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. 2018 Jun 12;2(4):e51. doi: 10.1097/HS9.0000000000000051

Figure 4.

Figure 4

iMLL-ENL preferentially transforms hematopoietic stem cells and pluripotent rather than more committed myeloid progenitor cells. (A) Ex vivo expansion of naive iMLL-ENL LSK- and GMP-derived cells in liquid cultures containing hIL-6 (10 ng/mL), mIL-3 (6 ng/mL), mIL-7 (10 ng/mL), mSCF (100 ng/mL), and mFlt-3L (100 ng/mL) in the presence of DOX. (B) Expression of iMLL-ENL mRNA of sorted LSK and GMP cells cultured ex vivo in MC for 3 rounds with growth factors in the presence or absence of DOX. Relative mRNA expression levels were normalized to Gapdh expression and are expressed as 2−ΔCt. Results the mean values ± standard deviation of duplicates. (C) Average survival of lethally irradiated mice transplanted with the indicated numbers (1000, 500, 250, and 125) of naive iMLL-ENL LT-HSCs or GMPs (5000, 2500, and 1250) into wild type recipients on DOX. Mice receiving 500 to 1000 LT-HSCs developed acute leukemia (median latency 169.6 ± 27.3 days, n = 10) whereas mice receiving 125 to 250 LT-HSCs (n = 10) or GMPs (n = 15) did not develop the disease within 400 days. Mice transplanted with the indicated cells but kept off DOX served as controls (n = 10). (D) Average survival of lethally irradiated mice transplanted with 104 of transgenic MPP4s or LMPPs or CMPs on DOX. Mice receiving MPP4s and LMPPs developed acute leukemia with a median latency of 61.0 ± 13.7 days and 54.4 ± 11.7, respectively (n = 5 per group), whereas 3 out of 5 mice receiving CMPs (n = 5) develop the disease within 120 days. (E) Wright-Giemsa-stained blood smear showing presence of leukemic blasts of different size; and histopathology of the spleen showing infiltration. (F) Quantification of flow cytometry analysis of “small” cell fraction showing the percentage of B220+ in BM of leukemic mice transplanted with different hematopoietic progenitor populations. Notably LT-HSC-derived disease was associated with a significantly higher fraction of B220+ BM cells than CMP-derived leukemia (P = 0.002, n = 2, unpaired t test). BM = bone marrow, CMP = common myeloid progenitor, DOX = doxycycline, GMP = granulocyte-macrophage progenitors, hIL-6 = human interleukin-6, LMPP = lymphoid primed multipotent progenitor cells, LSK = Lin− Sca-1+ c-Kit+, LT-HSC = long-term hematopoietic stem cells, MC = methylcellulose, MPP4 = multipotent progenitors, mSCF = murine stem cell factor.