Murine Mesenchymal Stem Cells Improve Haemopoietic Engraftment in a Murine Transplant Model and Is Maximised through Osteogenic Stimulation.

Several studies have preciously shown improved engraftment of human haemopoietic stem cells (HSC) following co-infusion of human mesenchymal stem cells (MSC). However, these studies have been criticised for using xenogeneic recipients which might support human haemopoiesis through the production of species-specific cytokines and growth factors by human MSC. To further investigate the potential for MSC to support HSC engraftment we used a murine to murine transplant model to co-infuse purified murine MSC and murine HSC.

The MSC were derived from collagenase-treated bone fragments of the Rosa26 murine strain, had the phenotype CD45- CD31-CD11b- Sca-1^bright VCAM1+ and could differentiate into osteocytes, chondrocytes and adipocytes. Donor and recipient HSC were obtained from unmanipulated bone marrow of either the C57Bl/6J murine strain, CD45 isotype CD45.2, or the PEP3b murine strain, expressing CD45.1. Following 10Gy irradiation to the recipients HSC and MSC were co-infused. Donor and recipient haemopoietic engraftment in the peripheral blood was quantified by FACS analysis of the CD45.1/CD45.2 ratio.

Successive transplant experiments persistently showed improved donor haemopoietic engraftment following co-infusion of 2 x 10⁵ BM cells and 1 x 10⁶ MSC (three seperate transplant series p=0.001, p=0.09 and p=0.13). The beneficial effect was maximal at the lower HSC dose of 1 x 10⁵ BM cells (p=0.004, p=0.045 and uninterpretable in the third transplant series due to excessive death observed in the non-MSC recipients).

Increasing the MSC dose to 2 x 10⁶ cells showed a non-significant increase in survival and donor haemopoietic engraftment but further MSC escalation resulted in fatal emboli post-infusion.

LacZ staining of tissue sections failed to show evidence of MSC outside the lungs but was detected at very low levels in the bone from one recipient.

As MSC are osteoblast precursors and recent literature suggests a role of osteoblasts as the HSC niche we investigated the effect of pre-culturing our MSC population in osteogenic media prior to transplant. Following 16 days of culture approximately 40% of colonies showed ALP activity. At this time point osteogenic-stimulated MSC (O-MSC) were removed from culture, washed to remove any residual osteogenic media, and infused in our standard method. O-MSC recipients receiving 1 x 10⁵ BM cells showed a mean engraftment of 79% compared to 21% in non-msc recipients receiving 2 x 10⁵ BM cells (p=0.000017), and 42% in recipients receiving standard MSC and 1 x 10⁵ BM cells (p=0.01).

In conclusion we confirm improved haemopoietic engraftment in a non-xenogeneic model and does not require significant MSC engraftment. The beneficial effect is maximised through osteogenic stimulation of MSC suggesting the possibility that the mechanism may be through priming of the HSC niche.