Mesenchymal stem cells/multipotent stromal cells (MSCs) are promising therapeutics for a variety of conditions. However, after transplantation, cell retention remains extremely challenging. Given that many hypoxic signals are transitory and that the therapeutic administration of MSCs is typically into tissues that are normally hypoxic, we studied the effect of hypoxic preconditioning (HP) prior to new exposure to hypoxia. We show that preincubation for 2 days or more in 1% oxygen reduces serum deprivation-mediated cell death, as observed by higher cell numbers and lower incorporation of EthD-III and Annexin V. Consistently, HP-MSCs expressed significantly lower levels of cytochrome c and heme oxygenase 1 as compared to controls. Most importantly, HP-MSCs showed enhanced survival in vivo after intramuscular injection into immune deficient NOD/SCID-IL2Rgamma 2/2 mice. Interestingly, HP-MSCs consume glucose and secrete lactate at a slower rate than controls, possibly promoting cell survival, as glucose remains available to the cells for longer periods of time. In addition, we compared the metabolome of HP-MSCs to controls, before and after hypoxia and serum deprivation, and identified several possible mediators for HP-mediated cell survival. Overall, our findings suggest that preincubation of MSCs for 2 days or more in hypoxia induces metabolic changes that yield higher retention after transplantation.

The online version of this article has a Supplementary Appendix. BackgroundHematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and 'stemness' of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and MethodsIn the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. ResultsPhase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34 + /CD38 -cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. ConclusionsOur data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system. Haematologica. 2010;95:542-550. doi:10.3324/haematol.2009 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n Hematopoietic stem cells in co-culture with mesenchymal stromal cells -modeling the niche compartments in vitro

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β1 blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.

Multipotent mesenchymal stromal cells (MSCs) are ideal candidates for different cellular therapies due to their simple isolation, extensive expansion potential, and low immunogenicity. For various therapeutic approaches, such as bone and cartilage repair, MSCs are expected to contribute by direct differentiation to replace the damaged tissue, while many other applications rely on the secretion of paracrine factors which modulate the immune response and promote angiogenesis. MicroRNAs (miRNAs), which target messenger RNA for cleavage or translational repression, have recently been shown to play critical functions in MSC to regulate differentiation, paracrine activity, and other cellular properties such as proliferation, survival, and migration. The global miRNA expression profile of MSC varies according to the tissue of origin, species, and detection methodology, while also certain miRNAs are consistently found in all types of MSC. The function in MSC of more than 60 different miRNAs has been recently described, which is the subject of this review. A special emphasis is given to miRNAs that have demonstrated a function in MSC in vivo. We also present in detail miRNAs with overlapping effects (i.e., common target genes) and discuss future directions to deepen our understanding of miRNA biology in MSC. These recent discoveries have opened the possibility of modulating miRNAs in MSC, in order to enhance their proregenerative, therapeutic potential. STEM CELLS