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. 2020 Sep 1;32(9):108093. doi: 10.1016/j.celrep.2020.108093

Figure 3.

Figure 3

Quiescent CD34+ HSPCs Perform Only NHEJ, but Cycling CD34+ HSPCs Perform Both NHEJ and HDR

(A) Editing outcomes in CD34+ subpopulations in different cell cycle status. One day post-electroporation and 2 days in culture. Percentage of reads positive for HDR or NHEJ by next-generation amplicon sequencing at the HBB site. G0 CD34+ HSPCs in 2-day culture do not accumulate HDR alleles. Representative data from experiments performed with three different mPB donors and n = 3 biological replicates per donor. Mean ± SD shown. ∗∗p < 0.01 by unpaired t test.

(B) Editing outcomes in CD34+ subpopulations sorted into different cell cycle statuses. Two days post-electroporation and 4 days in culture. Percentage of reads positive for HDR or NHEJ by next-generation amplicon sequencing at the HBB site. G0 CD34+ HSPCs sorted at 2 days in culture accumulate significant HDR alleles in 4-day culture. Representative data from experiments performed with three different mPB donors and n = 3 biological replicates per donor. Mean ± SD shown. ∗p < 0.05 by unpaired t test.

(C) Schematic of the workflow for mTOR and GSK-3 inhibition with rapamycin and CHIR99021 (XRC) for inhibition of cell cycle entry. Culture condition are SFEMII + CC110 (SC) and X-VIVO15 + rapamycin + CHIR99021 (XRC).

(D) Cell cycle profiles of CD34+ cells in SC or XRC at the time of electroporation (day 1) and two days post-nucleofection (day 3). XRC prevents cell cycle entry, but this can be reversed by placing the CD34+ cells in SC media. Representative data from experiments performed with three different mPB donors and n = 2 biological replicates per donor. Mean ± SD shown.

(E) Percentage of early and late apoptosis was assessed by staining the cells for annexin V (AnnV) and propidium iodide (PI) at 2 days post-nucleofection. AnnV−PI−, viable; AnnV+PI−, early apoptotic; AnnV+PI+, apoptotic. XRC does not induce apoptosis. Representative data from experiments performed with three different mPB donors and n = 2 biological replicates per donor. Mean ± SD shown.

(F) Editing outcomes in CD34+ cells kept in SC media or XRC in different cell cycle status 2 days post-nucleofection and 3 days in culture. Inhibition of cell cycle entry by XRC blocks HDR repair but is reversible. Representative data from experiments performed with three different mPB donors and n = 2 biological replicates per donor. Mean ± SD shown.

See also Figure S3.