Fig 2: iPSC-PC associate with mouse microvessels, reduces BBB leakage and improves neuronal retention in the hippocampus of pericyte-deficient mice.(a) Schematic showing the in vivo experimental setup. Vehicle (aCSF) or 105 CellTracker-labeled iPSC-PC were stereotaxically injected into the left (contralateral) and right (ipsilateral) hippocampus of 6–8-month old PdgfrbF7/F7 (F7/F7) mice, respectively. (b-c) Representative images and orthogonal views of CellTracker-labeled iPSC-PC (green) on host Lectin+ vessels of pericyte-deficient F7/F7 mice (magenta) counterstained with DAPI (blue) 24h (b) and 48h (c) post-transplantation. Scale bars = 5 μm (overview); 5 μm (cropped view). (d-f) Representative confocal microscopy imaging of extravascular fibrinogen and lectin-positive vasculature in the hippocampi and cortices in F7/F7 mice 5 days after vehicle or transplantation of iPSC-PC transduced with si.Control, si.PDGFRB or si.PTN, and wild type (WT) control mice (control) (d), and quantification of integrated extravascular fibrinogen signal in the hippocampus (e) and the cortex (f). Scale bar in (d) is 25 μm (g-i) Representative confocal microscopy images (g), and quantification of NeuN+ neurons (magenta; h) and SMI312+ neurofilaments (green; i) in the CA1 hippocampus in control mice and F7/F7 mice 5 days after vehicle or transplantation of iPSC-PC transduced with si.Control, si.PDGFRB or si.PTN. Scale bar in (g) is 50 μm. Data in e, f, h, i are mean ± SEM; n= 4 control mice in e,f, and n=5–6 mice for all other conditions in e, f, h, and i. Circles represent individual mice. Significance by one-way ANOVA followed by Tukey’s posthoc test.

Fig 3: iPSC-PC home to brain capillaries of pericyte-deficient mice to form hybrid human-mouse brain capillaries.(a) Schematic showing the ex vivo experimental model. CellTracker-labeled iPSC-PC were incubated with live brain slices from pericyte-deficient PdgfrbF7/F7 (F7/F7) mice for 24 h. (b) Immunoblotting for PDGFRB in iPSC-PC transduced with either control siRNA (si.Control) or human PDGFRB siRNA (si.PDGFRB). (c-d). Integration of CellTracker-labeled iPSC-PC (green) transduced with si.Control or si.PDGFRB with mouse brain capillaries (Magenta) after 24 h incubation with brain slices (c), and quantification of iPSC-PC that homed to capillaries and extended processes along vessels, homed without processes visible, and did not home to mouse brain capillaries from 3 independent experiments per treatment (d). Scale bar: 50 μm. (e) Schematic showing proximity-dependent Proximity ligation Assay (PLA) for protein interactions between iPSC-PC PDGFRB and endothelial (EC) PDGF-BB, and between iPSC-PC vitronectin (VTN) and EC integrin a5 (ITGA5). (f) High-magnification of representative confocal imaging of proximity interactions (red) between PDGFRB and PDGF-BB (left) and between VTN and ITGA5 (right) in CellTracker labeled iPSC-PC (green) and lectin-positive brain capillaries (magenta) after 24 h incubation with brain slices. Merged images show orthogonal views. Data in d are stack bars with mean ± SEM; significance by unpaired two-tailed Student’s t-tests between each homing category.