Osteoblast-Stem Cell Contact Is Required for B-Lymphopoietic Proliferation, Not Differentiation.

Abstract 3635

Poster Board III-571

Nonparenchymal stromal cells form the cellular constituents of tissue-specific stem cell niches. One key question in stem cell biology is the nature of signals provided by stromal cells in general, and via stem cell-stromal cell contact in particular, that direct the survival, proliferation and differentiation of tissue stem cells. We and others have previously reported that osteoblasts dictate the differentiation of hematopoietic stem cells (HSC) to B lymphopoiesis, supporting the acquisition of E2A, EBF1 and Pax5, as well as cell surface B220, CD18 and sIgM, with Flt3-ligand (FL) and interleukin 7 (IL-7) required for these activities. In this context, a fundamental unanswered question is whether, and if so why, B lymphopoiesis requires direct cell-cell contact between HSCs and osteoblasts. In the present study, we asked whether HSC contact with osteoblasts plays its principle role in B lymphopoiesis by supporting B differentiation, by supported proliferation, or both. Magnetically purified mouse Lin- Kit+ (LK) bone marrow cells were cultured for 11 days in the presence of 50 ng/ml FL plus 100 ng/ml IL-7, in direct contact with OP-9 or MS-5 osteoblastoid cells, or in proximity to OP-9/MS-5 cells but separated by a Transwell filter. Following co-culture, the hematopoietic cells were counted, the presence of B lineage-specific cell surface markers measured by flow cytometry, and the expression levels of B-lineage transcription factors E2A, EBF and Pax5 measured by quantitative PCR. LK cultured in FL + IL-7 acquired B220 as well as low levels of E2A expression, but did not express Pax5 expression nor acquire cell surface CD19 or IgM, suggesting differentiation to Pre-Pro B cells but not beyond; in contrast, LK cultured in direct contact with OP-9 differentiated fully to mature B cells, expressing Pax5 and cell surface CD19 and IgM. LK cultured above OP9 cells but with cell-cell contact prevented by an interposed Transwell also fully acquired the transcription factor and cell surface antigenic profiles of mature B cells, but the proliferation that accompanies OB-contact driven differentiation was blocked:

These data indicate that all the signals required to induce HSC differentiation to mature B cells are contained within the combination of cytokines and other molecules secreted by OP-9 cells, but that direct contact with OP-9 cells provides a proliferative stimulus not achieved by OP-9 secreted molecules, either via a direct receptor-ligand interaction or indirectly, by contact-induced secretion of specific soluble proliferative factors from osteoblasts. Experiments to distinguish these alternatives, and to identify whether such signals trigger nutrient influx, direct activation of Akt, MAPK or other key proliferative pathways, are now in progress. The proliferative role of stromal cell-parenchymal cell contact within the stem cell niche likely may have general implications to normal and neoplastic cell physiology.