Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34⁺ cells

Abstract

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34⁺ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1α was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34⁺ cells (cells which had divided thus becoming PKH2^dim) and cytokine nonresponsive (CNR) CD34⁺ cells (cells which had not divided and had therefore remained PKH2^bright). CR and CNR CD34⁺ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1α (MIP-1α/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1α was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1α/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1α/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34⁺ cells, an approach that may be clinically feasible for autotransplantation.