Fig 1.
Staging of natural bone marrow CD71+ erythroid cells.
A–Dot-plot for natural bone marrow CD71+ erythroid cells. B–Histogram of the distribution of bone marrow CD71+ erythroid cells by marker CD 45. C–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 71 and CD 235a from CD 45-negative natural bone marrow CD71+ erythroid cells, ProEB–proerythroblasts (CD235alowCD71high cells), BasoEB+PolyEB+OrthoEB+Ret–basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts/reticulocytes (CD235highCD 71pos cells), Erythrocytes–CD235ahighCD71neg. D–Histogram of the distribution of stages of development of erythroid cells with a phenotype CD235highCD 71pos depending on the FSC. Ret–reticulocytes (CD 235ahighCD71pos FSClow), Ortho–orthochromatophilic erythroblasts (CD235ahighCD71posFSClow cells), Poly–polychromatophilic erythroblasts (CD235ahighCD 71posFSCmiddle cells), Baso–basophilic erythroblasts (CD 235ahigh CD71pos FSChigh cells).
Fig 2.
Staging of induced CD71+ erythroid cells derived from CD34+ bone marrow cells.
A–Dot-plot for induced CD71+ erythroid cells derived from CD34+ bone marrow cells. B–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 45. C–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 71 and CD 235a from CD 45-positive induced CD71+ erythroid cells. D–Histogram of the distribution of stages of development of CD71+ erythroid cells with a phenotype CD45posCD235ahighCD 71pos depending on the FSC. E–Dot-plot of the distribution of bone marrow CD71+ erythroid by marker CD 71 and CD 235a from CD 45-negative induced CD71+ erythroid cells. F–Histogram of the distribution of stages of development of CD71+ erythroid cells with a phenotype CD45negCD235ahighCD 71pos depending on the FSC, ProEB–proerythroblasts (CD235alowCD71high cells), BasoEB+PolyEB+OrthoEB+Ret–basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts/reticulocytes (CD235ahighCD 71pos cells), Erythrocytes–CD235ahighCD71neg, Ret–reticulocytes (CD 235ahighCD71pos FSClow), Ortho–orthochromatophilic erythroblasts (CD235ahighCD71posFSClow cells), Poly–polychromatophilic erythroblasts (CD235ahighCD 71posFSCmiddle cells), Baso–basophilic erythroblasts (CD 235high CD71pos FSChigh cells).
Table 1.
Proportions (%) of CD45+ and CD45- cells among the induced CD71+ erythroid cells derived from CD34+ bone marrow cells and bone marrow CD71+ erythroid cells.
The data are presented as the mean ± standard derivation (n = 6).
Fig 3.
Comparison of the distribution of stages of development of bone marrow CD71+ erythroid cells and CD71+ erythroid cells induced from CD34+ bone marrow cells.
Kruskal-Wallis test ANOVA with Dunn’s correction for multiple comparisons was carried out to evaluate statistical significance of the differences between the cell groups. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.
Fig 4.
Expression of lymphoid cell markers on the surface of induced CD71+ erythroid cells.
A–Dot-plot for induced CD71+ erythroid cells derived from CD34+ bone marrow cells. B–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 45. С –Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 3 from CD 45-negative induced CD71+ erythroid cells. D–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 3 from CD 45-positive induced CD71+ erythroid cells. E–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 8 and CD 4 from CD 45-positive induced erythroblasts. F–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 16 and CD 19 from CD 45-positive induced erythroblasts.
Fig 5.
Heat map of studied cells’ transcriptome.
Fig 6.
Volcano-plots of differentially expressed genes.
The purple dots correspond to the samples of induced CD71+ erythroid cells, the orange dots correspond to the samples of natural CD71+ erythroid cells of bone marrow.
Fig 7.
MA-plots of differentially expressed genes.
The purple dots correspond to the samples of induced CD71+ erythroid cells, the orange dots correspond to the samples of natural CD71+ erythroid cells of bone marrow.
Fig 8.
Plot of Gene Set Enrichment Analysis (GSEA) results showing down-regulated differentially expressed genes.
Fig 9.
The PLS-DA plot of cytokine production by different types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34+ bone marrow cells).
An example distribution of data points for chemokines, interleukins, and growth factors is shown. (x)–ABM–bone marrow erythroblasts. (·)–CD 34 induced–induced erythroblasts from CD34+ bone marrow cells.
Fig 10.
The PLS-DA plot of cytokine production by different types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34+ bone marrow cells).
An example distribution of data points for colony-stimulating factors, interferons, and TNF family cytokines is shown. (x)–ABM–bone marrow erythroblasts. (·)–CD 34 induced–induced erythroblasts from CD34+ bone marrow cells.
Fig 11.
Cytokines that manifested significant differences in secretion of interleukins between the two types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34+ bone marrow cells).
The Mann–Whitney test was performed to compare the production of cytokines between the different types of CD71+ erythroid cells. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.
Fig 12.
Cytokines that manifested significant differences in secretion of chemokines and growth factors between the two types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34+ bone marrow cells).
The Mann–Whitney test was performed to compare the production of cytokines between the different types of CD71+ erythroid cells. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.
Fig 13.
Effects of the conditioned media from either the natural or induced bone marrow CD71+ erythroid cells on the mixed lymphocyte reaction (n = 4).
The results are presented as optical density of allogeneic cultures. Statistical analysis of the data was conducted in the GraphPad Prism 8.4 software. Two-way ANOVA with Tukey’s correction for multiple comparisons was carried out to evaluate statistical significance of the differences between the cell groups. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range. “Allostimulator”: culture of mononuclear cells with the addition of allogeneic donor cells treated with mitomycin C; “Allostimulator + BM EBs”: culture of mononuclear cells with the addition of mitomycin C–treated allogeneic donor cells and supplementation with the conditioned medium from natural bone marrow CD71+ erythroid cells; “Allostimulator + induced EBs”: culture of mononuclear cells with the addition of mitomycin C–treated allogeneic donor cells and supplementation with the conditioned medium from the induced CD71+ erythroid cells derived from CD34+ bone marrow cells of healthy donors.