Fig. 3: Human periventricular endothelial cells significantly promoted interneuron migration in vivo.

a Schema of transplantation strategy in adult NOD-SCID mouse. Human interneurons plus periventricular endothelial cells were co-transplanted in the striatum of 8-week-old adult NOD-SCID mice. Interneurons only, periventricular endothelial cells-only, or interneurons plus control endothelial cells were also transplanted in separate experiments. One month after transplantation, brains were collected and processed for histology or IHC. b–e H&E staining of co-transplanted brain showing the graft core (red asterisk) and the cell distribution close to graft (yellow arrows). Individual markers human β-tubulin (f), human mitochondria (g) and a co-labeled image of both with DAPI (h) illustrate grafted neurons in interneuron-only transplanted brain that remained mostly within the graft area (white arrows). Red arrow marks lateral ventricle boundary. i Magnified images of stalled neurons that are human mitochondria⁺ at graft sites. j In periventricular endothelial cells-only transplanted brains, grafted endothelial cells labeled with human vWF and CD31 markers showed robust migration within the striatum. Nuclei are stained with DAPI. k–m In interneuron + periventricular endothelial cell co-transplanted brains, interneurons showed significantly higher migration ability, and distributed widely in the striatum (white arrows). A co-labeled image of human β-tubulin, human mitochondria and DAPI is shown in m. Red arrows mark the lateral ventricle boundary. n Robust migration (white arrows) of periventricular endothelial cells in co-transplanted striatum. (o, o′) Co-labeling with isolectin B4 and anti-human CD31 shows how newly formed CD31⁺ vessels anastomose with host vessels. p In interneuron-only transplanted brains, grafted neurons showed very little migration into the cortex (red asterisks). The white dotted area marks the grafted site in the striatum where most of the neurons remained restricted. Additional stalled neurons (white arrows) from different brains are shown at ×20 (q) and ×40 (r) magnifications. s In neurons + control endothelial cells (without GABA and WNT7A) transplanted brain, grafted neurons remain stalled at the graft site (marked by white dotted circle). t In periventricular endothelial cells-only transplanted brain, endothelial cells migrated efficiently into the cortex. u In co-transplanted brains, grafted interneurons showed robust migration into cortical region (white arrows). v Magnified images (×40) of grafted human interneurons in the cortex. w Migrated cells in the cortex of co-transplanted brains show human-NKX2.1 labeling. x Robust distribution of human-CD31⁺ endothelial cells in the cortex of co-transplanted brains. y Cell count analyses of human-nuclei⁺ cells that have migrated into the cortex after 30 days. Data represent mean ± SD, (n = 20, ***P < 0.001, **P < 0.01, Student’s t test). z Quantification of β-tubulin⁺/human nuclei⁺ neurons in the cortex of transplanted brains 30 days after grafting. Data represent mean ± SD, (n = 20, ***P < 0.001, Student’s t test). Significantly higher percentage of interneurons in neuron + periventricular endothelial cell co-transplanted brains showed long-distance migration compared to those in interneuron-only condition or interneuron + control endothelial cells derived without GABA and WNT7A condition. For (y) and (z), cell numbers in cingulate, motor, somatosensory and piriform cortex at all bregma levels were analyzed. Scale bars: b 100 µm (applies to b–d, f–h, j, k–n, p, s–u, w, x), e 50 µm (applies to i, o, q), r 25 µm (applies to o′, v). Cpu striatum, cc corpus callosum, IN interneurons-only, CN EC: control endothelial cells derived without GABA and WNT7A, PVECs periventricular endothelial cells.