Two Distinct Populations of Enriched Non Mobilized Peripheral Blood Mononuclear Cells (PBMNCs) with Different Functional Capacities

Pluripotent stem cells are valuable sources for transplantation and tissue repair. The bone-marrow, umbilical cord blood and G-CSF mobilized peripheral blood are the main sources for adult stem cells. Non-mobilized peripheral blood contains mostly committed cells but recent studies suggest the presence of early progenitor and stem cells as well. Here we aim to develop a method to enrich and recover functional progenitor cell populations from non-mobilized peripheral blood. The ex-vivo enriched non-mobilized PBMNCs were tested by FACS analysis of specific cell surface markers, and by functional analysis of differentiation into different cells lineages. Non mobilized PBMNCs obtained from consenting healthy donors (n=18) cultured for 7 days in a defined cytokine cocktail media were analyzed by FACS using CD14, CD31, CD34, CD45, CD90, CD105, CD117 and CD133 and compare to non-manipulated PBMNCs. The enriched cells were also tested for their differentiation capacity into hematopoietic, endothelial and mesenchymal cells lineages compared to non-manipulated PBMNCs. We found that the enriched PBMNCs resulted in two distinct subpopulations, adherent and non adherent, that present different cell surface markers and have different differentiation capacities. Cell surface markers analysis showed that adherent cells possess high percentage of CD14 (28.1 ±6.13, 11.8 ± 3.9), CD90 (4.24±0.94, 1.53 ± 0.28), CD105 (42.19±8.42, 7.96 ± 4.8), CD117 (9.89±5.99, 1.75± 0.8) expression and reduction of CD45 (46.3±8.14, 62.74± 9.6), CD31 (5.9±1.9, 15.34 ± 4.9) and CD34 (0.18±0.03, 0.62 ±0.3) compare to non-manipulated PBMNCs, respectively. On the other hand, the non-adherent sub-population expresses more CD34 (1.2±0.02, 0.16±0.02), KDR (3.62±0.82, 0.49±0.2), CD105 (21.62±1.85, 7.96 ± 4.8) and CD45 (88.7±0.51, 62.74± 9.6) compared to non-manipulated PBMNCs, respectively. The adherent cells subpopulation showed higher differentiation potential into endothelial (116+23.1 EC colonies/10⁶ cells), mesenchymal (400±53.9 CFU-F/10⁶ cells) lineages compare to non-adherent cells (4.9±1.7 EC colonies/10⁶ cells, 200±29.5 CFU-F/10⁶ cells) and compare to non-manipulated PBMNCs (11.4±1.4 EC colonies/10⁶ cells and 2.5±0.08 CFU-F/10⁶ cells). The non-adherent subpopulations showed higher differentiation potential into hematopoietic colonies (445±75 CFU/10⁶ cells) compared to the adherent sub-population (41.2±24.8 CFU/10⁶ cells), and compared to the non-manipulated PBMNCs (373±39.7 CFU/10⁶ cells) of total colony numbers/10⁶ cells. All results (n=18) are presented as (mean ± SE). In summary, our ex-vivo enrichment methodology yields two different subpopulations with enriched hematological lineage in the non-adherent fraction and enriched endothelial and mesenchymal lineages in the adherent fraction. The ability to obtain enriched populations of endothelial, mesenchymal and hematopoietic progenitors from non mobilized peripheral blood cells may have an important clinical application.