[Expansion of bone marrow LTC-ICs in vitro by mouse fetal liver-derived stromal cell lines]

As main component of fetal liver hematopoietic microenvironment, different stromal cells may play distinct roles in the regulation of hematopoietic stem cell self-renewal, proliferation and differentiation. It is a unique approach to establish stromal cell lines for analyzing the interaction of hematopoietic cells with the stroma on the clonal level to dissect the function of hematopoietic microenvironment. In this study two immortal stromal cell lines-A4, B3 were established from mouse embryonic day 12.5 fetal liver by transfection of pSV3 neo plasmid. A4 exhibited a fibroblast-like morphology, 25 hours population doubling time as well as high levels of CD29, CD44, UEA-1 and low levels of CD105 expression. In contrast B3 displayed an epithelium-like morphology, 37 hours population doubling time along with high levels of CD105 and low levels of UEA-1 expression. In addition no or low levels of CD31, CD34, CD45 and CD144 expression were found in the two cell lines. These results indicate that A4, B3 are two discriminating cell lines in terms of morphological characters, growth behaviors and surface molecular expression types. Next functional assays using Limited-Diluted Assay(LDA) and Bulk-LTC-IC were done: both stromal cell lines had similar ability to maintain the survival proportion of inoculated mouse bone marrow-derived Long-Term Culture-Initiating Cells (LTC-ICs), and they could also support LTC-ICs expansion up to 4 weeks by co-culture in vitro. More strikingly, B3 could expand the absolute number of LTC-ICs over 13-fold at week 4 than that of week 0, and the ability of B3 to expand absolute number of LTC-ICs was over 8-fold of that of A4. Proportions of LTC-ICs in proliferating cell populations in two long-term culture systems was similar at week 4, no matter with or without extra cytokines. Further study indicated that the ability of LTC-ICs to yield CFCs was held as the number 6( +/- 1.2) after 4 weeks co-culture. Extra cytokines-SCF + IL-3 + IL-6 + Epo had no influence in the maintenance and expansion of LTC-ICs, but expanded the absolute number of CFCs and proliferating cell populations, and maintained similar proportions of LTC-ICs and CFCs in the end in two culture systems at week 4. Take together, these results implicate that B3 may act as an important functional component in embryonic day 12.5 fetal liver microenvironment to effectively expand primitive hematopoietic cells, yield more committed hematopoietic progenitor cells and mature hematopoietic cells to meet the need of quick development especially in the early phase of embryonic development. Alternatively A4 can probably function as being a structural element. Moreover the function of hematopoiesis-associated cytokines employed in this investigation was not to expand LTC-ICs, but to modulate the limited-proliferation and differentiation of CFCs. The maintenance and proliferation of LTC-ICs may depend on the type of the stromal cell as well as the interaction between stromal cells and LTC-ICs. It is suggested that B3 with cytokines including SCF, IL-3, IL-6, EPO in vitro can mimic embryonic day 12.5 hematopoietic microenvironment to investigate the mechanism of interaction between hematopoietic microenvironment and hematopoietic cells in clonal level.