Murine Adherent CD34+ Cell Population Expanded by Single Cell Cloning (Pages: 222-229)

Mohamadreza Baghaban Eslaminejad * *, Fardin Fathi , Poopak Eftekhari Yazdi , Takoyuki Asehara ,


Introduction: While human endothelial progenitor cells (EPCs) have been a subject of somehow extensive investigation, EPCs from adult mouse hematopoietic system were poorly studied. Present investigation is focused on FVB mouse endothelial progenitor cells in terms of their isolation, purification, and expansion. Materials and Methods: Mononuclear cells collected from murine peripheral blood were cultured in fibronectin coated plate for two weeks, at which point, the adherent cell population were lifted and analyzed in terms of some surface markers. Using FACS Vantage equipped with one-cell deposition unit, single CD34 positive cells were plated per well already containing medium optimized for single cell growth. Several clones were then emerged, expanded, and examined in terms of some surface markers. Furthermore, the cells were investigated regarding ability to uptake DiI-ac-LDL and form capillary network on matrigel surfaces. Results: Adherent population of mononuclear cells from mouse peripheral blood was appeared morphologically heterogeneous. About 5% of the adherent cells were CD34 positive. Having optimized their culture condition, several CD34 positive clones were expanded. The cells comprising the clones were DiI-ac-LDL+ and formed capillary-like tube when being seeded on matrigel surfaces. Conclusion: The primary culture of the mononuclear cells from murine peripheral blood contains a very limited number of cells positive for endothelial lineage markers. These cells (adherent CD34 positive) could be expanded by single cell cloning technique.