Isolation Methods for Human CD34 Subsets Using Fluorescent and Magnetic Activated Cell Sorting: an In Vivo Comparative Study

Introduction: Acute myocardial infarction (AMI) and resulting cardiac damage and heart failure are leading causes of morbidity and mortality worldwide. Multiple studies have examined the utility of CD34⁺ cells for the treatment of acute and ischemic heart disease. However, the optimal strategy to enrich CD34 cells from clinical sources is not known. We examined the efficacy of fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS) methods for CD34 cell isolation from mobilized human mononuclear peripheral blood cells (mhPBMNCs). Methods: mhPBCs were processed following acquisition using FACS or MACS according to clinically established protocols. Cell viability, CD34 cell purity and characterization of surface marker expression were assessed using a flow cytometer. For in vivo characterization of cardiac repair, we conducted LAD ligation surgery on 8–10 weeks female NOD/SCID mice followed by intramyocardial transplantation of unselected mhPBMNCs, FACS or MACS enriched CD34⁺ cells. Results: Both MACS and FACS isolation methods achieved high purity rates, viability, and enrichment of CD34⁺ cells. In vivo studies following myocardial infarction demonstrated retention of CD34⁺ in the peri-infarct region for up to 30 days after transplantation. Retained CD34⁺ cells were associated with enhanced angiogenesis and reduced inflammation compared to unselected mhPBMNCs or PBS treatment arms. Cardiac scar and fibrosis as assessed by immunohistochemistry were reduced in FACS and MACS CD34⁺ treatment groups. Finally, reduced scar and augmented angiogenesis resulted in improved cardiac functional recovery, both on the global and regional function and remodeling assessments by echocardiography. Conclusion: Cell based therapy using enriched CD34⁺ cells sorted by FACS or MACS result in better cardiac recovery after ischemic injury compared to unselected mhPBMNCs. Both enrichment techniques offer excellent recovery and purity and can be equally used for clinical applications.