Fig 2: Injection of IL-1a into the CNS of mice induces rapid activation of glial cells, neutrophil infiltration, and loss of mature oligodendrocytes in the spinal cord.a–d Quantification of the activation marker Fos in astrocytes (Fos+Sox9+ cells), oligodendrocytes (Fos+Olig2+CC1+), microglia (Fos+Iba1+), and neurons (Fos+NeuN+) in the spinal cord of C57BL/6 mice injected with either PBS or rmIL-1a i.c.m. and killed at 1, 4 or 24 h post-injection (n = 6 mice/group). e Quantification of the total number of Ly6G+ neutrophils that infiltrated the spinal cord at 4 and 24 h post-injection of PBS or rmIL-1a (n = 3 mice/group). f, g Representative confocal images showing Ly6G (a marker of neutrophils, red) and laminin (a marker of blood vessel basement membranes, green) immunostainings in the spinal cord of C57BL/6 mice injected with either PBS (f) or rmIL-1a (g) at 24 h post-injection. h Quantification of the total number of Olig2+CC1+ mature oligodendrocytes in the spinal cord white matter of C57BL/6 mice at 4 and 24 h post-i.c.m. injection of either PBS or rmIL-1a (n = 4–5 mice/group: n = 4 PBS 4 h, n = 4 rmIL-1a 4 h, n = 4 PBS 24 h, n = 5 rmIL-1a 24 h). i, j Representative confocal images showing immunostaining for the oligodendrocyte transcription factor 2 (Olig2, red cells), a nuclear marker of oligodendrocyte lineage cells, in the spinal cord of C57BL/6 mice at 24 h post-injection of either PBS (i) or rmIL-1a (j). Data are presented as mean values + /- SEM and statistical significance was determined by a two-way ANOVA followed by a Bonferroni post-hoc test (a–e, h). Pairwise comparisons and p-values are indicated in the graphs. Scale bars: (f, g, in g) 50 µm, (i, j, in j) 50 µm.

Fig 3: Effects of GFAP Mutations on Astrocyte Differentiation(A) Cartoon depicting relative locations of AxD mutations on a GFAP monomer along with an introduction of cell lines.(B) Sanger sequencing of patient-derived IPSCs and corrected isogenic lines.(C) Quantification of GFAP+ cells at 3 months (gray) and 6 months (black) during astrocyte differentiation (n = 4).(D) TaqMan SNP results showing relative RNA detection for each specific probe (n = 3).(E) Expression of astrocyte-associated markers by qPCR (n = 4).(F) Immunocytochemistry of 6 month astrocytes for GFAP, S100B, and Sox9. Scale bars, 100 µm.Data are represented as mean ± SD.

Fig 4: Generation of human pluripotent stem cell (hPSC)-derived multipotent pancreatic progenitors (MPCs). a Schematic summary of the protocols used to generate MPCs from hPSCs. hPSCs were differentiated through four stages, including definitive endoderm (DE), primitive gut tube (PGT), pancreatic foregut (PFG), and MPCs. b RT-PCR analysis of MPCs derived from human embryonic stem cells (hESCs) and induced hPSCs (hiPSCs) at the end of stage 4 (S4) using protocol 1 showing the expression of the MPC and early endocrine progenitor markers (PDX1, NKX6.1, SOX9, NKX2.2, GATA6, PAX6, NGN3, and HNF6). +RT indicates samples with reverse transcriptase, and –RT indicates samples without reverse transcriptase. c Real-time PCR analysis of MPCs differentiated using protocol 1 and protocol 2. Differential gene expression analysis was performed for the main MPC genes (PDX1, NKX6.1, and SOX9). Fold-expression for each TF obtained for protocol 2 was normalized to that of protocol 1. d Flow cytometry analysis for the expression of NKX6.1 at the end of stage 4 of differentiation. All data shown are representative results from at least three independent experiments

Fig 5: Characterization of ductal markers in hPSC-derived MPCs. a Immunofluorescence images of hPSC-derived MPCs showing the expression of SOX9 (green) and NKX2.2 (red) in 3D aggregates generated using protocol 2. Note the expression of SOX9 in the 3D aggregates. b, c Immunofluorescence images of hPSC-derived MPCs showing expression of cytokeratin 19 (green) and NKX6.1 (red). The NKX6.1 expression was noticed in the cells adjacent to cytokeratin-positive cells in a monolayer arrangement of pancreatic progenitors (b); however, cytokeratin 19 was completely absent in the 3D aggregates which were positive for NKX6.1 (arrowheads) (c). All data shown are representative results from at least three independent experiments. Scale bars (a, c) = 100 µm and (b) = 50 µm