Targeted activation of primitive neural stem cells in the mouse brain

Abstract

Primitive neural stem cells (pNSCs) are the earliest NSCs to appear in the developing forebrain. They persist into the adult forebrain where they can generate all cells in the neural lineage and therefore hold great potential for brain regeneration. Thus, pNSCs are an ideal population to target to promote endogenous NSC activation. pNSCs can be isolated from the periventricular region as leukaemia inhibitory factor-responsive cells, and comprise a rare population in the adult mouse brain. We hypothesized that the pup periventricular region gives rise to more clonal pNSC-derived neurospheres but that pup-derived pNSCs are otherwise comparable to adult-derived pNSCs, and can be used to identify selective markers and activators of endogenous pNSCs. We tested the self-renewal ability, differentiation capacity and gene expression profile of pup-derived pNSCs and found them each to be comparable to adult-derived pNSCs, including being GFAP(-) , nestin(mid) , Oct4(+) . Next, we used pup pNSCs to test pharmacological compounds to activate pNSCs to promote endogenous brain repair. We hypothesized that pNSCs could be activated by targeting the cell surface proteins C-Kit and ErbB2, which were enriched in pNSCs relative to definitive NSCs (dNSCs) in an in vitro screen. C-Kit and ErbB2 signalling inhibition had distinct effects on pNSCs and dNSCs in vitro, and when infused directly into the adult brain in vivo. Targeted activation of pNSCs with C-Kit and ErbB2 modulation is a valuable strategy to activate the earliest cell in the neural lineage to contribute to endogenous brain regeneration.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain

“…We previously observed that the number of pNSCs that can be isolated from the periventricular region in a neurosphere assay seems to decrease from the early postnatal period to adulthood , but that the absolute number of Oct4‐GFP + cells from a primary dissection of pup and adult brains is roughly equivalent when quantified by flow cytometry . We hypothesized that this is due to a decrease in the number of adult pNSCs that form neurospheres, rather than a decrease in the total number of adult pNSCs.…”

“…Despite reports of ependymal cells activated after injury to act as stem cells , pNSCs are not ependymal‐derived, based on the subependymal position of Tg(Oct4 IRES‐GFP ) cells in periventricular whole mounts and on the findings that pNSC‐derived neurospheres self‐renew and are not ciliated . We performed cell cycle analysis on pNSCs and dNSCs with the H2B‐GFP mouse model, which holds great benefit over BrdU or EdU label retention models, as the H2B model does not require a cell to be proliferating to take up the initial label.…”

“…We previously identified an additional type of NSC called the primitive NSC (pNSC) , which does not express GFAP and is therefore missed if purifying on the basis of this marker. pNSCs are self‐renewing, multipotent, GFAP – but Oct4 + NSCs that give rise clonal neurospheres in leukemia inhibitory factor (LIF) . pNSCs can be isolated from the mouse embryo at embryonic day (E)5.5, in advance of dNSCs that arise at E7.5 and give rise to clonal neurospheres in fibroblast growth factor (FGF)‐2 .…”

“…pNSCs uniquely express low levels of Oct4 based on quantitative polymerase chain reaction, Oct4 ‐driven antibiotic resistance, Tg (Oct4 IRES‐GFP ) flow cytometry, immunostaining, and morula aggregation experiments . Different cell surface markers are expressed on pNSCs than on dNSCs , which can be used to target either population independently .…”

Building a central nervous system: The neural stem cell lineage revealed

“…Most of the factors examined looked at their influence on dNSCs (EGF and FGF2 responsive). Leeder et al., (2016) are among the first to study the behavior of pNSCs in response to exogenous factors 46 . Basing their study on receptor expression on pNSCs derived from ESCs, they demonstrated that inhibitors of C-kit and ErbB2 selectively increased the size of pNSC pool (more neurospheres following in vitro exposure and in vivo exposure to specific receptor blockers) with no concomitant effect on the dNSC pool.…”