Fig. 1

NeuroTrace 500/525 selectively labels human brain pericyte-like cells in vitro. A NeuroTrace 500/525 staining was performed on human umbilical vein endothelial cells (HUVECs), human primary cultured brain vSMCs, brain microvascular endothelial cells (BMECs), and brain pericytes. Cellular uptake of NeuroTrace 500/525 appears as green fluorescence. Scale bars, 50 μm (5 μm in magnified area). B Schematic representation of pericyte differentiation. Brain pericyte-like cells were differentiated from hiPSCs (ASE9209) through a neural crest intermediate. Immunostaining images reveal changes in the expression of the NCSC marker p75^NTR and the pericyte markers PDGFRβ and NG2. Scale bars, 100 μm. C In vitro endothelial cord formation assay using HUVECs and hiPSC-derived brain pericyte-like cells. Representative bright field images of HUVECs alone or HUVECs co-cultured with hiPSC-derived pericyte-like cells. Scale bars, 500 μm. Immunostaining images of HUVECs (stained with CD31 antibodies, green) alone or HUVECs co-cultured with hiPSC-derived pericyte-like cells (stained with NG2 antibodies, red). Scale bars, 50 μm. D NeuroTrace 500/525 staining of hiPSC-derived NCSCs, pericyte-like cells, and BMECs. hiPSC-derived NCSCs, pericyte-like cells, and BMECs were incubated with NeuroTrace 500/525 (upper panels) for 20 min. hiPSC-derived NCSCs, pericyte-like cells, and BMECs were stained with antibodies against cell type-specific markers such as p75^NTR (magenta), NG2 (red), and ZO-1 (green), respectively (lower panels). Scale bars, 50 μm. E Schematic representation of pericyte-like cells and vSMCs differentiation from the same hiPSCs (ASE9209) via a common NCSC intermediate. Immunostaining of mural cell markers (i.e., PDGFRβ, NG2, and αSMA). Scale bars, 100 μm. NeuroTrace 500/525 staining of NCSC-derived pericyte-like cells and vSMCs (vSMCs-NCSC). Scale bars, 50 μm. F Schematic representation of neuroectoderm-derived vSMCs (vSMCs-NE) and NCSC-derived vSMCs (vSMC-NCSC) differentiations from the same hiPSCs (ASE9209). Immunostaining analysis of mural cell markers (i.e., PDGFRβ, NG2 and αSMA) in both cell types. Scale bars, 100 μm. G NeuroTrace 500/525 staining of vSMCs-NE and vSMCs-NCSC. Scale bars, 50 μm. H In vitro endothelial cord formation assay using HUVECs, vSMCs-NE, and vSMCs-NCSC. Representative bright field images (upper panels) and maps of cord networks analyzed by Angiogenesis Analyzer (lower panels) showing segments (magenta) and branches (green). Scale bars, 500 μm. I Quantification of average segment length from bright field images of vascular cord networks formed by HUVECs alone or HUVECs co-cultured with vSMCs-NE. p-values were calculated using an unpaired Student’s t test. Results are presented as means ± SEM. ** p < 0.01 (n = 5 for each group). All microscopy images are representative of at least three independent experiments