Use of Some Additives for Improving Mesenchymal Stem Cell Isolation Outcomes in Non-Mobilized Peripheral Blood

Background: The mesenchymal stem cells (MSCs) of peripheral blood (PB) have been recognized as a promising source for allogeneic cell therapy. The objective of the present study was to isolate and characterize MSCs derived from non-mobilized PB, and evaluate their differentiation potential. Methods: The buffy coat mononuclear fractions of the PB were concentrated using the Ficoll-Paque density gradient centrifugation and were grown on primary and secondary culture media, respectively. The isolated cells were characterized using a multidisciplinary approach which was based on morphology, immunophenotyping, gene expression, and multipotentiality. Flow cytometry and Reverse transcription polymerase chain reaction (RT-PCR) were used to identify the expression of different MSC markers. Finally, after culturing in osteogenic and adipogenic induction media, the isolated cells were stained by Alizarin red and Oil-Red O. Results: In spite of absence of any bone marrow stimulating factor, the isolation approach in this study yielded a rather homogeneous and spindle-shaped mononuclear cell population (the yield of passage 0 was 0.65 ± 0.15) that stained positive for CD90, CD105, and CD73, and were negative for CD45 and CD34. These cells have high proliferative capacity (confirmed by the expression of Oct-4, Nucleostemin, and Nanog genes) and were able to differentiate into lineage-specific committed cells, when exposed to the appropriate medium. Conclusions: Overall, it can be concluded that conventional, labour-intensive and time-consuming approaches are not necessary in isolating MSCs from PB. This relatively accessible and minimally invasive source, PB, represents a good alternative reservoir of homogeneous MSCs that could open a new era for practical exploitation in regenerative medicine.