Fig 2: Overexpression of TBC1D3 induces human cerebral organoid expansion.(A) Schematic diagram for human cerebral organoid culture. (B) Analysis for the size of organoids (Ctrl, 6 organoids in D12 and 15 in D18; TBC1D3, 11 organoids in D12 and 17 in D18). Scale bars, 100 µm. (C) Analysis for the percentage of KI67+ or P-VIM+ cells in D18 organoids (14 neuroepithelial rosettes from 6 control organoids; 16 rosettes from 8 TBC1D3 organoids). Scale bar, 20 µm. (D) Analysis for the percentage of PAX6+ (white arrowheads) or DCX+ (yellow arrowheads) cells among GFP+ cells in D40 organoids infected with adenovirus (AV) expressing shTBC1D3 or shCtrl. Scale bar, 20 µm. shCtrl, 23 rosettes from 15 organoids; shTBC1D3, 19 rosettes from 13 organoids. (E) TBC1D3 distribution in ReN cells. Scale bar, 10 µm. (F) Immunostaining for PAX6, TBR2, and TBC1D3 in GW15.5 fetal human cortex. Scale bars, 200 µm (left) and 5 µm (magnified). Histograms show the percentage of cells with TBC1D3 enriched in cytoplasm/membrane (Cyt/Mem; gray arrows) or nucleus (Nuc; red arrows). Five regions for cortical plate (CP) or OSVZ; six regions for VZ/ISVZ from three slices. Data are presented as means ± SD, unpaired Student’s t test. **P < 0.01; ***P < 0.001.

Fig 3: Effects of G9a inhibition on cortical expansion in human cerebral organoids.(A) Levels of H3K9me2 in D16 cerebral organoids treated with G9a inhibitor UNC0638 or DMSO control, determined by IB. (B) Representative images of D30 and D40 cerebral organoids treated with UNC0638 or vehicle control and quantification for the average diameter. Numbers of organoids analyzed: D30, 19 organoids for control and 20 organoids for UNC0638 group; D40, 11 organoids for control and 12 organoids for UNC0638 group. Scale bar, 1000 µm. (C) Immunostaining for signals of PAX6, TBR2, and DAPI in D30 organoids treated with UNC0638 or DMSO. Scale bar, 50 µm. (D) Immunostaining for signals of KI67, P-VIM, and DCX in D30 organoids treated with UNC0638 or DMSO control, with DAPI marking cell nucleus. Scale bar, 50 µm. (E and F) Quantification for the percentage of PAX6+ cells (E) or TBR2+ cells (F) among DAPI+ cells (20 rosettes from 14 control organoids; 31 rosettes from 17 UNC0638-treated organoids). (G to I) Quantification for the percentage of KI67+ (G), P-VIM+ (H), or DCX+ cells (I). Twenty-three rosettes from 15 organoids were analyzed in each group. Data are presented as means ± SD, unpaired Student’s t test. ***P < 0.001.

Fig 4: Immunofluorescence microscopy analysis of ILC1lc and enriched CD8/NKG2D cells in AA-induced xenotransplant.(A) EOMES+, CD49a+, and NKG2D+ around hair follicle (HF) in normal scalp skin, intrafollicular and perifollicular ILC1lc infiltrates in AA-induced xenotransplants (B) EOMES+,c-KIT-,CD49a+, and (C) NKp44+, CD103+, T-bet- ILC1lc. Absence of these cells in normal scalp xenotransplant. For each panel, yellow staining indicates double staining A-EOMES+, NKG2D+; B- EOMES+,CD49a+; C- NKp44+, CD103+. (D) Quantitation. (E) CD8+/NKG2D+ cells around HF in AA-induced xenotransplant versus absence of the cells in normal xenotransplant. (F) The quantitative data demonstrate the significant increased CD8+/NKG2D+ cells in HFs of alopecia areata (AA) humanized mice compared to normal scalp xenotransplants. N=6 xenotransplants/ group from three independent donors, three areas were evaluated per section. Following Shapiro-Wilk test, Student’s t-test: *p<0.05, **p<0.01, ***p<0.001. Mann Whitney U test: #p<0.05, ##p<0.01.Scale bar, 50 µm. DP - dermal papilla, HM - hair matrix, White arrow- c-KIT stained melanocyte. Figure 8—source data 1.Quantitative data for immunofluorescence microscopy analysis of ILC1lc and enriched CD8/NKG2D cells in AA-induced xenotransplant.

Fig 5: Immunofluorescence microscopy analyses of ILC1lc and CD8+/NKG2D+ cells in alopecia areata (AA) scalp skin.(A) ILC1lc (EOMES+, CD49a+, and NKG2D+) around HF in normal scalp skin, intrafollicular and perifollicular ILC1lc infiltrates in lesional and in non-lesional AA scalp patient. (B) EOMES+, c-KIT-,CD49a+, and (C) NKp44+, CD103+, T-bet- ILC1lc. For each panel, yellow staining indicates double staining A-EOMES+, NKG2D+; B- EOMES+, CD49a+; C- NKp44+,CD103+ (D) Quantitative immunohistomorphometry (qIHM) shows an increased number of ILC1lc in AA patients as compared to normal volunteers and increased number of the cells in lesional versus non-lesional areas of the patients. There is a significant increased perifollicular than intrafollicular ILC1lc in the lesional and non lesional areas. (E) CD8+/NKG2D+ cells around HF in AA scalp patient and absence of these cells in normal scalp skin of normal scalp skin. (F) There is an increased number of CD8+/NKG2D+ cells in HFs of AA patients compared to normal scalp skin and a significant lower number of ILC1lc versus CD8+/NKG2D+ cells in AA scalp skin. N=6 biopsies /AA patients and six biopsies /healthy donors from six independent donors, three areas were evaluated per section, and three sections per biopsy. Following Shapiro-Wilk test, Student’s t-test: *p<0.05, **p<0.01 or Mann Whitney U test: #p<0.05. Scale bars, 50 µm. CTS- connective tissue sheath, DP - dermal papilla, HM - hair matrix, White arrow- c-KIT stained melanocyte. Figure 1—source data 1.Quantitative data for immunofluorescence microscopy analyses of ILC1lc and CD8+/NKG2D+ cells in AA scalp skin.