Normal Short-Term but Reduced Long-Term Engraftment Capacity of CML Hematopoietic Cells with Skewed Myeloid Lineage Differentiation Is Seen in an Improved Mouse Model of Human Hematopoiesis.

Abstract

The NOD/SCID mouse model has been widely used to assay human hematopoietic cells capable of long-term multilineage engraftment (SCID-repopulating cells or SRC). Leukemia cells from CML patients can also engraft in NOD-SCID mice. However the utility of this model for studying CML stem cells is limited by inconsistent and low levels of engraftment, and lack of a leukemia-related phenotype of engrafted cells. Since NOD/SCID IL2Rγchain KO (NOG) mice support superior engraftment of human hematopoietic cells compared with NOD/SCID mice, they may allow for improved evaluation of CML stem cells. In the current study we investigated engraftment of CML CD34+ cells in the NOG mouse model. CD34+ cells were transplanted into irradiated (300 cGy) 8 wks old NOG mice via tail vein injection. Blood samples were obtained at 4 wks intervals to monitor human cell engraftment. At the end of the maintenance period of 16–18 wks, animals were euthanized and marrow content of femora, spleen cells and peripheral blood were obtained. Human cell engraftment was analyzed by flow cytometry after labeling cells with anti-human CD45 mAb and specific human cell subsets detected by staining with human CD34, CD33, CD14, CD11b, CD19 and CD3 mAbs. Establishment of human hematopoiesis was consistently seen 4 weeks after injection. Larger numbers of CML (1–2 x106) compared to normal bone marrow (1–8x105) CD34+ cells were required to establish engraftment, which is consistent with previous reports. Interestingly CML CD34+ cells established their highest levels of engraftment in blood 4 wks after transplantation (14.9±5.9% CD45+ cells, n=13) and engraftment levels were significantly lower at 8 wks (1.8±0.3%) and beyond. However engraftment was consistently detected in blood up to 16–20 wks. The engraftment kinetics of CML cells was strikingly different from that of normal CD34+ cells which while showing similar levels of engraftment at 4 wks (12.6±3.8%, n=16), showed considerably higher engraftment at 8 wks (19.2±3.8%) with a gradual decline subsequently (11.5±2.7% at 12 wks). Analysis after autopsy at 16–18 wks revealed higher levels of human cell engraftment in mouse BM and spleen compared with blood. CML CD34+ cells showed reduced BM engraftment compared with normal CD34+ cells (7.3±4.8% vs. 22.1±9.0% CD45+ cells, n=7, p<0.01). CML CD34+ cells engrafted with myeloid lineage cells and minimal lymphoid engraftment was seen (CD33: 5.2±3.7%, CD11b: 2.6±1.6% and CD19: 0.1±0.1%), whereas normal CD34+ cells engrafted with both myeloid and lymphoid cells (CD33: 7±3%, CD11b: 1.6±0.6% and CD19: 11±4.3%). We also observed reduced frequency of CD34+ cells in mice receiving CML compared with normal cells (2.6±2.5% vs. 6.2±3.0%, p<0.05). In conclusion we show, using an improved mouse model of human hematopoiesis, that CML SRC have similar short-term but reduced long-term engraftment capacity compared with normal SRC, possibly indicating a preponderance of short-term repopulating cells or increased HSC turnover. CML SRC demonstrated predominantly myeloid differentiation compared with balanced myeloid and lymphoid engraftment of normal SRC. These results support the utility of the NOG mouse model to investigate functional characteristics of CML stem cells and study the activity of specific therapeutic interventions against primitive CML cells.