Stem cell transplantation is a viable strategy for regenerative medicine. However, it is inevitable to have cells undergo storage for several hours or days due to processing and transportation. Therefore, it is crucial to have rigidly controlled conditions ensuring the therapeutic benefit of isolated stem cells. In the present study, we investigated the impact of short-term storage on human CD133(+) cells. CD133(+) cells were isolated from human bone marrow and kept at standardized nonfreezing storage conditions for up to 72 h. Cell viability (apoptosis/necrosis) and expression of CD133 and CXCR4 were analyzed by flow cytometry. Metabolic activity was determined using an MTT assay; colony-forming ability, as well as endothelial-like differentiation, was further evaluated. A qRT-PCR array was employed to investigate the expression of stemness genes. CD133 and CXCR4 expressions were preserved at all time points. After 30 h, cell number and metabolic activity decreased, although no significant changes were detected in cell viability and proliferation as well as endothelial-like differentiation. Cell viability and proliferation decreased significantly only after 72 h of storage. Our results indicate that storage of isolated human CD133(+) bone marrow stem cells in liquid allows for high viability and functionality. However, storage time should be limited in order to avoid cell loss.

Human bone marrow stem cell populations have been applied for cardiac regeneration purposes within different clinical settings in the recent past. The migratory capacity of applied stem cell populations towards injured tissue, after undergoing specific peri-interventional harvesting and isolation procedures, represents a key factor limiting therapeutic efficacy. We therefore aimed at analyzing the migratory capacity of human cluster of differentiation (CD) 133 + bone marrow stem cells in vivo after intraoperative harvesting from the sternal bone marrow. Human CD133 + bone marrow stem cells were isolated from the sternal bone marrow of patients undergoing cardiac surgery at our institution. Migratory capacity towards stromal cell-derived factor-1a (SDF-1a) gradients was tested in vitro and in vivo by intravital fluoresecence microscopy, utilizing the cremaster muscle model in severe combined immunodeficient (SCID) mice and analyzing CD133 + cell interaction with the local endothelium. Furthermore, the role of a local inflammatory stimulus for CD133 + cell interaction with the endothelium was studied. In order to describe endothelial response upon chemokine stimulation laser scanning microscopy of histological cremaster muscle samples was performed. SDF-1a alone was capable to induce relevant early CD133 + cell interaction with the endothelium, indicated by the percentage of rolling CD133 + cells (45.9 ± 1.8% in "SDF-1" vs. 17.7 ± 2.7% in "control," p < 0.001) and the significantly reduced rolling velocity after SDF-1a treatment. Furthermore, SDF-1a induced firm endothelial adhesion of CD133 + cells in vivo. Firm endothelial adhesion, however, was significantly enhanced by additional inflammatory stimulation with tumor necrosis factor-a (TNF-a) (27.9 ± 4.3 cells/mm 2 in "SDF-1 + TNF" vs. 2.2 ± 1.1 cells/mm 2 in "control," p < 0.001). CD133 + bone marrow stem cells exhibit sufficient in vivo homing towards SDF-1a gradients in an inflammatory microenvironment after undergoing standardized intraoperative harvesting and isolation from the sternal bone marrow.

Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.