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.

人类胚胎干细胞（human embryonic stem cells, hESCs）凭借其分化可塑性与增殖潜能，在再生医学（regenerative medicine, RM）中具备极高应用价值。然而，再生医学领域的核心挑战之一，在于构建可向新生合成组织输送营养的血管系统。本研究对现有方法进行了优化：采用化学成分明确的培养基与培养条件，将hESCs定向分化为CD34+血管祖细胞，以此构建致密血管网络；同时从同步产生的CD34-细胞扩增群中，选择性纯化得到上述细胞。
为解析上述分化过程，本研究采用高分辨率质谱、无标记定量技术与多变量分析方法，对比了hESCs、CD34+及CD34-细胞群体的蛋白质组。经免疫学实验验证，共有18种蛋白质标志物可用于确认分化表型；其中9种可通过蛋白质组学检测到，且丰度差异具有统计学显著性。另有225种蛋白质在三类细胞群体中呈现丰度差异，其中63种的功能已在CD34+与CD34-细胞中得到明确阐释。CD34+细胞所合成的蛋白质参与内皮细胞分化与平滑肌形成，这一结果佐证了该祖细胞的双潜能表型。CD34-细胞群体异质性更强，可合成肌源性、成骨源性、成软骨源性及成脂源性的谱系标志物。CD34+或CD34-细胞中剩余的150余种丰度差异蛋白质，为后续探索血管形态发生的研究提供了可验证的假说方向。