Vector Construction Of Hematopoietic Regulatory Factors And Optimization Of Infection Efficiency

Hematopoietic stem cells are adult stem cells that are capable of self-renewal and differentiation into all blood cell types.Although they are rare in vivo,they play an important role in hematopoiesis and the immune system.Currently,hematopoietic stem cell transplantation is the most effective method for the treatment and cure of hematological malignancies,severe aplastic anemia,and some solid tumors.However,the limitations of the hematopoietic stem cells,such as limited sources,insufficient number,and difficulty in amplification,are the difficulties in hematopoietic stem cell transplantation for the treatment of blood diseases.In recent years,a number of transcription factors that are closely related to the hematopoietic system have been identified through a large number of knockout mouse models and blood analysis of leukemia patients.These transcription factors play an important role in the self-renewal and maturation of hematopoietic stem cells.People think about whether they can transdifferentiate other cells of patient into hematopoietic stem cells through these transcription factors.It has been reported that transdifferentiation of mouse endothelial cells into hematopoietic stem cells can be performed with transcription factors.There are also reports that human iPSCs can be differentiated into hemogenic endothelium cells.The hemogenic endothelium cells are then induced to differentiate into hematopoietic stem cells through transcription factors.However,for humans,endothelial cells are a hard-to-get cells,and the safety of iPSCs cannot be guaranteed,so we hope to reprogramme the easily accessible fibroblasts into hematopoietic stem cells.Therefore,this article uses mice as a model to try to reprogramme mouse fibroblasts into hematopoietic stem cells,and then use this transdifferentiation system in human experiments of hematopoietic stem cell transdifferentiation.At present,two articles attempted to transdifferentiate mouse fibroblasts into hematopoietic stem cells by transcription factors,but ultimately only hematopoietic progenitor cells were obtained,probably because they did not find the true key transcription factors in the development and regulation of hematopoietic stem cells.According to the data of single cell sequencing to track mouse hematopoietic stem cell by Liu Bing's group,we intersected T1 pre-HSC signature genes,T2 pre-HSC signature genes,and BM HSC signature genes to obtain 34 genes that were highly expressed.Analysis of these 34 genes that were commonly expressed high and found thriteen transcription factors(Gfi1,Hif3 a,Hlf,Irf2,Meis1,Mllt3,Mpl,Mycn,Nfix,Nkx2-3,Scl,Stat4,Vdr)and one Epigenetic factor(Dnmt3b).After reviewing the related literature,we learned that many of these 14 regulatory factors play an important role in the generation and self-renewal of HSCs.Therefore,we established an overexpression plasmid vector for these 14 regulatory factors and tried to transdifferentiate mouse fibroblasts into hematopoietic stem/progenitor cells.At the same time,the plasmid vectors were also constructed by using the transcription factors Erg,Gata2,Lmo2,Runx1,and Bmi1 that have been used in the transdifferentiation of mouse fibroblasts into hematopoietic progenitors.We hope to find the best combination of regulatory factors.Firstly,the CDS sequences of these 19 regulatory factors were obtained by PCR.The obtained sequences were ligated into the Blunt vector,digested and verified by sequencing.Then,homologous sequences were obtained by PCR using primers containing homology arms,and the sequences were ligated into the pMXs expression vector by homologous recombination.Finally,specific PCR was used to identify the normal expression of genes in the pMXs vector in MEFs.In this study,Mouse Embryonic Fibroblasts(MEFs)were isolated from mouse embryonic day 13.5 embryos and used as the starting cells for transdifferentiation.The irradiation intensity and seeding density of OP9 cells were explored,and the best irradiation intensity and seeding density were obtained.At the same time,OP9-DL1 cells in good condition were constructed as supporting cells.In order to improve the quality of the virus,the method of calcium phosphate transfection was optimized in this experiment to find out the amount of plasmid with better calcium transfer,pH value of 2*HBS,method,and time point.After optimization of the infection efficiency,it was found that infection of MEFs with 14 ul of each of the 12 viruses that were concentrated 50 times could achieve an infection efficiency of more than 50%,indicating that using the discovered calcium transfection and infection methods can proceed the research of the transifferentiation of the mouse fibroblasts into hematopoietic stem/progenitor cells.Therefore,this study has established a preliminary platform for the transdifferentiation of fibroblasts to hematopoietic stem cells,which brings hope for the efficient retrieval of hematopoietic stem cells in vitro.