Fig 2: Novel reactive astrocyte markers are upregulated at the transcriptional level. (A) Transcript abundances in transcripts per million (TPM) for each of the six genes are plotted to visualize marker expression levels for each individual line (n = 3, line 3 = black, line 5 = yellow, line 6 = red from Barbar et al., 2020a). Individual dots are means of three technical replicates for each line; error bars represent standard error of means. p-value was calculated using a two-tailed Welch’s t-test. *, p-value < 0.05; **, p-value < 0.01. (B) Transcript abundances (in TPM) for each of the six genes are plotted to visualize marker expression in hiPSC-derived astrocytes from an independent cohort of 13 lines (individual replicates shown as dots). Error bars represent standard error of means. P-value was calculated using a Wald test through DESeq2 and adjusted for multiple comparisons using the Benjamini and Hochberg method. ****, p-value < 0.0001. (C) Feature plots of CD49f+ hiPSC-derived astrocyte scRNA-seq (Barbar et al., 2020a) demonstrates increased expression of each marker’s associated gene in TIC-induced reactive astrocytes compared to unstimulated astrocytes. Arrows highlight clusters most associated with TIC-stimulation and having the greatest upregulation of putative reactive astrocyte markers. (D) Dot plot showing genes enriched in TIC-induced reactive astrocytes and unstimulated astrocytes. Each row is an individual marker gene.

Fig 3: YBX1 depletion in primary human keratinocytes results in epidermal progenitor defects without affecting commitment to differentiation. a Colony formation capacity of primary human keratinocytes transfected either with a control siRNA or two different YBX1 siRNAs was assessed by a clonogenic assay; 400, 800, or 1600 cells per well were seeded in 6-well plates and incubated for 10 days. Cells were fixed and stained using Sulforhodamine B (SRB). % area with colonies was calculated using ImageJ. Average and SDs were calculated based on the two different siRNAs results. b YBX1 protein levels in keratinocytes transfected with control or YBX1 siRNAs (from a) were assessed using western blotting, and vinculin levels served as a loading control. c YBX1 depletion reduces the population of actively cycling epidermal progenitors: passage 2 of primary human keratinocytes pooled from three different donor cultures were transfected with either control siRNA or two different YBX1 siRNAs and sorted for expression of CD71 and Integrin-a6 (ITGA6) by a flow cytometer after staining with APC-conjugated anti-CD71, and Pacific blue-conjugated anti-ITGA6 antibodies. Quantification of the separate populations of cells is presented in the lower panel as % of the whole population. d Control and YBX1 siRNA transfected human keratinocytes were harvested 4 days after transfection and mRNA levels of the differentiation markers KRT1, KRT10, and INV were quantified by qRT-PCR using 36B4 mRNA as an internal control. Error bars represent mean SD, n = 3

Fig 4: Immunofluorescence analysis of novel TIC-reactive markers in CD49f+-sorted astrocytes. (A) Independent immunofluorescence analysis on CD49f-purified astrocytes using antibodies conjugated with fluorescent secondaries. All markers (with the exception of ICOSL) are enriched in TIC-stimulated astrocytes, validating the flow cytometry findings. Each marker is shown in AF488 (green) and nuclei are labeled with Hoechst 33342 (blue). Each marker was tested on astrocytes derived from three hiPSC lines (n = 3); line code numbers are shown in the bottom right corner of each image. (B) Quantification of marker-positive cells shown as percentage of total cells (all nuclei). Error bars represent standard error of means. p-value was calculated using ratio paired t-test. *, p-value < 0.05; **, p-value < 0.01.