Establishment of In vitro Culture System to Investigate the Role of STAT3 in Early B-cell Development

Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow. Previously, we have shown that STAT3KO mice displayed impaired B lymphopoiesis. Reduced pre-B, pro-B immature and mature B cells but accumulated pre-pro B stage of BM cells was found in mice lacking STAT3. The pro-B and pre-B population lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Rα. These data indicated that STAT3 played a critical role in early B cell development.
Here, we set up an in vitro culture system, to elucidate the mechanisms by which STAT3 regulates early B cell development. LSKs or common lymphoid progenitors (CLPs) were first isolated and co-cultured with γ-irradiated OP9 in the presence IL-7, SCF and Flt3L. We found that LSKs of both control and STAT3KO mice did not sufficiently support B cell development. Instead, myeloid cells such as granulocytes and macrophages were preferentially developed in these culture conditions. In contrast, CLPs had higher potential to develop into B cell lineages in both mice. Interestingly, the absence of STAT3 greatly reduced the numbers of B cell developed from CLPs despite that relative percentage was similar between control and STAT3KO mice. Moreover, we found the expression of genes that were critical for B cell development such as E2A, EBF, pax5, Igα, Igβ,VperB, λ5, RAG1 and RAG2 was comparable in control and STAT3KO cells, suggesting that STAT3 might affect on cell proliferation rather than on cell differentiation.
In limiting dilution assay, we investigated the frequency of B progenitor cells in the presence or absence of STAT3. We found that a decreased frequency of progenitor cells was observed in STA3KO mice. Moreover, left out exogenous IL-7 but not Flt3L substantially reduced the developmental potential of STAT3KO CLPs, suggesting that IL-7 dependent B lymphopoiesis was increased and that Flt3L-mediated B lymphoipoesis was impaired in the absence of STAT3.
Taken together, these results were agreed with our previous in vivo studies, that formation progenitor cell was dependent on STAT3.