Characterization of the human SVZ at single-cell level
We have recently confirmed the progenitor identity of NGFR+ (i.e. CD271) cells33 from the human SVZ by assessing their transcriptome and proteome signature. To further characterize the dorsal SVZ of the aged human brain at single-cell level, we isolated progenitors, astrocytes, and microglia by fluorescently labelling the different populations for CD271 (progenitors)32, GLT-1 (astrocytes), and CD11b (microglia), followed by FACS (Figure S1a-b). We also sorted the negative fraction. We obtained the profile of 1074 cells from the SVZ of the aged human brain. After QC-analysis, 728 cells remained for further analysis (Figure S1c-h). We performed unbiased cluster analysis using the Louvain algorithm and the Uniform Manifold Approximation and Projection (UMAP)36 identifying seven clusters (Figure 1a and Figure S2a). Cell types clustered based on biological cell type, rather than donor or technical artefacts (Figure S1d-e). We identified three microglia clusters, viz. Microglia 1, Microglia 2, and Microglia 3 as they expressed canonical microglia markers (e.g. CX3CR1 and AIF1) (Figure 1). These three clusters contained cells that were CD11b+. We identified two clusters as progenitor clusters (i.e. Progenitors 1 and Progenitors 2), which expressed markers for progenitors (e.g. SOX2 and SOX10), and lacked expression of markers for ependymal cells (FOXJ1 and AQP4), radial glial cells (HOPX), or astrocytes (VIM, GFAP and ALDH1A1) (Figure 1c). These two clusters contained the cells that were sorted based on CD271 expression and some cells from the negative fraction (Figure S1e-h). They expressed the marker for early progenitor/astrocyte CD912, but did not express markers for activated progenitors NES and EGFR23, and neither PROM1 (not shown) or markers for late neuronal progenitors (e.g. PAX6 and ASCL1). Both clusters also expressed markers for the oligodendrocyte lineage including the oligodendrocyte progenitor cell (OPC) markers SOX10 and RGCC37. The cluster Neuronal was negative for all the above markers, and instead, expressed SOX6 and neuronal markers (e.g. MAP2, RBFOX1, NRXN1 and CTNNA2) (Figure 1b-c and Figure S3). SOX6 is a transcription factor expressed in early OPCs, but has also been associated with the development of interneurons37–39. The final cluster that we identified only expressed LYZ and SPINT2 as cluster marker genes (Figure S3 and Supplementary Data 5). These two clusters only contained CD271-CD11b-GLT1- sorted cells.
To further substantiate the identity of the two Progenitor and the Neuronal clusters we performed Gene ontology (GO) analysis on all highly expressed genes (adj P-value < 0.01) (Supplementary Data 5) within the clusters: Progenitors 1, Progenitors 2, and Neuronal. Cluster Progenitors 1 and 2 showed enrichment for GO terms related to central nervous system development, axonogenesis, and glial cell development (Figure S2b). Cluster Neuronal showed enrichment for terms related to protein modification, cell adhesion, and glutamate receptor binding. These GO analyses corroborate the identities of the three clusters as progenitors and neuronal.
Progenitors isolated from the aged human SVZ are OPCs
As the gene signature of aged human SVZ progenitors suggested an OPC identity we compared our data to the dataset from Zhong, et al., 201840 and Jäkel, et al., 201941. Zhong et al., isolated cells from the fetal human brain at different gestational stages and Jäkel et al., isolated white matter cells from healthy donors aged between 35 and 82 years (mean age 60 years). We used Seurat v3.2.2 to run an integrative analysis on the three datasets. This revealed several clusters including early progenitors, late progenitors, and migrating neurons (Figure 2a-c). Moreover, we observed substantial mixing of cells from the different datasets, arguing against clustering due to batch effects. Neuronal lineage clustered together, and include mostly fetal cells and mid-aged cells from Jäkel et al. Microglia from our dataset clustered with microglia from the two other studies. Cells from the OPC lineage from our dataset formed clusters with OPC lineage cells from Jäkel et al., and fetal OPCs (Figure 2a-b and Supplementary Data 6). We next performed clustering analysis on cells from the OPC lineage only, which revealed seven subclusters (Figure 2d-e). These subclusters corresponded to early OPCs (PDGFRA and SOX6), late OPCs (SOX10 and SOX2), and oligodendrocytes (KLK6 and OPALIN) (Figure 2f). Our analysis showed that from the 395 progenitor cells that we analyzed, 138 cells corresponded to late OPCs and the remaining cells to oligodendrocytes (Supplementary Data 6). We performed SOX10 immunofluorescence staining on post-mortem human brain tissue, which showed that only a few SOX2 progenitors in the SVZ are SOX10 positive (Figure S4).
