CD133 Positive Mesenchymal Stem Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression and Plasticity.

Abstract

Background: Mesenchymal Stem Cells (MSC) can be isolated from bone marrow, adipose and fetal tissues, but their presence in Mobilized Peripheral Blood (MPB) and in Umbilical Cord Blood (UCB) remains controversial.Methods: In this study we evaluated whether MPB (n=6) and UCB (n=8) could be two other sources of MSC beside Bone Marrow (BM). CD133 positive cell fraction was isolated through immunomagnetic system and MNC were seeded in medium supplemented or not with 5% of conditioned medium durong the first 48 hours of adhesion. MSC derived from MPB or UCB were identified by their expression of mesenchymal (SH2, SH3) and hematopoietic markers (CD14, CD34, CD45 and CD62-E). MPB and CB-MSC were also tested for their capacity to generate CFU-F (Fibroblast colony-forming units) and to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells after specific induction. Finally we tested through RT-PCR the gene expression of Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity.Results: Through CFU-F, we observed that the selection of CD133 positive cells allows to obtain a great amount of MSC in comparison with the MNC fraction seeded (MPB-MSC, 139 versus 72 and UCB-MSC 165 versus 8 after the primoculture). After four passages, more than 1.108 cells were obtained by the cell expansion assays from 1.106 cells seeded in primoculture. During the first culture, whatever the medium used or cell fraction seeded, the cell population was composed of osteoblasts, osteoclasts, marcophages and hematopoietic cells with less than 5%SH3 positive cells. After two passages, cells derived from MPB or UCB expressed mesenchymal markers (SH3 and SH2) and Oct4. Under appropriate conditions these cells were able to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells.Conclusion: These results indicate that MSC are present in MPB and UCB, with similar characteristics to bone marrow (self-renewal capacity and multi-diffentiation potential), and may have a major clinical importance due to their accessibility. They could be an attractive target for cellular and gene therapiesMesenchymal marker expression (%)T0 (%)second passage (%)MPBSH22,7±1,196,1±2,9SH34,9±1,595,7±3,3UCBSH21,4±0,698±0,7SH35,2±1,896,5±2,5