37 Limited Ability of Long-Term Cultured Murine Marrow-Derived Mesenchymal Cells to Support Hematopoiesis

Abstract

We studied the growth, differentiation and ability to support hematopoiesis of long-term cultured murine marrow-derived mesenchymal cells (MC) cultured in serum-containing medium but without additional cytokines. Unfractionated marrow from C57Bl6/J (Bl/6; CD45.2+) mice was plated in DMEM with 10% bovine serum; adherent cells were passaged when >80% confluent. During early (<6 weeks) culture, adherent cells were ∼50% CD45+ and showed variable morphology. After 6-8 weeks of slow growth (<1 passage/week) a population of fibroblast-like, rapidly dividing (∼24h doubling time) cells emerged, which we have continuously cultured for at least 8 months. These cells express a surface phenotype (<2% CD45+; >90% CD29, 90, 105, 106+) consistent with MC, and are capable of osteogenic and adipogenic differentiation in vitro. Analysis of five clones derived from one long-term MC culture showed similar growth characteristics as the parental culture, but the clones displayed variability in surface phenotype and the ability to undergo adipogenic differentiation. Furthermore, 2/5 clones were unable to undergo osteogenic differentiation, indicating that long-term MC cultures are comprised of heterogeneous subpopulations of cells. Transplantation of 106 long-term cultured MC with 5x105 unfractionated B6.SJL-PtrcaPep3b/BoyJ (BoyJ; CD45.1+) marrow cells into ablated BoyJ hosts revealed <2% MC-derived CD45.2+ blood cells four months post-transplant, demonstrating that long-term cultured MC lack inherent hematopoietic potential. Marrow cells co-cultured on long-term cultured MC feeder layers in alpha-MEM with 20% serum produced ∼5-fold more hematopoietic colonies in CFU-C assays than marrow cultured without MC feeders. Marrow cells co-cultured on MC displayed >5-fold lower repopulating ability, however, compared to marrow cells cultured without MC feeders, four months post-transplant in two independent competitive repopulation assays. These data suggest that long-term cultured MC, though morphologically and phenotypically homogeneous and lacking hematopoietic potential, may not support ex vivo culture/expansion of primitive hematopoietic cells, at least under these stringent (i.e., without additional cytokines) conditions. Assays comparing the hematopoiesis-supporting capacity of long-term cultured MC to classic Dexter-type stroma, and studies to determine optimal co-culture conditions are ongoing.