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AF4-MLL in human embryonic hematopoietic development
glycophorin A, anti-CD43-FITC, anti-CD45-APC antibodies (all from BD Biosciences) and 7-actinomycin D, and analyzed using a FACS Canto flow cytometer.9,29,39-41 Colony-forming unit assays were performed on days 10 and 15 of EB differentiation by plating 60x104 EB cells onto serum-free methylcellulose H4435 (Stem Cell Technologies). Colonies were scored after 12 days.9,29,42-44
Cell cycle and apoptosis analysis
For cell cycle analysis of hESC-derived HEP and CD45+ cells, day 15 EB were dissociated and harvested cells were fixed overnight in 70% ice-cold ethanol. Cells were then washed in phosphate-buffered saline and incubated with anti-CD31-FITC, anti-CD34-PE-Cy7 and anti-CD45-APC antibodies for 15 min.
AB
Cells were then suspended in propidium iodide-containing buffer and acquired and analyzed on a FACS Canto-II using Modfit LT4.0 software, discriminating between quiescent cells (G0/G1), cycling cells (S-phase) and G2/M cells.45,46 Apoptosis was assessed with an Annexin-V Apoptosis Detection kit (BD Biosciences).16
Human embryonic stem cell-OP9 co-cultures
hESC-OP9 co-cultures were performed as described else- where.47,48 OP9 stroma was prepared by plating OP9 cells in gela- tin-coated dishes, and allowing them to overgrow as a monolayer. hESC were prepared as a suspension of small aggregates using col- lagenase IV:dispase. One-tenth of this suspension was plated on top of the 8-day overgrown OP9 stroma. Media were replaced on
C
D
Figure 1. Characterization of transgenic human embryonic stem cells expressing the reciprocal fusion A4M together with MA4. (A) RNA-sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) valida- tion revealed that ~45% (11/25) of the patients with t(4;11)+ B-cell precursor acute lymphoblastic leukemia do not express the reciprocal fusion A4M.18 (B) Left, Phase-con- trast morphology of representative colonies from each transgenic human embryonic stem cell (hESC) line. Right, Reverse transcriptase polymerase chain reaction analysis (RT-PCR) confirming expression of both fusions in undif- ferentiated hESC. (C) qRT-PCR expression of the pluripotency genes OCT4, SOX2, NANOG, CRIPTO, and DNMT3B. (D) Representative FACS data confirming expression of the pluripo- tency surface markers SSEA-3, SSEA-4, TRA-1- 60, and TRA-1-81. BCP-ALL: B-cell precursor acute lymphoblastic leukemia; RNA-seq: RNA- sequencing; pos: positive; neg: negative; EV: empty vector; C+: positive control.
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