two types of astrocytes, and 527 (10.2%) genes were only “Present” in one type of astrocyte; (c) Among the 3,394 common genes shared by the astrocytes, 3,231 genes are also shared with ASF.

Fig 2. Gene expression profiles of sample candidate genes were confirmed using real time quantitative PCR

(a) IL-6; (b) IL-1β; (c) VCAM-1; and (d) IGFBP6. The relative mRNA levels of each candidate genes were obtained by normalizing to an internal control, GAPDH. PCR results were consistent with the results obtained by microarray analysis.*, p<0.05; **, p<0.01; ***, p<0.001 (unpaired t-test).

Fig. 3. Factors expressed at higher levels in neurogenesis-inhibiting astrocytes and non-neurogenic regions inhibit NSPC neuronal differentiation promoted by NBH astrocytes

(a, b, c) GFP-expressing NSPCs (green) differentiate into TuJI+ neurons (red) when co-cultured with NBH astrocytes for 4 days, in the absence (a,b) or presence of 50ng/ml IGFBP6 (c). (d) Quantification of immunofluorescent staining indicated that IGFBP6, decorin (Dcn) and enkephalin (Enk) significantly reduced neuronal differentiation (represented by the percentage of TuJI+ neurons) of NSPCs that were co-cultured with NBH astrocyte; (e) Dose-dependent inhibition of NSPC neuronal differentiation by IGFBP-6. *, p<0.05; **, p<0.01, ***, p<0.001 (unpaired, 2-tailed student's t-test).

Fig 4. Inflammatory cytokines, IL-1β and IL-6, could modulate affect NSPC differentiation

(a) Both NRG/ED (p<0.05, n=8) and IL-6 (P<0.05, n=14) increased NeuroD1 promoter activity in NSPCs; (b) IL-6 treatment increased GFAP promoter activity in NSPCs (p<0.05, n=6); (c) Both IL-1β and IL-6 promote the neuronal differentiation of NSPCs (p<0.01, n=7 for both) and such effect could be blocked by their specific blocking antibodies (p<0.05, n=3 for both); (d) Antibodies to IL-6 and IL-1β blocking the neurogenic effects of NBH astrocytes on co-cultured NSPCs, control un-treated NSPCs co-cultured with NBH astrocytes. (e) IL-6, but not IL-1β also increased GFAP+ astrocyte differentiation of NSPCs (p<0.05). *, p<0.05; **, p<0.01, ***, p<0.001 (unpaired, 2-tailed, student's t-test).

Fig 5. A combination of factors expressed at higher levels by neurogenesis-promoting astrocytes and tissues promote neuronal differentiation of NSPCs

(a) The combination of IL-1β, IL-6, VCAM1, IP-10, Cathepsin S, and TGFβ2 (Combo) enhanced NeuroD1 promoter activity in NSPCs, analyzed by luciferase activity assays (p<0.01, n=14); (b) Immunofluorescent images showing TuJI+ neurons (red) differentiated from NSPCs that were treated by IL-1β, IL-6, Combo and RA in the absence of FGF-2. Such fluorescent staining results were quantified using unbiased stereology quantification to generate data shown in Fig 4c and Fig 5d. (c) A Z-stack confocal image of a TuJI+ neurons differentiated from IL-6 treated NSPCs; (d) IL-1β, IL-6 and Combo could promote neuronal differentiation of NSPCs indicated by the percentage of TuJI+ neurons. Combo was significantly more potent than IL-1β and IL-6 alone. *, p<0.05; **, p<0.01, ***, p<0.001 (unpaired, 2-tailed, student's t-test).