Differences In Hematopoietic Induction And Differentiation Of HiPSC With Different Mutation Types In Thalassemia

Background:In C hina,thalassemia has a high incidence in Guangxi,Guangdong and Hainan.Currently,the treatment of thalassemia mainly relies on blood transfusion,but the long-term cost of blood transfusion treatment is high,and the patient’s medical compliance is poor,leading to the poor treatment effect of thalassemia.In addition,thalassemia can also be treated by transplantation of hematopoietic stem cells,but the source of stem cells is insufficient and the cost is extremely high.The proportion of globin in mature red blood cells of thalassemia patients is unbalanced,causing insufficient synthesis of normal hemoglobin and excess globin chains in red blood cells forming unstable products,leading to ineffective red blood cell production.However,it is still unclear whether the stem cell lines of patients with different gene mutations in thalassemia have differences in the process of early hematopoietic differentiation.Therefo re,this study intends to use hiPSCs from patients with different thalassemia gene defects and different phenotypes to induce hematopoietic differentiation experiments,to study the differences in the early hematopoietic differentiation of stem cell lines with different types of thalassemia gene mutations,and to study the related mechanisms,which is expected to provide a certain theoretical basis for the clinical transformation of thalassemia treatment.Objective:To explore the differences of hiPSCs with different types of thalassaemia gene mutations in the process of hematopoietic differentiation,to clarify the influence mechanism of hiPSCs with different gene mutations of thalassemia on the early hematopoietic differentiation,and to provide mechanism and theoretical basis for the diagnosis and treatment of thalassemia.Methods:In this study,experimental group and control group were set up.The experimental group:hiPSCs derived from amniotic fluid cells of patients with minor and major thalassemia.Control group:hiPSCs derived from normal human amniotic fluid cells.We performed complete transcriptome sequencing on hiPSCs with different gene mutations and analyzed their differential genes related to hematopoiesis.At the same time,the hematopoietic differentiation of hiPSCs was induced by feeder-free and serum-free monolayer culture containing various cytokines.By observing the cell morphology at different stages of the induction process,flow cytometry and q-PCR were observed to compare the differences of stem cell factors,hematopoietic related factors and hematopoietic related target genes during induction.Results:First,the whole transcriptome sequencing of hiPSCs from normal people,minor and major thalassemia was performed,and the genes with log2>2 were analyzed and screened the literature related to hematopoiesis.Four differential target genes（CAT,EGR1,FOS,KLF4）were screened to be related to hematopoiesis.Compared with hiPSCs in normal people,the expression of CAT were up-regulated in minor and major thalassemia,and the expression of EGR1,FOS and KLF4 were down-regulated in minor and major thalassemia.This research group verified the above 4 genes by q-PCR,and found that the expression of KLF4 was consistent with the sequencing results,while CAT,EGR1 and FOS were contrary to the sequencing results.At the same time,feeder-free and serum-free monolayer culture containing multiple cytokines were used to induce hematopoietic differentiation of hiPSC s.It was found that compared with AF888 cells,AF076 and AF091 cells had no significant differences in cell morphology during hematopoietic induction.The results of flow cytometry showed that CD34⁺CD45⁺,CD34⁺CD31⁺,CD34⁺,CD31⁺,CD45⁺expressed similar percentages of D10 induced by the three cell lines.The q-PCR results showed that the expression of OCT4,SOX2 and NANOG gradually decreased with the progress of induction,indicating that the cells were clearly differentiated.There is no statistical difference in the expression of Brachyury at D0.The expression increased in both D3 and D10,especially in D3,indicating that D3mainly differentiated into the mesoderm,and the expression decreased over time.The expression of RUNX1 decreased at D0,but gradually increased the expression of D3 and D10,indicating that as the induction progressed,hematopoies is continued to increase.The expression of C D34 and CD31 was increased in D10 induced by the three stem cell lines,and the expression of D10 in the hematopoietic-induced differentiation of hiPSCs derived from major thalassemia is more,indicating that hematopoietic differentiation is successful.The severity of globin gene defects may affect the hematopoietic induction and differentiation process of hiPSC s and increase hematopoietic capacity.Compared with D10 induced by AF888 cells,AF076 cells induced D10 globin expression,while the expression ofε-globin,Aγ-globin,Gγ-globin,α-globin,β-globin-N,β-globin-M in AF091 cells induced D10 increased significantly.In the early stage of hematopoietic induction,the expression ofβ-globin did not decrease but increased in the mutation type of major thalassemia.Moreover,the expression of globin increased in both embryonic and fetal stages,indicating that the mutation type of major thalassemia may cause the compensatory increase of globin in embryonic and fe tal stages.The study found that the expression of hematopoietic-related differential target genes CAT,EGR1,FOS and KLF4 increased in the cells of major thalassemia of the hematopoietic-induced D10.Conclusions:Using feeder-free and serum-free hematopoietic induction and differentiation conditions,hematopoietic induction and differentiation were successfully achieved in three hiPSCs of different genotypes.We found that compared with the normal hiPSCs（αα/αα,β^N/β^N）,the major thalassemia gene mutation(αα/αα,β^41-42M/β^41-42M)increased in hematopoietic ability,while there was no significant difference in the hiPSC s of minor thalassemia gene mutation(αα/αα,β^41-42M/β^N).In major thalassemia,what are the factors that lead to the high expression of KLF4?It remains to be further studied.