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. 2018 Nov 29;11(6):1462–1478. doi: 10.1016/j.stemcr.2018.11.001

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Crown Copyright © 2018.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Forward Programming of hPSCs with GATA1, FLI1, and TAL1 Generates a Bipotent Megakaryocyte/Erythroid Population

(A) Schematic representation of the experimental setup. Following transduction with three lentiviruses and an initial mesoderm-promoting 2-day culture, cells were divided into an erythroid (EPO) or MK (TPO) culture.

(B and C) Cells from the EPO and TPO culture conditions were analyzed by flow cytometry at day 9 post-transduction. (B) Representative dot plots for CD41a and CD235a expression and (C) chart summarizing the percentages of cells belonging to the non-programmed (NEG) (CD41a−/CD235a−), bipotent (BPP) (CD41a+/CD235a+), erythrocyte (ERY) (CD41a−/CD235a+), and megakaryocyte (MK) (CD41a+/CD235a−) populations, respectively (n = 5, mean ± SEM); EPO culture (red bars), TPO culture (blue bars).

(D) Boxplots over five replicates of the log(e) of the number of ERYs (left plot) and MKs (right plot) divided by the number of BPPs as detected by flow cytometry at day 9. The log odds of cells being ERY rather than BPP (“mean effect”) in EPO (versus TPO) was 1.6, while the log odds of a cell being an MK rather than BPP in TPO (versus EPO) was 0.5 (^∗∗∗p < 0.001).

(E and F) The colony potential of day 9 cells was assessed by colony-forming unit (CFU) assays. Cells were cultivated in duplicate in semi-solid medium and the colony outcome (number and morphology) analyzed blindly by at least two operators after 14 days. (E) Exemplars of the three types of colonies recorded, including erythroid (ERY), megakaryocytic (MK), or mixed colonies containing both erythroid and MK cell types (MIXED). (F) The pie charts show CFU distribution depending on culture condition (EPO versus TPO; n = 4, mean% ± SEM); ERY in red, MK in blue and MIXED in gray. Poisson regression analysis shows that the distribution of the number of colonies per cell type depends on cytokine exposure (^∗∗∗p < 0.001).

(G) Colony potential of day 9 cells sorted according to their cell surface markers. Only cells expressing both CD235a and CD41a showed colony-forming potential.