Human Primary CD271⁺/CD45⁻/CD146^−/Low and CD271⁺/CD45⁻/CD146⁺ Bone Marrow Cells Are Developmentally Closely-Related Stroma Stem Cells with Similar Functional Properties but Different In-Situ Localization.

Abstract 2594

Mesenchymal stem cells (MSC) are of central importance for the hematopoietic microenvironment. However, the exact contribution of specified MSC populations to bone marrow stroma anatomy and function is unknown. We have previously characterized the phenotype of primary human bone marrow MSC and found that all assayable CFU-F were highly and exclusively enriched not only in the lin⁻/CD271⁺/CD45⁻/CD146⁺ cell fraction, but also in lin⁻/CD271⁺/CD45⁻/CD146^−/low cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype and gave rise to typical cultured stroma cells. However, we observed that CD146 expression was up-regulated in normoxia and down-regulated in hypoxia, which correlated to in situ localization differences: CD146 co-expressing reticular cells were located in perivascular regions, whereas bone-lining MSC expressed CD271 alone (Tormin et al, Blood 2009, 114[22]:107). We now went on to further characterize the two populations with regard to in-situ localization and function. Multicolor confocal microscopy analysis of normal human bone marrow sections revealed that CD34⁺ hematopoietic stem/progenitor cells were located in close proximity to CD271⁺ MSC in perivascular as well as endosteal regions. Ongoing experiments address whether particular HSC subsets localize specifically with certain stroma stem cell populations. To further investigate possible functional differences between lin⁻/CD271⁺/CD45⁻/CD146⁺ and lin⁻/CD271⁺/CD45⁻/CD146^-/low cells, FACS-sorted single cells were clonally expanded, loaded overnight on hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder and transplanted s.c. into NOD-SCID mice. Eight weeks post transplantation, bone, adipocytes, fibroblastic tissue, and capillaries could be detected in both transplants. Orthotopic intrafemoral transplantations into irradiated NSG mice were performed with GFP-labeled MSC generated from either lin⁻/CD271⁺/CD45⁻/CD146^-/low or lin⁻/CD271⁺/CD45⁻/CD146⁺ cells. After 8 weeks, GFP⁺ cells could be detected in the perivascular regions surrounding the endothelium of vessels, and as cells lining the surface of cortical and trabecular bone, surrounding adipocytes, or as reticular cells in the marrow space. Some of the bone-lining GFP⁺ MSC were found to express N-cadherin. Interestingly, this anatomical distribution is similar to the localization of primary MSC in human marrow in situ. No differences were observed between transplanted cells from lin⁻/CD271⁺/CD45⁻/CD146^-/low MSC compared to lin⁻/CD271⁺/CD45⁻/CD146⁺ derived cells. Secondary colony-formation capacity was investigated by harvesting bone marrow cells 8 weeks post intrafemoral transplantation and plating them for CFU-F in standard MSC culture medium. GFP-positive fibroblastic colony growth was detected in the bone marrow of mice transplanted with lin⁻/CD271⁺/CD45⁻/CD146^-/low as well as in the marrow of mice transplanted with lin⁻/CD271⁺/CD45⁻/CD146⁺ derived MSC. Taken together, our findings indicate that lin⁻/CD271⁺/CD45⁻/CD146^-/low and lin⁻/CD271⁺/CD45⁻/CD146⁺ bone marrow cells are developmentally closely-related stroma stem cells with similar functional properties but different in-situ localization, which might be the first step towards a better characterization of the human hematopoietic microenvironment.