Maintenance of neural stem cell regional identity in culture

Abstract

Neural stem cells (NSCs) are distributed throughout the ventricular-subventricular zone (V-SVZ) in the adult mouse brain. NSCs located in spatially distinct regions of the V-SVZ generate different types of olfactory bulb (OB) neurons, and the regional expression of specific transcription factors correlates with these differences in NSC developmental potential. In a recent article, we show that Nkx2.1-expressing embryonic precursors give rise to NKX2.1+ NSCs located in the ventral V-SVZ of adult mice. Here we characterize a V-SVZ monolayer culture system that retains regional gene expression and neurogenic potential of NSCs from the dorsal and ventral V-SVZ. In particular, we find that Nkx2.1-lineage V-SVZ NSCs maintain Nkx2.1 expression through serial passage and can generate new neurons in vitro. Thus, V-SVZ NSCs retain key aspects of their in vivo regional identity in culture, providing new experimental opportunities for understanding how such developmental patterns are established and maintained during development.

Keywords: Nkx2.1; Regional identity; Stem cell heterogeneity; V-SVZ; adult neurogenesis.

Figure 1.

Establishment of regional V-SVZ NSC cultures. (A) Schematic depiction of regional V-SVZ NSC microdissection. Blue dots represent location of dorsal dissection. Pink dots represent location of ventral dissection. (B) Representative image of 300 μm vibratome-derived coronal section prior to microdissection. (C) Immunocytochemistry of dorsal and ventral cultures under proliferation conditions for Nestin (green), NKX2.1 (red) and DAPI (blue). (D) Bar graph depicting percentage of NKX2.1 immunopositive cells in culture after 1 (black), 3 (white), and 5 (gray) passages in vitro. Error bars depict standard deviation. (E) Immunocytochemistry of dorsal and ventral cultures after 4 d of differentiation for Tuj1 (green), Calbindin (red), and DAPI (blue). Scale bars = 10 μm (C, D), 400 μm (B).

Figure 2.

V-SVZ NSCs cultures retain regional transcriptional differences in vitro. (A and B) qPCR of ventral and dorsal cultures under proliferative conditions. Ventral gene expression (cyan), Dorsal gene expression (yellow). Error bars depict standard deviation. * = P < 0.05, **= P < 0.01. ND= not detected.

Figure 3.

Maintenance of Nkx2.1 expression in neurogenic V-SVZ NSCs. (A) Top, Nkx2.1-CreER:Ai14 fate-labeling schema in ventral culture under proliferative conditions. Bottom, immunocytochemistry for NKX2.1 (green), tdTomato (red), EdU (magenta), and DAPI (blue). Arrows depicts NKX2.1+/tdTomato+ cell. Arrowhead depicts EdU+/NKX2.1+/tdTomato+ cell. (B) Top, Nkx2.1-CreER:Ai14 fate-labeling followed by differentiation schema in ventral culture. Bottom, immunohistochemistry for Tuj1 (green), tdTomato (red), and DAPI (blue). DIV= days in vitro. Scale bars = 10 μm (A), 25 μm (B).

Figure 4.

V-SVZ precursors maintain Nkx2.1 expression in a stable and heritable manner. (A) Nkx2.1^E12.5-labeling and culture schema. In vivo time points depicted with red bar, culture time points depicted with green bar. p1-5 = passages 1-5. (B) Bar graph depicting percentage of Nkx2.1^E12.5-labeled cells in culture at passages 1 (black), 3 (white), and 5 (gray). Error bars depict standard deviation. Immunocytochemistry for Nkx2.1^E12.5-labeled cells (red), DAPI (blue), and (C) Ki67 (green), (D) EdU (green), and (E) NKX2.1 (green). Scale bars = 25 μm (C-E).