The enucleation efficiency of bone marrow erythroblasts from the MDS group that were cultured in vitro was lower than that from the normal group

Erythroblast differentiation was induced in vitro by the three-stage erythroid culture system in both the MDS patient group and the normal group [15]. In this culture system, a small number of cells in the normal group began to undergo enucleation from day11. Then, the enucleation gradually increased and reached the highest rate at day15. After that, a relatively high enucleation rate was maintained for a long period of time. According to our experimental data, there is a certain degree of delay in the differentiation of erythroblasts in MDS. Usually, the enucleation rate is highest at day17, and then the enucleation rate remains at this higher stage, forming a plateau. Therefore, we tested the enucleation rate of the normal group and MDS group at day17. At this time, the enucleation rate of erythroblasts in both the MDS and normal groups was maintained at the highest stage.

The results showed that 2 cases of MDS patients had a enucleation rate lower than 10%, 11 cases had a rate of 10–20%, 20 cases had a rate of 20–30%, 13 cases had a rate of 30–40%, and only 7 cases had a rate higher than 40%. Additionally, the rate of enucleation in the 17 samples from the normal group was 38–48%. The typical results of the enucleation rate of each subtype of the MDS and normal groups are shown in Fig. 1A. Compared with that in the normal control group, the rate of enucleation in the MDS group was significantly decreased (Fig. 1B). Meanwhile, We also examined the difference between MDS and normal group on cells growth. The growth curve shown in Supplementary Fig. 1 demonstrate that erythroid cells growth in MDS group was significantly lower than that in normal group. Furthermore, Pearson correlation analysis was conducted between the rate of enucleation and the hemoglobin concentration of MDS patients at initial diagnosis. The results showed that the rate of MDS enucleation was positively correlated with the hemoglobin concentration (Fig. 1C).

Fig. 1

The enucleation of erythroblasts in the MDS group was lower than that in the normal group. A Gating strategy for erythroblast enucleation. (A:a) All events are displayed on this plot of forward scatter area vs side scatter area, exclusion of dead cells and debris. (A:b-c) Exclusion of doublets on a plot of forward scatter area vs height and side scatter area vs height. (A:d) Singles are displayed. Hoechst negative cells are gated, these are mature red blood cells. B Flow cytometry detection of erythroblast enucleation in normal control individuals and patients with different subtypes of MDS. (B: a-d) Normal group of erythroblast enucleation. (B: e-j) Erythroblast enucleation in the MDS subtype was RA, RARS, RCMD, RAEB-1, RAEB-2 and MDS-U. C Quantitative analysis statistics of the rate of enucleation in the MDS and normal groups. D Pearson correlation analysis showed a positive correlation between enucleation and hemoglobin concentration in MDS patients (r = 0.5992, p < 0.01). The results are expressed as the mean ± SD, and * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001

The enucleation efficiency of erythroblasts and the expression levels of pERK and pAKT were significantly different in MDS patients with different prognosis stratifications

The International Prognostic Score System (IPSS) evaluates risk based on a patient's karyotype, the proportion of blasts in the bone marrow, and the degree of hemocytopenia [16]. High risk patients have more karyotype changes (gene mutations) or more blasts than low-middle risk patients. To determine whether the rate of enucleation of MDS in different stratifications was different, risk stratification of MDS was performed to further compare the rate of enucleation among all groups. The results showed that the rate of enucleation of MDS in the high risk group and low-middle risk group was significantly lower than that in the normal group, but the enucleation of MDS in the high risk group was significantly higher than that in the low-middle risk group (Fig. 2A). These results suggest that MDS patients with different risk levels may differ in the biological behavior of erythroblast enucleation due to differences in chromosome or gene mutations in hematopoietic stem cells. In view of the important role of the MAPKs/ERK and PI3K/AKT pathways in the occurrence of hematologic malignant diseases, we further detected the changes in pERK and pAKT of erythroblasts at day17 between individuals in the normal group and MDS patients with different stratifications. Typical western blotting results of pERK and pAKT are shown in Fig. 2B. The expression levels of pERK and pAKT in the high risk group was significantly higher than those in the normal group and the low-middle risk group, and the expression levels of of pERK and pAKT in the low-middle risk group was lower than those in the normal group and the high risk group (Fig. 2C and D).

