Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages in vitro, but their limited capacity to reconstitute hematopoiesis in irradiated mice raises the question of whether a definitive hematopoietic stem cell (HSC) develops from ES cells in culture. The relationship between primitive embryonic yolk sac and definitive adult-type hematopoiesis remains controversial, and to date an ES-derived HSC has not been identified and subjected to clonal analysis in a manner comparable to bone marrow-derived HSCs. The Chronic Myeloid Leukemia-associated BCR/ABL oncogene enables the adult HSC to achieve clonal dominance while allowing lymphoid and myeloid differentiation. We have introduced BCR/ABL into differentiating cultures of ES cells to enhance reconstitution potential and to facilitate clonal analyses of hematopoietic progenitors. Our initial efforts generated a stable BCR/ABL-transformed cell line (EB57) that when injected into irradiated mice generated acute leukemia with modest granulocytic differentiation. Because accommodation to cell culture may have limited the differentiation potential of this cell line, we infected differentiating cultures of ES cells with a BCR/ABL retrovirus and tested low passage cells for multi-lineage hematopoietic reconstitution in irradiated mice. CCE ES cells were differentiated into embryoid bodies, disrupted and infected with the BCR/ ABL retrovirus, and plated on OP9 stroma. Cells from mock-infected cultures failed to proliferate under these conditions, but BCR/ABL cultures generated abundant semiadherent cells. When plated in methylcellulose containing VEGF, SCF and D4T-conditioned medium, these cells gave rise to primitive hematopoietic blast colonies, which when replated, gave rise to secondary primitive and definitive erythroid colonies as well as CFU-GEMM and macrophage/mast cell colonies. Flow cytometry of cells expanded in liquid culture demonstrated expression of stem cell markers, including Sca-1, cKit, and CD34. When transplanted into sublethally irradiated 129 Sv/Ev or Nod/Scid mice, these cells differentiated into GR-1+ granulocytes and Mac-l+ myeloid cells as well as CD4/8+ and B220+ lymphocytes. Engrafted mice ultimately succumbed to a myeloproliferative disorder. These data demonstrate enhanced lymphoid-myeloid reconstitution of adult mice with hematopoietic derivatives of ES cells. We are currently conducting a clonal analysis of reconstituted mice to determine whether the myeloid and lymphoid elements derive from a single HSC.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|