Establishing murine hematopoietic progenitor HPC^LSKlines. (A) Schematic workflow of HPC^LSK cell line establishment. LSKs were sorted from murine BM, transfected with Lhx2 including a puromycin selection marker and kept in SCF and IL-6 on 1% agarose-coated plates. StemPro-34 SFM: serum free media. (B) Principal component analysis (PCA) of the expression profiles of HPC^LSKs (n = 3) compared with murine HSCs (batch-corrected top 500 variance genes are plotted). (C) Immunoblot of lysates from 3-hour starved HPC^LSK cells followed by treatment with IL-7, EPO, TPO, GM-CSF, SCF, IL-6, or IL-3 (100 ng/mL each) for 15 minutes. The presence of total and phosphorylated STAT5, STAT3, AKT, and ERK was detected. HSC70 serves as a loading control. st, starved. A representative blot of 2 independent experiments is shown. (D) Colonies with different morphologies were counted. Seeding density of 1250 HPC^LSKs or 240 000 BM cells per 35-mm dish. Error bars represent mean ± standard deviation (SD), n ≥ 3. (E) Images of colonies formed by HPC^LSK cells 10 days after cytokine cocktail treatment (EPO, GM-CSF, holo-transferrin, IL-7, SCF, IL-6, IL-3) in semisolid methylcellulose gels. BFU-E, burst-forming unit-erythroid; CFU-GEMM, CFU-granulocyte erythrocyte monocyte megakaryocyte; CFU-GM, colony-forming unit-granulocyte macrophage.