Purification and Functional Characterization of CD34-Expressing Cell Subsets Following Ex Vivo Expansion of Umbilical Cord Blood-Derived Endothelial Colony-Forming Cells

Abstract

Fluorescent-activated cell sorting (FACS) remains a powerful tool to enrich blood-derived progenitor cells for the establishment of highly proliferative endothelial colony-forming cells (ECFC). Further investigation remains necessary to determine whether the retention of progenitor cell phenotypes after expansion can identify ECFC with enhanced proangiogenic and regenerative functions. This study employed FACS purification to segregate umbilical cord blood-derived ECFC using conserved provascular progenitor cell markers CD34 or aldehyde dehydrogenase (ALDH) activity. ECFC FACS purified for high versus low ALDH activity formed single cell-derived colonies and demonstrated tubule formation in Matrigel at comparable rates. Surprisingly, FACS purification of ECFC for CD34 enriched cells with enhanced colony-forming capabilities and tubule formation within the CD34^- population. CD34 expression was enriched on early ECFC populations; however, steady-state expression of CD34 rapidly declined and stabilized on expanded ECFC after serial passage. CD34 expression on ECFC was shown to be cell density dependent and coincided with a loss of progenitor cell characteristics in vitro. Silica-bead surface membrane capture followed by proteomic analysis by label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) identified >100 distinctions (P < 0.05) associated with the plasma membrane of CD34^- versus CD34⁺ ECFC, including a significant enrichment of CD143 (angiotensinogen converting enzyme) on CD34⁺ cells. Despite an enrichment for traditional endothelial cell markers on the CD34⁺ ECFC in vitro, implantation of both CD34⁺ and CD34^- ECFC within Matrigel plugs in immunodeficient NOD.SCID mice promoted the formation of vessel-like structures with equivalent integration of human cells at 7 days post-transplantation. Although positive selection of CD34 enriched ECFC establishment before culture, FACS-purified CD34⁺ ECFC demonstrated reduced colony and tubule formation in vitro, yet demonstrated equivalent vessel formative function in vivo compared to CD34^- counterparts. The knowledge will support future studies aiming to identify ECFC subsets with enhanced vessel forming functions for applications of regenerative medicine.

Keywords: CD34; aldehyde dehydrogenase; blood vessels; endothelial cells; proteomics.