Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells

Abstract

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…For significance test, a two‐side t ‐test was used without assuming equal variance. Calculated P ‐values were adjusted for multiple hypothesis testing error using the “Benjamini–Hochberg” (Benjamini & Hochberg, ) method, as previously described (Tsolis et al , ; Tsolis & Economou, ).…”

Embryonic stem cells are devoid of macropinocytosis, a trafficking pathway for activin A in differentiated cells

“…6E). Moreover, both Dynasore and EIPA exhibited a statistically significant inhibition of activin A-induced SMAD2/3 phosphorylation when administered to mature ECs generated by differentiating H1 hESCs via CD34 + vascular progenitor cells (VPCs) (Tsolis et al, 2016) (Fig. 6B), to a comparable level to that observed in HUVECs (Fig.…”

“…Indeed, whereas H1 cells internalised activin A-receptor complexes via CME, the mature ECs derived by differentiation of H1 cells (Tsolis et al, 2016) internalised the ligand-receptor complexes by both CME and MP. Moreover, HFF cells that were capable of internalising activin-receptor complexes by both CME and MP, upon reprogramming to hiPSCs (Kyrkou et al, 2016) retained only the former.…”

“…Since MP is a property shared by most differentiated cells, we addressed whether MP is established early or late during the differentiation process. Towards this purpose, using a method for the differentiation of hiPSCs to ECs via VPC progenitors (Tsolis et al, 2016), we observed that during days 3-5 of the differentiation process, Activin-Alexa488 and Dextran-70 kDa internalisation increased and internalised activin A-Alexa488 colocalised with Dextran-70 kDa and rabankyrin-5 (Fig. 8A).…”

Tissue Engineering Using Vascular Organoids From Human Pluripotent Stem Cell Derived Mural Cell Phenotypes

“…Flow cytometry analysis of the cells was performed as previously described (Tsolis et al, 2016). Briefly, 2.0 × 10 5 cells were incubated with FITC/PE/APC-conjugated anti-human primary monoclonal antibodies ( Supplementary Table S1).…”