Differentiation Of Human Pluripotent Stem Cells Into Red Blood Cells

Preparation of red blood cells in vitro has provided new ideas for medical research of blood,and there are two traditional methods of induction,embryoid body induction and co cultured with stromal cells induction.However,these two methods have some defects,like the procedure is cumbersome,the induction period is long,the efficiency is low,and it contains animal-derived substances.Our study has improved the previous methods,establish a two induction steps to obtian red blood cells.The first step is adherent culture of hiPS cells and hUCMSC^Rh-A cells to differentiate into mesoderm,and induced into CD31⁺and CD34⁺cells.The second induction step,the obtained CD31⁺and CD34⁺cells were differentiated into red blood cells under the addition of various growth factors.Finally we obtained hiPS cells derived red blood cells,and the induced red blood cells'size and morphology are similar to normal red blood cells by Giemsa staining,and examed the expression of mature?-globin by real time RT-PCR.However,hUCMSC^Rh-Ah-A cellsderived red blood cells are immature red blood cells.As the study subjects,hUCMSC^Rh-Ah-A cells were used to generate Rh negative red blood cells,and our study provides a new idea for the preparation of"universal blood"?Rh negative O type?in vitro.The main results from our study are as follows:1.In vitro culture and identification of human pluripotent stem cellsDuring the process of hiPS culture in vitro,the morphology and proliferation rate of cell clones were normal,and there were no signs of cell aging and differentiation.Immunofluorescence assay showed that multipotent markers SOX2,NANOG and OCT4 were positive in hiPS cells.hUCMSC^Rh-A cells always show the spindle shape during the process of culture in vitro,and there were no signs of cell aging and differentiation.Through RT-PCR,we found that hUCMSC^Rh-A cells always expressed genes CD29,CD90 and CD117 of mesenchymal stem cells,and did not express the genes related to hematopoiesis,such as CD31,CD34 and CD45.And we found that CD44 and CD71 were highly expressed in hUCMSC^Rh-A cells,but only 0.2%of the cells expressed CD31,CD34 and CD45 markers by flow cytometry.2.Differentiation of human pluripotent stem cells into CD31⁺and CD34⁺cellsThe first step of two induction steps is human pluripotent stem cells were differentiated into CD31⁺and CD34⁺cells through the adherent culture.hiPS cells were induced for 6 days,and we found that CD31⁺and CD34⁺cells were 31.2%and 8.2%,respectively through flow cytometry.And after 10 days induction,hUCMSC^Rh-A cells derived CD31⁺and CD34⁺cells were 5.3%and 22.7%,respectively through flow cytometry.After 14 days induction of hUCMSC^Rh-A cells,the CD31⁺and CD34⁺cells were only 1.2%and 5.6%,respectively through flow cytometry.It was demonstrated that the cells expressing CD31 and CD34gradually decreased as the induction progressed,and this result is confirmed by RT-PCR.3.CD31⁺and CD34⁺cells differentiate into red blood cellsAt the seconed induction step,the hiPS derived CD31⁺and CD34⁺cells were differentiated into reed blood cells for 36 days suspension culture.Through Giemsa staining,we found that the induced red blood cells have been denucleated,and their morphology is similar to that of human normal red blood cells.Quantification of globin gene expression by real time RT-PCR revealed that the expression ratios of?-globin,?-globin,and?-globin were6%,74%,and 20%,respectively.The induced red blood cells are collected into the centrifuge tube,and then these cells were naturally settled for 2 hours and showed the red color.The second step of induction with hUCMSC^Rh-A cells derived CD31⁺and CD34⁺cells can only differentiation into immature red blood cells.This immature cells did not express mature?-globin and only expressed?-globin.Our study has optimized the previous red blood cell inducing system and simplified it into a sticking-suspension two-step induction method.And we successfully obtained the CD31⁺and CD34⁺cells from hiPS cells and hUCMSC^Rh-A cells,and hiPS cells derived red blood cells from hiPS cells and hUCMSC^Rh-A cells.Our research provides a new technique for the large-scale generation of red blood cells in vitro.