Fig. 2

The enucleation efficiency of erythroblasts and the expression levels of pERK and pAKT were significantly different in MDS patients with different prognosis stratifications. A Quantitative analysis statistics of erythroblast enucleation in the normal group, low-middle risk MDS group and high risk MDS group. The expression levels of pERK and pAKT in erythroblasts in the high risk MDS group were higher than those in the normal and low-middle risk MDS groups, and the expression levels of pERK and pAKT in the low-middle risk MDS group were lower than those in the normal and high risk MDS groups. B Western blotting was used to detect the expression levels of pERK and pAKT in day17 erythroblasts from MDS patients and the normal group. GAPDH served as the loading control. C Quantitative analysis of pERK protein expression statistics. D Quantitative analysis of pAKT protein expression statistics. The results are expressed as the mean ± SD and * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001

Correlation analysis of erythroblast enucleation and the phosphorylation of ERK and AKT in MDS patients

We have previously found that the dephosphorylation of ERK and AKT can inhibit the enucleation of erythroblasts [13, 14]. The results of the upper section showed that the activation degrees of ERK and AKT in MDS erythroblasts with different risk levels were different, and the enucleation was different. These results suggest that the phosphorylation degree of ERK and AKT in MDS patients may be one of the factors affecting their enucleation. Pearson correlation analysis was performed to verify the relationship between the enucleation of MDS and the phosphorylation degree of erythroblast ERK and AKT. The results showed that the enucleation efficiency of MDS was weakly positively correlated with the phosphorylation of ERK (r = 0.4654, p < 0.01) and AKT (r = 0.3615, p < 0.01) (Fig. 3A and B).

Fig. 3

Pearson correlation analysis of the erythroblasts enucleation rate of MDS and phosphorylation of ERK and AKT. A The enucleation rate of MDS patients was positively correlated with the degree of ERK phosphorylation (r = 0.4654, p < 0.01). B The rate of enucleation in MDS patients was positively correlated with the phosphorylation of AKT (r = 0.3615, p < 0.01)

Morphological analysis of erythroblasts from MDS patients cultured in vitro

Morphological changes in orthochromatic erythroblasts from the normal group, low-middle risk group and high risk group were further observed. The results showed that the frequency of nuclear malformation in the high risk group was approximately 30%, which was significantly higher than that in the low-middle risk group (15%) and normal group (5%) (Fig. 4B). Nuclear malformations often manifest as binucleation, nuclear budding, multinucleation, and megaloblastosis (Fig. 4A). These findings suggest that although orthochromatic erythroblast pERK and pAKT expression is increased in high risk MDS, there may be more chromosome malformations or gene mutations that could lead to abnormal cell division, resulting in increased nuclear malformations. These inhibiting factors offset the promoting effect of ERK and AKT phosphorylation on the enucleation of erythroblasts. The rate of enucleation was still lower than that of the normal group.

Fig. 4

The abnormality rate of MDS erythroblast nuclei in the high risk group was higher than that in the normal group and low-middle risk group. A The morphology of erythroblasts from MDS patients with different risk stratifications cultured in vitro (the black arrow indicates malformed nucleus), with scale bar = 10 μm. B Quantitative analysis of the enucleation efficiency of erythroblasts from MDS patients with different risk stratifications. Nuclear malformations include binucleation, nuclear budding, multinucleation, and megaloblastosis, 100 cells of each group from three independent experiments were used for the quantification. The results are all expressed as the mean ± SD; * indicates p < 0.05,** indicates p < 0.01,*** indicates P < 0.001