Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells

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

Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells

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

Total distance covered in the Open Field per 5 minute bin (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age and test time during the dark phase of the diurnal cycle.

<p>Total distance covered in the Open Field per 5 minute bin (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age and test time during the dark phase of the diurnal cycle.</p

Qualitative Polymerase Chain Reaction (qPCR) information.

<p>Qualitative Polymerase Chain Reaction (qPCR) information.</p

Test ages for behavioral work, broken down by test cohort.

<p>Test ages for behavioral work, broken down by test cohort.</p

Proportion of mice climbing in the rearing climbing assay in the wild type, heterozygous and homozygous group during the dark phase of the diurnal cycle.

<p>Proportion of mice climbing in the rearing climbing assay in the wild type, heterozygous and homozygous group during the dark phase of the diurnal cycle.</p

Kaplan-Meier survival curve in WT vs. homozygous mice as a function of genotype and age.

<p>Kaplan-Meier survival curve in WT vs. homozygous mice as a function of genotype and age.</p

Rearing frequency in the Open Field (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age during the dark phase of the diurnal cycle.

<p>Rearing frequency in the Open Field (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age during the dark phase of the diurnal cycle.</p

Grip strength (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age during the light phase of the diurnal cycle.

<p>Grip strength (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age during the light phase of the diurnal cycle.</p

Body weight (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age for female (A) and male (B) mice.

<p>Body weight (mean ± SEM) of wild type, heterozygous and homozygous mice as a function of age for female (A) and male (B) mice.</p