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Updated: Aug 17, 2026
Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood
Published on: September 14, 2017
In vitro differentiation characteristics of cultured human mononuclear cells-implications for endothelial progenitor
Katrin Walenta1, Erik B Friedrich, Falko Sehnert
1Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, 66421 Homburg/Saar, Germany.
Insights
Human mononuclear cells (MNCs) differentiate slowly into endothelial progenitor cells (EPCs) in culture. Most cultured cells remain committed to leukocyte or lymphocyte lineages, highlighting the need for careful surface marker analysis in EPC research.
Area of Science:
- Cardiovascular Research
- Cell Biology
- Immunology
Background:
- Endothelial progenitor cells (EPCs) are crucial in cardiovascular disease pathogenesis and treatment.
- Current characterization of EPCs using DiLDL uptake and lectin staining is reliable, but their precise nature and function remain unclear.
- Understanding EPC surface markers is key to clarifying their role.
Purpose of the Study:
- To investigate the cell surface characteristics of EPCs derived from human blood.
- To analyze the differentiation patterns of mononuclear cells (MNCs) into EPCs over a 10-day culture period.
- To identify specific surface markers for accurate EPC identification in vitro.
Main Methods:
- Isolation of mononuclear cells (MNCs) from human blood.
- Flow cytometry (FACS) analysis of cell surface marker expression before and after 1-10 days of cell culture.
- Quantitative assessment of EPC-specific markers (CD34, CD133, VEGFr2, CD31, CD146) and lineage markers (CD45, CD14, CD3).
Main Results:
- EPC differentiation markers (VEGFr2, CD146, CD31) co-expressed with stem cell markers in <2% of cultured cells.
- Expression of EPC markers like CD34 and CD133 showed dynamic changes during culture.
- A majority of cultured MNCs retained leukocyte/lymphocyte lineage markers (CD45, CD3, CD14), with decreasing expression over time.
Conclusions:
- Human MNCs differentiate into EPCs at a very low rate in vitro.
- Most cells in cultured MNC populations remain committed to leukocyte or lymphocyte lineages.
- Precise surface marker analysis is essential for reliable identification and study of in vitro-derived EPCs.
Abstract:
Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis and treatment of cardiovascular disease. By use of quantitative uptake of DiLDL and lectin staining, EPCs have been characterized reliably. However, the exact nature and function of this cell population still remains poorly defined. In an attempt to further clarify the cell surface characteristics of EPCs, mononuclear cells (MNCs) were isolated from human blood and cell surface expression patterns were defined by FACS analysis before and after differentiation for 1-10 days in cell culture. "Classical" double staining for DiLDL and Ulex europaeus increases to 89.2 /- 0.05 after 10 days in culture. Looking at EPC-specific markers by FACS analysis, 0.18 +/- 0.11% of freshly isolated MNCs express CD34, 0.13 +/- 0.08% CD133, 0.59 +/-0.51% VEGFr2, 0.01 +/- 0.02% CD34/VEGFr2, 0.09 +/- 0.05% CD34/CD133, 0.58 +/- 0.13% CD34/CD31, and 0.02 +/- 0.01% CD34/CD146, respectively. Induction of the endothelial phenotype is evidenced by positive staining for VEGFr2, CD146, and CD31, and occurs in co-expression with stem cell markers in less than 2 +/- 0.52% of cultured cells. Expression of CD34 increases to 0.38 +/- 0.10% after 10 days, whereas the CD133(+) cell population shows an initial peak at 24h (0.29 +/- 0.18%) before decreasing to 0.15 +/- 0.02% at day 10. EPCs co-expressing CD34/CD133 increase to 0.19 +/- 0.09% after 10 days, and EPCs double-positive for CD34/VEGFr2 increase to 1.45 +/- 1.03%. Looking at leukocyte, lymphocyte, and monocyte lineage markers, 56.27 +/- 0.15% of freshly isolated MNCs express CD45, 7.13 +/- 0.02% CD14, and 38.65 +/- 0.01% CD3. Over the 10-day culture period, expression of CD45 decreases to 28.48 +/- 0.18%, CD3 to 23.11 +/- 0.02%, and CD14 to 0.09 +/- 0.02%. Cells co-expressing CD3/CD45 decrease from 38.88 +/- 0.33% to 24.86 +/- 2.49% after 10 days in culture. These findings extend present knowledge by showing that human MNCs differentiate at a very low rate to EPCs, while a majority of the cultured cell population remain committed to the leukocyte or lymphocyte lineage. Careful surface marker analysis might be necessary when using in vitro EPC differentiation systems.
