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Endothelial Identity of Porcine Double-sorted CD31+ Kidney Cells

ABSTRACT: Endothelial cells of xenotransplanted gene-edited porcine kidneys are important due to the initial and prolonged exposure to host immune cells. To better study this cell population kidney endothelial cell lines (KECs) were generated by double CD31+ sorting. In order to ensure the endothelial identity of the cells RNA-seq was performed on KECs and the concomitant CD31- population from the initial sort. Analysis of gene expression in KECs showed a depletion of markers associated with tubular identity (SLC34A1, SLC2A2, SLC12A1), and enrichment for markers of endothelial (PECAM1, CDH5, VWF) and kidney endothelial (EHD3, HOX87, HOX89) identity.

ORGANISM(S): Sus scrofa

PROVIDER: GSE211431 | GEO | 2023/04/25

REPOSITORIES: GEO

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Project description:A major challenge in realizing regenerative treatment using freshly isolated Adipose Derived Regenerative Cells (ADRCs) is the cellular heterogeneity and donor variability. Ex vivo expanded ADRCs (=ASCs) are available at larger quantities and represent a more homogeneous population suitable for allogenic use. Emerging pre-clinical and clinical data however suggest similar, or even superior efficacy of ADRCs as compared to ASCs for indications involving (micro)vascular deficiency. Since CD31+ ADRCs lack in cultured ASCs, we hypothesized that these cells account for improvement of vascular function in the heterogenous ADRCs. Herein, we found that in patients with erectile dysfunction (ED), the number of injected CD31+ ADRCs correlates positively with erectile function 12 months after one bolus of autologous ADRCs. Comprehensive in vitro and ex vivo analyses confirmed superior pro-angiogenic and paracrine effects of human CD31+ enriched ADRCs compared to the corresponding CD31- and parent ADRCs. CD31+, CD31- and ADRCs were co-cultured in aortic ring-, as well as in ED-relevant corpus cavernousum tube formation assays, both showing a significantly higher ability of the CD31+ ADRCs to support tube development. This effect was corroborated using conditioned medium (CM), while quantitative mass spectrometric analysis suggested that this is explained by secretory pro-angiogenic proteins such as DKK3, ANGPT2, ANAX2 and VIM, all being enriched in CD31+ADRC CM. To gain further specification of the ADRC subpopulations expressing these proteins, single-cell RNA sequencing was performed and showed that transcripts of the upregulated and secreted proteins were present in 9 endothelial ADRC subsets including endothelial progenitor cells in the heterogenous non-cultured ADRCs. Our data thus suggest that the vascular benefit of using ADRCs in regenerative medicine is dictated by CD31+ ADRCs, which likely represent an improved alternative to heterogenous ADRCs as well as ASCs when aiming for vascular repair in cell therapy.

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Endothelial Identity of Porcine Double-sorted CD31+ Kidney Cells

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