Isolation Methods for Human CD34 Subsets Using Fluorescent and Magnetic Activated Cell Sorting: an In Vivo
Himi Tripathi1, Hsuan Peng1, Renee Donahue1
1Gill Heart and Vascular Institute and Division of Cardiovascular Medicine, University of Kentucky and the Lexington VA Medical Center, Lexington, KY, USA.
Insights
Both fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS) effectively isolate CD34+ cells for cardiac repair after myocardial infarction. These methods improve cardiac function and reduce scarring, offering viable options for cell-based therapies.
Area of Science:
- Cardiovascular Research
- Regenerative Medicine
- Cell Therapy
Background:
- Acute myocardial infarction (AMI) leads to significant cardiac damage and heart failure globally.
- CD34+ cells show promise for treating ischemic heart disease, but optimal isolation methods are unclear.
- Mobilized human peripheral blood mononuclear cells (mhPBMNCs) are a potential source for these therapeutic cells.
Purpose of the Study:
- To compare the efficacy of fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS) for isolating CD34+ cells from mhPBMNCs.
- To evaluate the therapeutic potential of FACS- and MACS-enriched CD34+ cells in a mouse model of myocardial infarction.
Main Methods:
- CD34+ cells were isolated from mhPBMNCs using either FACS or MACS.
- Cell viability, purity, and surface marker expression were assessed via flow cytometry.
- In vivo efficacy was tested in NOD/SCID mice post-myocardial infarction, with intramyocardial transplantation of unselected cells or enriched CD34+ cells.
Main Results:
- Both FACS and MACS yielded high purity and viability of CD34+ cells.
- Transplanted CD34+ cells were retained in the peri-infarct region, promoting angiogenesis and reducing inflammation.
- FACS and MACS groups showed reduced cardiac scarring and fibrosis, leading to improved cardiac function recovery.
Conclusions:
- Cell-based therapy using FACS- or MACS-enriched CD34+ cells significantly improves cardiac recovery post-ischemic injury.
- Both FACS and MACS are effective and comparable methods for CD34+ cell enrichment for clinical applications.
- Enriched CD34+ cell therapy represents a promising strategy for treating myocardial infarction and heart failure.
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.