Increased expression of cell cycle inhibitors in OPCs from the aged human SVZ
Analysis of the expression of a panel of markers for the oligodendroglial cell lineage further confirmed the OPC identity of the CD271+ progenitor cells (Figure 3a). We next identified genes that were differentially expressed over time using Monocle3 v0.2.3.0 (Supplementary Data 7). One of the genes that was differentially expressed over time was SFRP1, which increased in expression with age (Supplementary Data 7 and Figure 3c). SFRP1 is an antagonist of the Wnt pathway, thereby inhibiting cell proliferation42,43. This is interesting as the mechanisms that regulate quiescence of progenitors from the human SVZ are unclear. Therefore, we compared the expression of several proliferation and cell cycle markers in fetal, mid-aged, and aged OPC lineage cells. As expected, markers for proliferation and cell cycle progression were mostly absent in the mid-aged and aged OPC lineage cells (Figure 3b), while markers for quiescence and cell cycle arrest were highly expressed in mid-aged and aged OPC lineage cells, in particular CDKN1B (i.e. P27), CDKN1C (i.e. P57) and SFRP1 (Figure 3c).
Cell cycle inhibitors are expressed in the aged human SVZ
P57 is a known marker for stem cell quiescence in rodents9,24 and SFRPs are a family of biphasic regulators of Wnt signaling expressed in the nucleus or cytoplasm of the cell42–44. SFRP1 is mainly expressed in late OPCs (Figure 4b) and is the only member of the SFRP family that is expressed in aged OPCs (Figure S5a). In contrast, P57 is expressed in both late OPCs as well as oligodendrocytes (Figure 4a). To characterize the expression pattern of both P57 and SFRP1 in the aged SVZ, we performed immunofluorescence staining on post-mortem human brain tissue (Supplementary Data 2). In the aged SVZ around 25% of SFRP1+ cells in the SVZ expressed SOX2 (Figure 4c-d). SFRP1 expression is not limited to progenitors, as it is also highly expressed in ependymal cells, cortical neurons (Figure S6a) and OLIG2+ cells in the SVZ (Figure S6b-c). While SFRP1 is expressed in the nucleus of progenitors (Figure 4c) and ependymal cells, it is also expressed in the cytoplasm of neurons (Figure S6a).
SFRP1 inhibits the Wnt pathway by binding to Wnt ligands and by directly binding to β-catenin in the nucleus42. To determine whether SFRP1 expression correlated with a quiescent state, we assessed the expression of P57 in SFRP1+ cells in the SVZ only. Our results showed that around 78% of the SFRP1+ cells in the SVZ expressed P57 (Figure 4e-f). The majority of P57+ cells were positive for SFRP1 (87.81 ± 10.39, not shown). This suggests that in the adult SVZ, SFRP1 is mostly expressed by quiescent/primed-quiescent stem cells. We confirmed that SFRP1 is also expressed by post-mitotic progenitors of the fetal human brain at nine gestational weeks (Figure 4g). Immunofluorescence staining of SFRP1 expression in the SVZ from aged, mid-aged, and fetal post-mortem brain shows an increase in the number of SFRP1+ cells from mid-aged (mean age of 61 years) to aged (mean age of 91 years) (Figure 4h) and from GW9 to GW16-17 (Figure 4i). We also confirmed in our bulk RNAseq dataset32 that SFRP1 has the highest expression from the SFRP family members, in both CD271+ cells and SVZ homogenate isolated from post-mortem brain tissue from healthy donors (Figure S5b).
Inhibition of SFRP1 stimulates proliferation in iPSC-derived NSCs
A previous study showed that proliferation and differentiation increases during early corticogenesis in Sfrp1-/- mouse embryos43. Therefore, we assessed the effect of inhibiting SFRP1 function on proliferation of human NSCs by using a human iPSC-derived neural stem cell line to model human NSCs in vitro. This was done with the small molecule WAY-316606, which is known to sequester SFRP1 in vitro. This molecule prevents SFRP1 from binding to Wnt ligands, thereby stimulating the Wnt pathway45. We first confirmed the expression of SFRP1 protein in human iPSC-derived NSCs (Figure 5a). Most cells expressed SFRP1 protein in the cytoplasm and nucleus, while in some cells cytoplasmic expression was absent. Sequestration of SFRP1, stimulated proliferation of iPSC-derived NSCs 72 hours after stimulation in vitro (Figure 5b-e). This effect is dosage-dependent (not shown). Stimulation with WAY increased the number of cells by two fold (Figure 5b). While we observed an increase in SOX2+ cells, the percentage of KI67+ iPSC-derived NSCs did not increase when compared to control condition (Figure 5d-f). Our data therefore, suggest that, this increase in cell number is mediated by a shortening of the cell cycle rather than an increase in cell activation. This can be explained by the fact that iPSC-derived NSCs do not exit the cell cycle, and instead remain actively cycling. To confirm that the observed effect is mediated by increased activity of the canonical Wnt pathway we performed a Topflash luciferase reporter assay on HEK293 cells. Our results confirmed that the small molecule WAY-316606 activates the canonical WNT pathway through inhibition of SFRP1 (Fig S7). WAY-316606 acts specifically on SFRP1 and does not activate the Wnt pathway when in presence of SFRP5, an SFRP isoform that promotes NSC quiescence in the mouse SVZ7.
