Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism

Objective Direct molecular and cellular studies of hematopoietic stem cells (HSCs) are hampered by the low levels of HSCs in hematopoietic tissues. To address these issues, we generated immortalized multipotent hematopoietic precursor cell (HPC) lines by expressing the LIM-homeobox gene Lhx2 (previously LH2) in hematopoietic progenitors derived from embryonic stem cells differentiated in vitro. Materials and Methods To validate further the relevance of the HPC lines as a model for normal HSCs, we analyzed in detail the growth requirements of HPC lines in vitro. Results Lhx2 immortalized the HPC lines by a putatively novel and cell nonautonomous mechanism. Self-renewal of the HPC lines is dependent on functional Lhx2 expression. Most early-acting hematopoiesis-related growth factors show synergistic effects on the HPC lines, whereas late-acting factors do not induce differentiation by themselves. Transforming growth factor-β1 is a potent inhibitor of proliferation of the HPC lines. HPC lines form cobblestone areas with high efficiency when seeded onto stromal cell lines, and the cobblestone area-forming cell can be maintained in these cultures for several months. Conclusions Our data show that, in many respects, HPC lines are similar to normal hematopoietic progenitor/stem cells on the cellular level, in contrast to most previously described multipotent hematopoietic cell lines. The cell nonautonomous mechanism for immortalization of the HPC lines suggests that Lhx2 regulates, directly or indirectly, soluble mediators involved in self-renewal of the HPC lines.