A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells

Abstract

Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers.

Keywords: gliogenesis; human astrocytes; neural progenitor cells; neurodevelopment; radial glial cells; transcriptomic.

Figure 1. Astroglial differentiation of human Neural Progenitor Cells (NPCs)

A) Immunostaining of human neurospheres stained with anti-NESTIN (red), anti-VIMENTIN (green) and anti-BLBP (magenta) antibodies. Nuclei were stained with DAPI (blue). B) RT-qPCR analysis of GFAP expression changes in NPCs differentiated for 1 week using different culturing conditions. On the Y axe is depicted the expression of GFAP relative to the housekeeping gene β-ACTIN. GFAP expression level was normalized to NPCs (EGF + FGF). C) Western blot analysis of GFAP protein expression in NPCs and in cells differentiated for 1 week in the presence of FBS and CNTF/BMP4. The expression of the housekeeping gene GAPDH was used to as loading control. D) Immunostaining of NPCs differentiated for one 1 week in the presence of FBS and CNTF/BMP4. Cells were stained with anti-GFAP antibody (Green) and with DAPI (blue). E) RT-qPCR analysis of astrocytes (GFAP, CD44 and AQP4) and progenitor cells (BLBP, NESTIN and VIM) specific-genes in NPCs differentiated for 1 week using FBS (upper panel) and CNTF/BMP5 (lower panel). On the Y axe is depicted the expression of the analyzed gene relative to the housekeeping gene β-ACTIN. Gene expression is normalized to non-differentiated NPCs (-FBS and –CNTF/BMP4). Error bars are S.E.M.; **p<0.01.

Figure 2. Transcriptomic analysis of differentiated astrocytes

A) Venn diagram depicting the number of genes that are differentially expressed in NPCs differentiated for one 1 week in the presence of FBS and CNTF/BMP4. B) Heatmap showing log₂(FPKM) values and hierarchical clustering analysis of differentially expressed genes in NPCs, FBS-astrocytes and CNTF/BMPB4-astrocytes. C) Graphical representation of Gene Ontology (GO) enrichment analysis showing biological processes that are overrepresented in the 398 genes differentially expressed in both FBS- and CNTF/BMP4-astrocytes. D) Heatmap showing log₂(FPKM) values of genes differentially expressed with both culturing conditions (FBS and CNTF/BMP4) and belonging to the most enriched biological processes from GO enrichment analysis (Table1).

Figure 3. Quantitative RT-qPCR validation of RNAseq differential expression analysis

Technical validation of RNAseq differential expression analysis of FBS (A) and CNTF/BMP4 (B) differentiation using RT-qPCR showing high degree of correlation between log2 fold change differences from RNAseq and RT-qPCR data for 13 different genes.

Figure 4. Comparison of FBS-astrocytes and CNTF/BMP4-astrocytes

A) Heatmap showing log2(FPKM) values and unsupervised hierarchical clustering analysis of all the genes expressed in FBS- and CNTF/BMP4-astrocytes. B) Heatmap displaying log2(FPKM) and hierarchical clustering analysis of differentially expressed genes between FBS- and CNTF/BMP4 astrocytes. Graphical representation of Gene Ontology (GO) enrichment analysis showing biological processes that are overrepresented in FBS-astrocytes enriched genes (C) and CNTF/BMP4-astrocytes enriched genes (D).

Figure 5. Enrichment of reactive astrocytes genes in FBS-astrocytes

A) Gene set enrichment analysis (GSEA) performed in pre-ranked mode using differentially expressed genes in NPCs differentiated with FBS. A) Enrichment plot for reactive astrocytes genes signature. Genes related to reactive astrocytes genes signature most strongly associated with the FBS-astrocytes are represented on the far left. B) Heatmap showing FPKM expression profile of the core enrichment of genes from GSEA. C) RT-qPCR analysis of FBS-astrocytes-expressed reactive astrocytes genes in the spinal cord of mice control mice (CTRL) or mice after 1 or 2 weeks from injury. RNA was extracted from the site of injury of the spinal cord. On the Y axe is depicted the expression of the analyzed gene relative to the housekeeping gene β-Actin. Gene expression is normalized to CTRL. Error bars are S.E.M.; *p<0.05; **p<0.01.; n=4.