Fig 2: Quality check for neural progenitors and neurons isolated from the neocortex of wild-type embryos on the basis of CD133 staining(A–D) Isolated CD133-high, -medium, -low, or -negative cells at E12 (A), E14 (B), E16 (C), or E18 (D) were subjected to RT-qPCR analysis of the indicated mRNAs. Data were normalized by the amount of Gapdh mRNA, are expressed relative to the higher value for each mRNA, and are means + SEM from three (E12, E18, and Pax6 and Tbr1 at E14) or four (E16 and Eomes, Neurod1, and Tubb3 at E14) independent experiments.

Fig 3: Gating Strategy for Defining Cell Populations. Dot plots show representative data from pw0 unwounded skin, which represented our control situation. (A) The analysis using flow cytometry started by including negative singlet events for dead cell markers such as PI or Zombie NIR (so-called vital cells). Singlet and vital cells were divided into 3 compartments (1: CDH1(CD324)+/EpCam(CD326)+ Epithelial (Epi); 2: CDH1-/EpCam-/PTPRC(CD45)- dermal non-immune cells (Imm-/Epi-); 3: PTPRC+ immune cells (Immune)). (B) Epithelial cells were further divided into IFE, Inf, Ist, Bul and HG using the markers Ly6A, EpCam, CD34, CDH3(P-Cam) and ITGA6(Integrin alpha 6). (C) Dermal non-immune cells were classified as PECAM1(CD31)+ endothelial cells (endothel), PROM-1(CD133)+ DP, or Fib (PECAM1-/PROM-1-). Immune cells were further divided into myeloid derived cells and T cells. (D) Myeloid derived cells expressing ITAM(CD11b) were subdivided into ITAX(CD11c)+ DC, ITAX-/FCGR1(CD64)- mono, and ITAX-/FCGR1+ mac. Mac cells were classified into MRC1(CD206) + or - cells. Highly granular (SSC-A high) PTPRC+ cells in myeloid derived compartments were also analyzed regarding Ly6G expression to define neutrophils. (E) T cells in immune compartments were defined as CD3+ cells and further divided regarding CD4, CD8 and d?TCR expression, while CD4+ cells were also analyzed regarding expression of FOXP3 to identify Treg.

Fig 4: Luminal HS-AV Cells Show Age-Dependent Abundance(A) Relative proportion of HS-AV cells in young (n = 3) and aged (n = 4) mice by scRNA-seq (Fisher’s exact test).(B) Markers distinguishing HS-AV cells from both HS cells and AV cells in young mice. See also Table S5.(C) Expression pattern of select HS-AV markers in (B).(D) Dimensionality reduction plot from STREAM lineage trajectory analysis of luminal cells in young mice (n = 3). See also Figure S5D.(E and F) Representative immunofluorescence staining of HS-AV cells (indicated by arrows) co-expressing HS marker (PR or ER) and AV marker (LTF or MFGE8) in young mice.(G–J) Immunofluorescence staining of HS-AV cells (indicated by arrows) co-expressing PR and MFGE8 (G) or ER and LTF (I) in young and aged mice. Scale bar, 10 µm. Quantifications are shown in (H) and (J), respectively (n = 6 mice per age group, Student’s t test). In (G) and (I), MFGE8 and LTF are also detected as secreted proteins in the lumen. KRT8 marks luminal cells.(K) FACS quantification of HS-AV cells co-expressing HS marker CD133 and AV marker CD14 in an independent cohort of young (n = 8) and aged (n = 6) mice. In the representative FACS plots, 10,000 cells are displayed. Statistical significance is determined by Student’s t test.

Fig 5: Gating strategies for isolation of neural progenitors and neurons from the neocortex of Nestin-d4Venus or wild-type embryos(A) Neocortical cells of Nestin-d4Venus–/– or Nestin-d4Venus+/– embryos at E14 were dissociated and subjected to FACS analysis as described previously (Sakai et al., 2019). The gates for dVenus-high, -medium, -low, and -negative cells are shown.(B) Neocortical cells of wild-type embryos at E14 were dissociated, stained (or not) with PE-conjugated antibodies to CD133, and subjected to FACS analysis as described previously (Sakai et al., 2019). The gates for CD133-high, -medium, -low, and -negative cells are shown.(C) Neocortical cells of C57BL/6 embryos at E15 were dissociated, stained (or not) with both APC-conjugated antibodies to CD133 and PE-conjugated antibodies to CD24, and subjected to FACS analysis. Nonneural cells determined as those negative for both CD133 and CD24 were removed. The gate for CD133-high cells is shown.