Induced differentiation and molecular characterization of monocyte-derived multipotential cells (MOMCs) generated from commonly discarded leukapheresis filters

Abstract

Background & Aim Μonocyte-derived multipotential cells (MOMCs), easily isolated from the peripheral blood, include progenitors capable of differentiating into endothelial lineages in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis (Kuwana M et al, 2012). Methods, Results & Conclusion discarded leukapheresis filters (T.P.H. Meyer et al, 2005)and estimate the expression of several stem cell factors that play a key role in their pluripotency and differentiation capacity. A leukocyte elution medium composed of Citrate phosphate dextrose (CPD), hypertonic SAGM and Phosphate Buffered Saline was prepared and used for back-flushing of the filters, opposite to the primary blood flow during the leukocyte depletion step. Upon centrifugation in density gradient, mononuclear cells were collected and subsequently incubated in 37οC 5% CO2conditions for 10 days in poly-L-lysine+gelatin coated plates in the presence of 50 ng/ml SDF-1a chemotactic factor enabling the MOMCs generation. Aiming the characterization of the harvested cells, flow cytometry was performed for the detection of typical surface marker expression (CD45, CD14, CD34, Collagen type I and CD184). The induced differentiation capacity of MOMCs towards bone and endothelium in specific culture mediums was estimated via alizarin red staining and flow cytometry, respectively. Finally, the expression levels of genes related with self-renewal and differentiation were estimated with Real-time PCR. The generated MOMCs were characterized by high expression of the CD45/CD14/CD34/CD184/Col I markers while progressively lost their native markers and gained endothelial or bone features upon induced differentiation. The MOMCs expressed several stem cell factors like Nanog, Oct4, c-Myc, Sox2, Klf4, Sall4, ESRRB and ZNF214. All these factors are responsible to activate the networks related with the pluripotency, differentiation and reprogramming of cells while are highly expressed in embryonic stem cells. To conclude, we successfully optimized conditions to elute leukocytes adsorbed to leukapheresis filters resulting in leukocyte suspensions collection for multiple uses. The easy and quick generation of MOMCs from monocytes derived from leukapheresis filters, makes them potential candidates for future therapeutic application in tissue regeneration.