Growth of p210Bcr-Abl Transformed Human Hematopoietic Stem Cells in Immunodeficient Mice.
Authors: Modi Hardik V , Shultz Leonard , Bhatia Ravi 
Published: April 2020

Abstract
Expression of the p210Bcr-Abl fusion gene in primary hematopoietic stem and progenitor cells by transduction with Bcr-Abl-encoding retroviruses is a useful approach to investigate CML pathogenesis. We have previously shown that human CD34+ cells expressing BCR/ABL demonstrate several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1alpha. Here we investigated the growth of Bcr-Abl transformed human hematopoietic stem cells (HSC) in immunodeficient NOD/SCID IL2Rγ chain KO (NSG) mice. Human cord blood (CB) CD34+ cells were transduced with retroviral vectors expressing the p210Bcr-Abl gene and transplanted via tail vein injection into sub-lethally irradiated (300 cGy) 8 weeks old NSG mice. Mice were euthanized at different times after transplant and human cell engraftment was analyzed by flow cytometry. We observed a progressive increase in human CD45+ve cells in bone marrow of injected mice over time (% human CD45+ve cells in BM: Wk 2, 28±5,n=3; Wk 4, 57±6,n=3; Wk 8, 79±3,n=6; Wk 12, 80±5,n=7; Wk16–20, 95±2,n=3; total number of human CD45+ cells in both femora in millions: Wk 2, 0.1±0, Wk 4, 2.1±0.5, Wk 8, 9.7±1.8, Wk 12, 6.5±2.4, Wk 16–20, 14.5±6.2). The number of GFP expressing cells also increased over time up to 20 weeks after transplantation (GFP+ cells in both femora in millions: Wk 2, 0.01±0, Wk 4, 0.38±0.08, Wk 8, 0.81±0.27, Wk 12, 0.91±0.39, Wk16–20, 2.58±2.27). We confirmed using quantitative RT-PCR and Western blot analysis that engrafted cells expressed the Bcr-Abl gene, and that Bcr-Abl expression was restricted to GFP+ cells. We further observed that mice started developing splenomegaly 12 weeks after transplantation (spleen weight in grams: Wk 8, 36±4, Wk12, 196±56, Wk16–20, 207±55; spleen cell counts in millions: Wk 8, 2.6±1.0, Wk 12, 11.2±4.2, Wk 16–20, 18.6±10.5). Analysis of the phenotype of engrafted GFP+ human cells indicated that both myeloid and lymphoid GFP+ cells were generated. The majority of engrafted cells expressed B-lymphoid markers, consistent with the usual engraftment pattern of CB CD34+ cells in this mouse model, (% CD19+ cells in BM at Wk 8, 75±6, Wk 16–20, 51±17, % CD33+ cells in BM at Wk 8, 34±12, Wk16–20, 27±14). Two mice demonstrated very high percentages of GFP expressing B-lymphoid cells, consistent with clonal outgrowth of Bcr-Abl expressing B-cell progenitor population. To confirm that Bcr-Abl-expression was achieved in HSC, we transplanted one million BM mononuclear cells obtained from primary mice at weeks 4, 6 and 8 post-transplantation to secondary recipients. GFP+ human CD45+ cells were detected in secondary hosts 6 weeks after transplantation (Wk 4, 14.93±14.63, n=3; Wk 6, 0.87±0.44,n=3; Wk 8, 1.1±0.83,n=3). Engraftment of Bcr-Abl expressing cells in secondary hosts was confirmed by RT-PCR. In conclusion, we show that retroviral transduction of human CD34+ cells results in expression of the p210Bcr-Abl gene in cells capable of long-term multi-lineage engraftment of NSG mice and engraftment of secondary hosts, indicating the utility of this approach to study Bcr-Abl-mediated transformation of human HSC. Mice receiving transduced HSC develop progressive splenomegaly and increasing human cell engraftment in BM, but do not show selective proliferation of myeloid cells up to 20 weeks after transplantation. These results are in contrast to the vigorous myeloid progenitor proliferation seen when Bcr-Abl transduced human CD34+ cells are cultured in vitro, and the efficient and rapid induction of a myeloproliferative disease seen in mice transplanted with p210Bcr-Abl-transduced murine BM cells. Development of a myeloproliferative disorder following Bcr-Abl-mediated stem cell transformation may therefore be dependent on specific microenvironmental factors not available in the mouse host or may require the acquisition of additional abnormalities in Bcr-Abl expressing HSC.