Figure 7
Figure 7. Defective erythropoiesis in RPL5-haploinsufficient iPSCs is restored by gene correction. (A) Hematopoietic cells released from EBs generated by RPL5+/p.R23X and 3 different WT lines at day 14 of differentiation. Cultures contained roughly equal numbers of EBs that were of similar size. **P < .01; n = 4 independent experiments performed in parallel between WT1 and RPL5+/p.R23X, 2 of them included WT2 and WT3. All the results are shown as a ratio relative to WT1, which was used as an internal standard in every experiment and is assigned an arbitrary value of 1. (B) Hematopoietic cells released by EBs from RPL5+/p.R23X (designated as “–”) and gene-corrected (+GFP control and +RPL5) iPSCs at day 14 of differentiation. Cultures contained roughly equal numbers of EBs that were of similar size. *P < .05. All experiments were performed in parallel. n = 5 independent experiments. (C) Ery cells released by day 14 EBs from RPL5+/p.R23X and WT iPSCs. Results are shown as relative ratio to WT1 present in each experiment and used as an internal standard. RPL5+/p.R23X EBs released fewer Ery cells than WT1 EBs (P < .01, n = 4), but not WT2 or WT3 EBs. The increased proportion of erythroblasts produced by WT1 iPSCs reflects clonal variability in Ery potential that we observe consistently between different WT iPSC lines. (D) Relative frequency (%) of each hematopoietic lineage present in day 14 EB cultures represented in experiment from panel B, as determined by flow cytometry according to Figure 3C. The proportion of Ery cells in WT RPL5-rescued RPL5+/p.R23X iPSCs was not significantly increased compared with nonrescued clones; n = 5 independent experiments. (E) Hematopoietic cells (2 × 105) released by 14-day-old EBs from WT and RPL5+/p.R23X iPSCs were incubated with the Ery cytokine combination EPO, SCF, and IGF-1 and cultured for 14 days. (F) Expansion of iPSC-derived Ery progenitors after 14 days in liquid culture according to the protocol described in panel E. Data are shown for 3 WT control cell lines and the RPL5+/p.R23X clone (**P < .01). (G) Expansion of Ery progenitors derived from RPL5+/p.R23X iPSCs with GFP or WT RPL5 cDNAs, assessed according to the strategy described in panels E and F. Correction of RPL5 haploinsufficiency increased Ery expansion by about sevenfold (*P < .05). (H) May Grünwald-Giemsa–stained cells from experiment in panel G at day 14 of culture. The cultures derived from RPL5+/p.R23X + GFP iPSCs express a greater proportion of myeloid cells (*) compared with cultures derived from RPL5+/p.R23X + RPL5 iPSCs, which are predominantly Ery (right panel). Images were obtained with a Zeiss Axioskope 2 microscope, Axiocam camera, and AxioVision 4.8 software (Carl Zeiss). The black bars in the graphs (A,C,F) represent mean values. For iPSC lines examined in 3 or more independent experiments, the error bars show SD. For iPSC lines examined twice, individual data points from each experiment are shown as open squares.

Defective erythropoiesis in RPL5-haploinsufficient iPSCs is restored by gene correction. (A) Hematopoietic cells released from EBs generated by RPL5+/p.R23X and 3 different WT lines at day 14 of differentiation. Cultures contained roughly equal numbers of EBs that were of similar size. **P < .01; n = 4 independent experiments performed in parallel between WT1 and RPL5+/p.R23X, 2 of them included WT2 and WT3. All the results are shown as a ratio relative to WT1, which was used as an internal standard in every experiment and is assigned an arbitrary value of 1. (B) Hematopoietic cells released by EBs from RPL5+/p.R23X (designated as “–”) and gene-corrected (+GFP control and +RPL5) iPSCs at day 14 of differentiation. Cultures contained roughly equal numbers of EBs that were of similar size. *P < .05. All experiments were performed in parallel. n = 5 independent experiments. (C) Ery cells released by day 14 EBs from RPL5+/p.R23X and WT iPSCs. Results are shown as relative ratio to WT1 present in each experiment and used as an internal standard. RPL5+/p.R23X EBs released fewer Ery cells than WT1 EBs (P < .01, n = 4), but not WT2 or WT3 EBs. The increased proportion of erythroblasts produced by WT1 iPSCs reflects clonal variability in Ery potential that we observe consistently between different WT iPSC lines. (D) Relative frequency (%) of each hematopoietic lineage present in day 14 EB cultures represented in experiment from panel B, as determined by flow cytometry according to Figure 3C. The proportion of Ery cells in WT RPL5-rescued RPL5+/p.R23X iPSCs was not significantly increased compared with nonrescued clones; n = 5 independent experiments. (E) Hematopoietic cells (2 × 105) released by 14-day-old EBs from WT and RPL5+/p.R23X iPSCs were incubated with the Ery cytokine combination EPO, SCF, and IGF-1 and cultured for 14 days. (F) Expansion of iPSC-derived Ery progenitors after 14 days in liquid culture according to the protocol described in panel E. Data are shown for 3 WT control cell lines and the RPL5+/p.R23X clone (**P < .01). (G) Expansion of Ery progenitors derived from RPL5+/p.R23X iPSCs with GFP or WT RPL5 cDNAs, assessed according to the strategy described in panels E and F. Correction of RPL5 haploinsufficiency increased Ery expansion by about sevenfold (*P < .05). (H) May Grünwald-Giemsa–stained cells from experiment in panel G at day 14 of culture. The cultures derived from RPL5+/p.R23X + GFP iPSCs express a greater proportion of myeloid cells (*) compared with cultures derived from RPL5+/p.R23X + RPL5 iPSCs, which are predominantly Ery (right panel). Images were obtained with a Zeiss Axioskope 2 microscope, Axiocam camera, and AxioVision 4.8 software (Carl Zeiss). The black bars in the graphs (A,C,F) represent mean values. For iPSC lines examined in 3 or more independent experiments, the error bars show SD. For iPSC lines examined twice, individual data points from each experiment are shown as open squares.

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