(Isolation And Functional Characterization Of Human Erythroblasts Of Terminal Differentiation)

Erythropoiesis is a continuous process which hematopoietic stem cells develop and differentiate into mature red blood cells. The appearance of Glycophorin A indicates the starts of the terminal differentiation that the cells start to differentiate from proerythroblast to basophilic erythroblast, polychromatic erythroblasts and orthochromatic erythroblasts. Orthochromatic erythroblasts expel its nuclei and become reticulocytes。Finally, reticulocyte mature into red blood cell, and enter the circulation thru bone marrow.In current study, we established an in vitro culture system that mimic the erythropoiesis process in vivo, and examined dynamic changes of several skeleton proteins and transmembrane proteins by western blot and flow cytometric analysis. The results shown that Band3displayed the most dramatic change in all the proteins that increase their expression during erythropoiesis, and a.4integrin, an adhesion molecule, stayed high at first, and decreased its expression level to almost negative. Therefore, we picked Band3and a4integrin, in combination with Glycophorin A, an red cell lineage marker to monitor the erythropoiesis process in our in vitro culture system, and picked day7and day14cells to sort different stages of erythroblast. On day7, we are able to obtain cells that morphologically resembles proerythoblasts, early basophilic erythroblasts, late basophilic erythroblasts, and polychromatic erythroblasts; on day14, we are able to get polychromatic erythroblasts and orthochromatic erythroblasts, the sorted cells has reached more than90%purity. To examine whether these cells carry the function of different stages, we cultured the sorted cells, count them every day and did growth curve. The results have shown that proerythroblasts can divide4times, early basophilic erythroblasts can divide3times, late basophilic erythroblast can divide2times, polychromatic erythroblasts can divide1time, and orthochromatic erythroblasts cannot divide at all. This implies that our sorted cells carry their function as they displayed their mitotic capacity.We applied these markers to primary normal human bone marrow, and sorted different stages of erythroblasts in primary normal human bone marrow, which obtained different stages of erythroblasts that reached purity of more than90%. Then we analyzed and quantified7primary normal human bone marrow, and found that the ratio of proerythroblasts:early basophilic erythroblasts:late basophilic erythroblasts:polychromatic erythroblasts:orthochromatic erythroblasts has reached our predicted1:2:4:8:16. Then we further applied this method into the bone marrow of patients of myelodysplastic syndromes, but only found altered erythropoiesis files in these patients.In this study, we established a stable in vitro culture system that can observe the process of erythropoiesis, and found that specific markers that can distinguish different stages of erythroblasts in terminal differentiation. Finding these markers enabled us to obtain different stages of erythroblasts in large quantity, which will shed new light into the study of erythroblasts differentiation as well as developmental biology, and we can also use these cells for molecular biology and bio informatic research, such as for RNA sequence, DNA sequence, etc. Last but not the least, the finding of erythroid specific markers can be applied for clinical approach, which will be helpful in diagnosis of stage specific disease and diseases in the blood system that has relation to erythroid differentiation such as bone marrow failure.