Journal: Cardiovascular Research

Article Title: Endothelial to haematopoietic transition contributes to pulmonary arterial hypertension

doi: 10.1093/cvr/cvx161

Figure Lengend Snippet: BM-derived ZsGreen+ c-kit+ CD45− progenitor cells are haemogenic. (A) FACS sorting strategy to isolate BM-derived ZsGreen+ c-kit+ CD45− EPCs from the VE-cadherin-cre; ZsGreen double transgenic mice (n = 15). Sorted cells were immediately used for haematopoietic gene expression analysis on a RT2 Profiler PCR Array or for transplantation into lethally irradiated CD45.1 recipient mice (n = 15). The ratio between ZsGreen+ c-kit+ CD45− donor cells from CD45.2 mice (3 × 103) to radio-protective total BM cells from CD45.1 mice (3 × 105) was 1: 100 so that the maximal donor chimerism in the recipient mice would be 1%. (B) FACS sorting strategy to isolate BM-derived haematopoietic stem and progenitor enriched LSK cells from same age C57BL/6J mice (n = 15). Isolated LSK cells were immediately used for haematopoietic gene expression analysis on a RT2 Profiler PCR Array. (C) Relative expression of key haematopoietic genes including transcription factors and markers by the ZsGreen+ c-kit+ CD45− putative haemogenic EPCs (ZsG, blue bars) and by the haematopoietic stem and progenitor enriched LSK cells (LSK, red bars). (D) Heat map of quantitative RT-PCR based gene array comparing 84 haematopoietic gene expression levels of the ZsG cells with the LSK cells. Red indicates up-regulation and green indicates down-regulation of the genes. Black suggests no significant changes in gene expression level. Grey indicates either undetectable or very low level of gene expression in both ZsG and LSK cells, and therefore no reasonable comparison can be made for those genes. Those genes that had most dramatic difference between these two cells are tabulated on the right of the heat map. The data shown in (C) and (D) are mean values of three experiments (n = 15 mice for ZsG cell isolation and n = 15 for LSK cell isolation). (E) Long-term engraftment and reconstitution capability of the BM-derived ZsGreen+ c-kit+ CD45− progenitor cells. Representative FACS analyses of chimerism in peripheral blood of the CD45.1 recipient mice are shown to indicate donor-derived CD45.2+ cells at 8 weeks (n = 14) and 16 weeks (n = 13) but not at 2 weeks (n = 4) or 4 weeks (n = 13) post-transplantation (Tx). Quantitative summary of all recipients are also shown on the right. Data in (C) and (E) are mean ± SEM.

Article Snippet: The Ct values for all wells were exported to a Microsoft Excel spreadsheet for use with the PCR Array Data Analysis software [ www.SABiosciences.com/pcrarraydataanalysis.php (25 August 2017, date last accessed)] according to the manufacturer’s instruction.

Techniques: Derivative Assay, Transgenic Assay, Expressing, Transplantation Assay, Irradiation, Isolation, Quantitative RT-PCR, Cell Isolation