Strategy to study the initial effects of BCR-ABL expression during hematopoietic progenitor development.

(A) BCR-ABL expression was turned on at day 5 of ES cell in vitro differentiation when the majority of the cells were mesodermal cells and hematopoietic precursors as defined by FLK-1 and VE-cadherin expression. Effects were analyzed on day 7 ES in vitro–differentiated hematopoietic progenitors by harvesting nonadherent hematopoietic progenitors with gentle pipetting and not harvesting adherent endothelial cells. In this system, removal of TET on day 5 of differentiation induced BCR-ABL IRES EGFP expression in 40% to 60% of day 7 ES-derived hematopoietic progenitors as measured by flow cytometry. BCR-ABL effects were analyzed in EGFP⁺ cells and EGFP⁻ cells both in BCR-ABL–induced and uninduced cultures, used as controls. (B) Exclusive BCR-ABL activation in EGFP-expressing ES-derived day 7 hematopoietic progenitor cultures without TET. EGFP⁺ and EGFP⁻ES-derived hematopoietic progenitors from BCR-ABL–induced cultures without TET were sorted to more than 95% purity using the FACSVantage along with EGFP⁻ cells from a control BCR-ABL uninduced culture with TET. Purified cell extracts were prepared and immunoblotted with anti-ABL and antiphosphotyrosine antibodies to determine BCR-ABL expression and activity, respectively. (C) SCF is expressed in OP9 cells and ES-derived hematopoietic progenitors. RNA was extracted from OP9 cells and day 7 ES-derived hematopoietic progenitors expressing BCR-ABL and control day 7 ES-derived hematopoietic progenitors. cDNA was generated from these samples and PCR using SCF and HPRT-specific primers were conducted. SCF primers would generate a 592-bp cDNA fragment and an about 1500-bp genomic band; HPRT primers would generate a 249-bp cDNA fragment and an approximately 1100-bp genomic band.