Journal: bioRxiv

Article Title: Identification of Human Pluripotent Stem Cell Derived Astrocytic Progenitors that Correlate with Glioblastoma Subtypes

doi: 10.1101/2025.08.26.672421

Figure Lengend Snippet: ( A ) Immunostaining of CD51, CD63 and CD71 in LBF treated cells at 160 DIV ( n =3). ( B ) Quantification of the percentage of LBF stage cells with CD51, CD63, and CD71 immunostaining and different combinations of co-expression ( n =3). ( C ) Immunostaining of CD51, CD63 and GFAP in LBF treated cells ( n =3). ( D ) Quantification of the percentage of LBF stage cells with CD51, CD63 and GFAP immunostaining and different combination of co-expression ( n =3). ( E ) Histogram of flow cytometry gating strategy for sorting CD51, CD71, and CD63 expressing or co-expressing cells after LBF treatment. ( F ) Proportion of FACS-isolated cells with each possible combination of CD51, CD71, and CD63 co-expression ( n =6). ( G ) Labeling of the four predominant populations identified based on CD51, CD71, and CD63 expression. Population 1 (CD51+CD71+CD63+, green), population 2 (CD51+CD71-CD63-, red), population 3 (CD51+CD71+CD63-, purple), and population 4 (CD51+CD71-CD63+, blue) account for 70% of FACS-isolated cells. ( H ) Principal component analysis (PCA) plot illustrating the overall sample distribution based on transcriptomic profiles of populations 1-4 cells used for RNA-seq. ( I ) Table depicting the number of differentially expressed genes from pairwise and 3 versus 1 comparison analyses based on greater than 1.5-fold difference and false discovery rate of 0.05. ( J ) Heatmap representation of relative expression of fetal and mature astrocyte enriched genes in each population. n represents the number of independent differentiation experiments from one ESC line and two iPSC lines. Scale bar, 50µm.

Article Snippet: Primary antibodies used in this study are as follow: Acsbg1 (Abcam #ab65154, IHC at 1:100), CD51 (Biolegend #327910, FACS at 1:500), CD51 (Cell Signaling Technology #13208 1CC at 1:1000), CD63 (Millipore Sigma #C7930, FACS and ICC at 1:500), CD71 (Biolegend #334208, FACS and ICC at 1:1000), Doublcortin (Novus Bio.

Techniques: Immunostaining, Expressing, Flow Cytometry, Isolation, Labeling, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Identification of Human Pluripotent Stem Cell Derived Astrocytic Progenitors that Correlate with Glioblastoma Subtypes

doi: 10.1101/2025.08.26.672421

Figure Lengend Snippet: ( A ) Heatmap representation of relative expression of GBM subtype-enriched genes in FACS isolated populations 1-4 cells GBM subtype enriched gene list obtained from . ( B ) Pathway analyses of differentially expressed genes identified in populations 1 and 2 highlighted enrichment in TGF-β signaling in population 1 and Notch signaling in population 2. ( C ) Expression of select TGF-β pathway members and associated genes in population 1-4 cells as detected in the RNA-seq analysis (n=3)( D ) Expression of mesenchymal GBM subtype signature genes and proneual GBM subtype signature genes as detected in the RNA-seq of surface marker identified cell populations. ( E ) UMAP feature plot of CD51, CD63, and CD71 expressing cells illustrating the distribution of cells expressing each surface maker. ( F ) Distribution of CD51+CD63+CD71+ population 1 cells and CD51+CD63-CD71-population 2 cells in the UMAP plot illustrating enrichment in of triple positive cells in cluster Ast.3. ( G, H ) Heatmap of population 1 enriched genes ( G ) and population 2 enriched genes ( H ) among the 10 cell clusters identified in the scRNA-seq. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001 followed by Tukey’s post-hoc test.

Article Snippet: Primary antibodies used in this study are as follow: Acsbg1 (Abcam #ab65154, IHC at 1:100), CD51 (Biolegend #327910, FACS at 1:500), CD51 (Cell Signaling Technology #13208 1CC at 1:1000), CD63 (Millipore Sigma #C7930, FACS and ICC at 1:500), CD71 (Biolegend #334208, FACS and ICC at 1:1000), Doublcortin (Novus Bio.

Techniques: Expressing, Isolation, RNA Sequencing, Marker

Journal: bioRxiv

Article Title: Identification of Human Pluripotent Stem Cell Derived Astrocytic Progenitors that Correlate with Glioblastoma Subtypes

doi: 10.1101/2025.08.26.672421

Figure Lengend Snippet: ( A ) Schematic of sequential FACS experimental setup and subsequent assays performed on the cell populations isolated based on CD51, CD63, and CD71. ( B ) Phase-contrast images of populations 1 and 2 cells at 1, 3, and 7 days in vitro (DIV) after sorting. ( C ) Quantification of the percentage of cell in populations 1 to 4 after sorted populations 1 and 2 were subsequently FACS isolated on day 3 and 7 ( n =3). ( D, E ) Immunostaining representative images ( D ) and quantifications ( E ) of Nestin, GFAP, Ki67 and DCX-expressing cells in population 1 and population 2 at 1 DIV after FACS. ( F ) Immunostaining representative images and quantifications of cells expressing lineage markers GFAP, Ki67, and DCX after re-sorting populations 1 and 2 cells by FACS after 3 DIV and 7 DIV. n represents the number of independent differentiation experiments from one ESC line and two iPSC lines. Data are represented as means ± s.e.m. Scale bar, 50µm.

Article Snippet: Primary antibodies used in this study are as follow: Acsbg1 (Abcam #ab65154, IHC at 1:100), CD51 (Biolegend #327910, FACS at 1:500), CD51 (Cell Signaling Technology #13208 1CC at 1:1000), CD63 (Millipore Sigma #C7930, FACS and ICC at 1:500), CD71 (Biolegend #334208, FACS and ICC at 1:1000), Doublcortin (Novus Bio.

Techniques: Isolation, In Vitro, Immunostaining, Expressing