BACKGROUND: With the aging of the population and the continuous updating of medical technology, the demand for red blood cells is increasing and blood supply intends to be increasingly tight. The preparation of blood products for clinical application in vitro has become a hot spot of many experts, and hematopoietic stem cells differentiating into mature red blood cells in vitro may be an approach for new blood sources.
OBJECTIVE: To explore the practicability of using three-stage suspension culture system to induce the differentiation of peripheral blood hematopoietic stem cells from the abandoned buffy-coat into mature erythrocytes in vitro.
METHODS: The study protocol was approved by the ethics committee of Henan Red Cross Blood Center, China. CD34+ cells were isolated from peripheral blood buffy-coat, and were subsequently induced to differentiate into mature red blood cells in vitro by 21-day three-stage suspension culture system. Cell counts were performed on 4, 7, 9, 11, 13, 15, 17, 19, and 21 days of culture, and cell growth curves were then drawn. Cell smear and May-Giemsa staining were performed on 7, 11, 15, 19, and 21 days of culture, for cell morphology observation under a microscope. Flow cytometry was used to detect early erythroid differentiation on 7 days of culture and to detect terminal erythroid differentiation on 7, 11, 15, and 17 days of culture. Denucleation rate of erythroblasts was measured on 15, 17, and 19 days of culture.
RESULTS AND CONCLUSION: (1) With the increase of culture days, the number of cells showed an increasing trend, reaching a peak on 17 days of culture. The cells expanded to about 1 300 times, and then tended to be stable. (2) With the increase of culture days, the cells differentiated from proerythrocytes to basophilic erythrocytes, polychromatic erythrocytes and positive-stained erythrocytes, and almost all of the cells differentiated into denucleated erythrocytes on 21 days of culture. (3) The percentage of IL-3R/GPA double-negative cells was (15.8±0.21)%, of which erythroid burst-forming units accounted for (0.98±0.21)% and colony-forming units accounted for (8.13±1.42)%. (4) The GPA expression increased gradually with the increase of cell culture days, and about 90% of cells expressed GPA on 17 days of culture, indicating a successful erythroid differentiation of hematopoietic stem cells. (5) With the increase of culture days, the non-nuclear red blood cells increased in number, and accounted for over 80% of the cells until the 19th day of culture. To conclude, in the 21-day three-stage suspension culture system, peripheral blood hematopoietic stem cells from the abandoned buffy-coat can be induced to differentiate into mature red blood cells in vitro, with a high denucleation rate.