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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Nat Neurosci. 2019 Apr 1;22(5):741–752. doi: 10.1038/s41593-019-0366-7

Fig. 3. Astroglial cell populations isolated from 8.3-astroglia mouse CNS and human cortex have unique transcriptomes as validated with immunohistochemistry (IHC) and RNA in situ hybridization.

Fig. 3

(A) The cortex microarray heatmap displays unique genes differentially expressed between all rodent astroglia (GLT1-eGFP), 8.3 kb labeled astroglia (8.3-astroglia) and remaining non-labeled cells (negative). (B) Microarray expression of astroglia markers highly expressed in both rodent astroglia populations. (C) Selected markers highly enriched in 8.3-astroglia microarray data in the cortex. (D) qPCR validation of candidate markers from the microarray analyses in the cortex of the three cell populations. (E) KCNJ10 enhanced immunoreactivity in 8.3-astroglia compared to GLT1-eGFP astroglia in the mouse motor cortex (i,ii scale bars 15μm). (F) LGR6-GFP expression is restricted to 8.3-astroglia in the adult mouse cortex as evaluated in double transgenic LGR6-GFP-ires-CreERT2/8.3-astroglia mice (i,ii scale bars 15μm). (G) Analysis of Lgr6 mRNA expression in the human motor cortex in a subset of cells (i scale bar 2.5μm), arrow indicates RNA. (H) LGR6 protein in the human motor cortex colocalizes with GFAP and ALDH1L1 protein (i scale bar 7.5μm). (I) A subset of cultured primary mouse cortical astroglia are LGR6-positive (i scale bar 4μm). (J) A subset of cultured and matured human iPSC-astroglia are LGR6-immunoreactive (i scale bar 4μm). Three cell populations were isolated from the adult mouse whole cortex and subjected to FACS, microarray, and qPCR analyses from a total of three mice. For histological sections, n = five mice were imaged with a minimum of 3–5 images per mouse, representative images are shown. Human post-mortem tissue was evaluated in n = three non-neurological diseased cases with at least 3–5 images taken per case for both RNA ISH and immunohistochemistry. For mouse immunocytochemistry, n = five different cultures of mouse primary astroglia were generated and imaged at least 3–5 times per astroglia generation. For iPSC astroglia a minimum of three control human iPSC lines were generated. All images are representative of the total amount imaged. Statistics were performed by two-sided Student’s T-test of 8.3-astroglia vs negative cell population with represented values showing the mean and error bars representing SEM, *p<0.05, **p<0.01, ***p<0.001.