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Objective To explore the phenotypes of erythroblastic island-like (EBI-like) macrophages during erythroid differentiation from human embryonic stem cells in vitro and the function of EBI-like macrophages in the differentiation of erythroid. Methods To study the function of EBI- like macrophages in the differentiation of erythroid, we used the Spin-EB method to induce human embryonic stem cells (hESCs) to differentiate into red blood cells. The expression of specific surface markers of erythrocytes and macrophages and the cell morphology were tested by flow cytometry and imaging flow cytometry. Multiple gene expressions were detected by qRT-PCR. An immunofluorescence assay was used to identify the phenotype of EBI-like macrophages. Results Erythroid cells were induced by embryonic stem cells in vitro. Giemsa staining revealed that EBI-like structures derived from the hESCs were similar to natural EBI. Immunofluorescence staining confirmed that the erythroid cells (CD235a) were surrounded by macrophages (CD68); CD45+CD235a+CD163+CD169+CD106+ EBI-like macrophage (0.092%±0.013%) resembles in vivo EBI macrophage. Imaging flow cytometry observed that CD45+CD235a+CD163+CD106+CD169+ EBI-like macrophages were surrounded by CD235a+ erythroid cells. The proportion of induced CD71+CD235a+ cells was 37.37%±1.68% after removing the EBI structure, which was significantly lower than the untreated group (46.97%±4.16%). We found that central macrophages of EBI-like may play a role in promoting erythroid differentiation through the interaction of CD169 and CD43. Conclusions EBI-like structure in the erythroid induction system of hESCs is similar to the natural EBI. The central EBI-like macrophages maybe promote the differentiation of erythrocytes in vitro through the interaction of CD169 and CD43. We provided a theoretical and practical reference for optimizing the erythroid induction system in vitro.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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