The effect of bone morphogenetic protein 4 on the differentiation of mouse embryonic stem cell to erythroid lineage in serum free and serum supplemented media

Abstract

This study was done to compare the effects of bone morphogenetic protein-4 (BMP-4) on mouse embryonic stem cells (ESC) differentiation to erythroid lineage in serum free and serum supplemented media. The embryoid bodies (EBs) cells were seeded in semisolid serum free and serum supplemented media in the presence of different concentrations of BMP-4. The erythroid colonies were assessed morphologically, ultrastructurally and by benzidine staining. The expression of the epsilon (ε), βH1 and βmajor globins, Runx1 and β2m genes was evaluated by Real time PCR. The colony size and the percent of benzidine-positive colonies increased in both BMP-4 supplementd groups but the number of colonies were lower in these groups than control. Erythropoiesis related genes were expressed in both serum free and serum supplemented groups. There were not significant differences between the ratios of genes expression to β2m in these groups except the ratio of Runx1 was significantly higher in serum free group (P<0.05). The ratio of ε and βH1 to β2m in EBs was higher than both BMP-4 containing groups (P<0.05) and βmajor was not expressed in EB cells. These findings showed in serum free condition the effects of BMP-4 on the erythroid differentiation was prominent than serum supplemented group.

Keywords: bone morphogenetic protein-4; embryonic stem cells; erythropoiesis; serum free media.

Figure 1

A, The undifferentiated colony of CCE embryonic stem cells; 3100 magnification; B, Four-day-old embryoid body; 3400 magnification; C, The photograph of erythroid colony; 3100 magnification; (D) Benzidine-positive colony; 3200 magnification.

Figure 2

Total number and benzidine positive colonies formed after differentiation of EBs cells in serum free medium with different concentrations of BMP-4. Data are given as mean ± SD. a, Significant difference with control in the same column (P<0.05); b, Significant difference with 5 ng/ml group in the same column (P<0.05); c, significant difference with 40ng/ml group in the same column (P<0.05).

Figure 3

Total number and benzidine positive colonies formed after differentiation of EBs cells in semisolid media supplemented with FBS and different concentrations of BMP-4. Data are given as mean ± SD; a, Significant difference with control in the same column (P<0.05); b, Significant difference with 5ng/ml group in the same column (P<0.05); c, significant difference with 10ng/ml group in the same column (P<0.05).

Figure 4

A, The light microscopic photograph of semi-thin section of a hematopoietic colony derived from ESCs treated with BMP-4 in serum free condition. Arrow shows the heterochromatin nucleus and its extrution from the cells and astretics showed the mitotic figures in this colony; B, Electron micrograph of proerythroblast cells differentiated from BMP-4-treated ESCs, showing the euchromatin nucleous and hemoglobin in its cytoplasm (less dense background of hemoglobin; N: nucleus).

Figure 5

The ratio of genes expression erythropoiesis related genes (epsilon and beta H1 globin, Runx1, beta major globin genes) to β2m were shown in three groups of study (EBs; embryoid bodies cells (control), serum free media with BMP-4 and serum supplemented media with BMP-4). a, Significant differences with EBs or control group (P<0.05); b, Significant differences with serum free group (P<0.05).