SFRP1 is expressed in the postnatal mouse brain
To determine whether SFRP1 inhibition also stimulates proliferation of progenitors in vivo, we first assessed the expression pattern of SFRP1 over time in the embryonic and postnatal mouse brain. In situ hybridization (ISH) data from Allen Brain Atlas showed a gradual increase in SFRP1 expression from E11.5 to E18.5 (Figure 6a). During the embryonic period, SFRP1 is mainly expressed in the germinal regions. Following birth, SFRP1 expression decreases in the SVZ, while increasing in regions outside the SVZ. We confirmed the ISH data by performing immunofluorescence staining for SFRP1 on P1 and P67 mouse brains. This showed expression of SFRP1 in the SVZ, striatum, and cortex in P1 mouse brain (Figure 6b-d), and a strong decrease in SFRP1 expression in the SVZ in P67 mouse brains (Figure 6e-g). Kalamakis et al., 20197 showed that from all members of the SFRP family, only SFRP5 expression increased with time in the mouse SVZ, while SFRP1 expression decreased (Figure S5c). Thus, in contrast to the expression pattern of SFRP1 in the human SVZ, its expression is highest in the early postnatal mouse SVZ.
Inhibition of SFRP1 promotes proliferation and differentiation through stimulation of the Wnt and Notch pathways
Previous studies showed that SFRPs are multifunctional proteins that regulate both Wnt and Notch signalling42,43, through which they regulate dopamine neuron development46 and cortical expansion43. We first assessed whether inhibiting SFRP1 function increased activation of the Wnt and Notch pathways in vivo. SFRP1 was prevented from binding to Wnt ligands by the administration of the small molecule WAY-316606 to two days old mouse pups. We assessed this in the early postnatal mouse brain, as SFRP1 levels are highest in the SVZ at this age (Figure 6, Figure S5c). The entire SVZ was dissected 72 hours after treatment with WAY-316606 for RT-PCR analysis, focusing on Wnt and Notch pathway related genes. Our results show a 3.5-fold increase in Cyclin d1 (Ccnd1) (P = 0.0079), which promotes cell proliferation47 (Figure 7a). p57 (Cdkn1c) expression did not change (P = 0.4812). Moreover, some key genes of the Wnt signaling (Fzd7 P = 0.0025, Ctnnb1 P = 0.0203, and Lef1 P = 0.0497) and the Notch signaling (Hes5 P = 0.0131, and Nrarp P = 0.0041) were also increased following administration of the small molecule WAY-316606 (Figure 7a). Administration of WAY also enhanced the expression of Dcx (P = 0.0007) and CNPase (P = 0.0466) genes, suggesting increased specification towards neuronal and OPC lineages (Figure 7b).
Sequestration of SFRP1 increases activation of progenitors in the mouse SVZ
We next determined whether WAY-316606 administration would stimulate progenitor proliferation also in vivo. To determine if inhibiting the function of SFRP1 increases the number of GFP+ cells and their migration away from the SVZ, we specifically labelled progenitors from the dSVZ by dorsal electroporation of a GFP plasmid at P2 and terminated the pups 72 hours after administration of WAY-316606. Our results show a 1.6-fold increase in the number of GFP+ cells in the dSVZ (Figure 8a-b). We did not see a significant increase in migration towards the cortex, nor to the olfactory bulb (not shown). The increase in the number of GFP+ cells in the dSVZ correlated with a 2-fold increase in proliferating cells in the dSVZ (Figure 8c-d). There was also a 1.6-fold increase in Ki67+ cells in the lSVZ (Figure 8c-d). Our results show that while the number of Sox2+ progenitors remains constant in the lSVZ, and increases with 2-fold in the dSVZ, there was a 3-fold decrease in the mSVZ after administration of WAY-316606 (Figure 8e-f). There was a significant increase in the number of Olig2+ cells in both the dorsal and lateral SVZ (Figure 8g-h).