Figure 4
Figure 4. Ex vivo cultures of Eklf−/− erythroblasts recapitulate enucleation and cell cycle defects observed in vivo during terminal differentiation. (A) E12.5 fetal liver definitive erythroid cells were grown in expansion media to derive ESREs. Representative growth curves of Eklf+/+, Eklf−/−, and Eklf+/− fetal liver cultures are shown (from a total of 15). (B) MGG staining of Eklf+/+ and Eklf−/− ESREs at day 0 and at day 3 of terminal differentiation. The red arrow shows enucleated cells present at day 3 of differentiation in Eklf+/+ cultures but absent in Eklf−/− cultures. The green arrow shows representative orthochromatic erythroblasts, which are present in both cultures at day 3 of differentiation. Scale bars correspond to 50 µm length. Original magnification ×40. (C) (Left) Cells were stained with CD71, Kit, and DAPI and analyzed using imaging flow cytometry to identify progressive stages of erythroid differentiation. Representative images of Eklf+/+ kit+ (CFU-Es and proerythroblasts), basophilic, polychromatic, orthochromatic, enucleating orthochromatic erythroblasts and pyrenocytes at day 2 of differentiation obtained by imaging flow cytometry are shown. (Right) Quantification of the progressive stages of erythroid differentiation in ESRE cultures using imaging flow cytometry at day 0 and day 2 of differentiation. Eklf+/+ and +/− (n = 8), Eklf−/− (n = 4). (D) Enucleated cells were measured by flow cytometry using DRAQ5 nuclear stain in Eklf+/+, Eklf−/−, and EKLF wild-type rescue in Eklf−/− cells at day 3 of terminal differentiation. (Left) Flow cytometry gating for the DRAQ5-negative population. (Right) Quantification of biological replicates (n = 3) for all 3 cultures show almost complete absence of enucleation in Eklf−/− erythroid cultures. (Bottom) MGG staining was also performed on Eklf−/− cells with wild-type EKLF rescue at day 3 of terminal differentiation. Red arrows show enucleated cells present at day 3 of differentiation of Eklf+/+ cultures and in Eklf−/− cultures with wild-type EKLF rescue, whereas absent in Eklf−/− cultures. Scale bars correspond to 50 µm length. Original magnification ×40. (E) Cell cycle flow cytometric analysis was performed using ethanol-fixed cells stained with PI before (day 0; top) and after (day 2; bottom) erythroid differentiation in ESRE cultures (n = 3). Eklf−/− cultures show a decrease in percentage of cells at G0/G1 and an increase of cells in S phase in Eklf−/− cultures compared with Eklf+/+ cultures. *P < .05, **P < .01, ***P < .001.

Ex vivo cultures of Eklf−/− erythroblasts recapitulate enucleation and cell cycle defects observed in vivo during terminal differentiation. (A) E12.5 fetal liver definitive erythroid cells were grown in expansion media to derive ESREs. Representative growth curves of Eklf+/+, Eklf−/−, and Eklf+/− fetal liver cultures are shown (from a total of 15). (B) MGG staining of Eklf+/+ and Eklf−/− ESREs at day 0 and at day 3 of terminal differentiation. The red arrow shows enucleated cells present at day 3 of differentiation in Eklf+/+ cultures but absent in Eklf−/− cultures. The green arrow shows representative orthochromatic erythroblasts, which are present in both cultures at day 3 of differentiation. Scale bars correspond to 50 µm length. Original magnification ×40. (C) (Left) Cells were stained with CD71, Kit, and DAPI and analyzed using imaging flow cytometry to identify progressive stages of erythroid differentiation. Representative images of Eklf+/+ kit+ (CFU-Es and proerythroblasts), basophilic, polychromatic, orthochromatic, enucleating orthochromatic erythroblasts and pyrenocytes at day 2 of differentiation obtained by imaging flow cytometry are shown. (Right) Quantification of the progressive stages of erythroid differentiation in ESRE cultures using imaging flow cytometry at day 0 and day 2 of differentiation. Eklf+/+ and +/− (n = 8), Eklf−/− (n = 4). (D) Enucleated cells were measured by flow cytometry using DRAQ5 nuclear stain in Eklf+/+, Eklf−/−, and EKLF wild-type rescue in Eklf−/− cells at day 3 of terminal differentiation. (Left) Flow cytometry gating for the DRAQ5-negative population. (Right) Quantification of biological replicates (n = 3) for all 3 cultures show almost complete absence of enucleation in Eklf−/− erythroid cultures. (Bottom) MGG staining was also performed on Eklf−/− cells with wild-type EKLF rescue at day 3 of terminal differentiation. Red arrows show enucleated cells present at day 3 of differentiation of Eklf+/+ cultures and in Eklf−/− cultures with wild-type EKLF rescue, whereas absent in Eklf−/− cultures. Scale bars correspond to 50 µm length. Original magnification ×40. (E) Cell cycle flow cytometric analysis was performed using ethanol-fixed cells stained with PI before (day 0; top) and after (day 2; bottom) erythroid differentiation in ESRE cultures (n = 3). Eklf−/− cultures show a decrease in percentage of cells at G0/G1 and an increase of cells in S phase in Eklf−/− cultures compared with Eklf+/+ cultures. *P < .05, **P < .01, ***P < .001.

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