Characterization Of Bone Marrow Derived CD34+ Cells With Different Mobility Potentials By Micro RNA Fingerprinting

AMR-001, an autologous CD34⁺ cell product derived from mini-marrow harvest, is currently undergoing Phase II trials to treat acute myocardial infarction (AMI). AMR-001 is administered to the patient by infusion via the infarct related artery within five to ten days following coronary artery stenting post AMI. At the time of infusion, it is believed that the infarct-region SDF-1 (stromal derived factor) levels are peaked and scar formation has not yet occurred. It was found that, in addition to the quantity of CD34⁺ cells infused, improvement in cardiac perfusion and infarct size correlated with the mobility potential of CD34⁺ cells mediated by a SDF-1 gradient (Quyyumi et al, Am Heart J 2011, 161:98–105).

We have developed a cell based in vitro mobility assay as a potential potency release assay for AMR-001. However, this assay is not suitable for a Phase III or commercial scale release assay due to the length of the assay, high skill level required to perform, and variability. To develop a more robust assay, we have initiated a study to identify potential microRNAs (miRNAs) that may be used as biomarkers for CD34⁺ cell SDF-1 driven migration. Our preliminary results suggest CD34⁺ cells with different mobility potentials may be characterized by miRNA fingerprinting.

Cryopreserved purified CD34⁺ cells derived from bone marrow of healthy donors were purchased from a commercial vendor. Thawed CD34⁺ cells were washed and the cells were assayed in an in vitro transwell system (Jo et al, J Clin Invest 2000, 105:101-111). The trans-membrane migration of CD34⁺ cells into the lower chamber in the presence of SDF-1, as well as the non-mobilized CD34⁺ cells in the upper chamber, were collected after 4 hours incubation at 37°C. Total RNA of the cells was isolated and the miRNA expression profile was analyzed using SurePrint G3 Human v16 microRNA 8x60K microarray slide (Agilent, Santa Clara, CA). A normalization algorithm was used to generate miRNA expression profiles (SistemQC™, Sistemic, Ltd) for the characterization of untreated cells, the mobilized population that migrate towards SDF-1, and non-mobilized population; from two independent donors.

Two hundred and four (204) miRNAs were reliably detected across the cell samples. The mobilized cells had different miRNA profiles compared with non-mobilized/untreated cells. Hierarchical cluster analysis showed that mobilized cells grouped separately from the non-mobilized/untreated cells.

Analysis of the miRNA profiles of the CD34⁺ cells across two independent donors, identified a number of key miRNAs (kmiRs™) that represent possible markers for a mobility phenotype. Additional samples will be analyzed to confirm these preliminary findings. This approach will enable the identification of markers associated with mobility potential of CD34⁺ cells and the potential development of a molecular biomarker assay for potency.

Warbington:Progenitor Cell Therapy, LLC: Employment. Weinstein:Progenitor Cell Therapy, LLC: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Lin:Progenitor Cell Therapy, LLC: Employment. LeBlon:Progenitor Cell Therapy, LLC: Employment. Fong:NeoStem, Inc.: Employment. Chan:Progenitor Cell Therapy, LLC: Employment.