Obtaining Of Polycistronic Entivirus Induced Pluripotent Stem Cells From Mongolian Horse Embryonic Fibroblasts

Mongolian horse is one of the oldest horse breeds in the world and an excellent horse breed resource in our country.It has the characteristics of strong endurance to cold and dry environment and roughage,as well as strong adaptability.Moreover,because especially in terms of the anatomy and physiology of joint and tendon and after-injury situation of horse is highly similar to that of human,horse is one of the traditional model for investigating cartilage injury or degenerative cell therapy.Induced pluripotent stem cells(iPSCs)are a type of stem cells similar to embryonic stem cells obtained by introducing specific transcription factors into somatic cells for reprogramming.iPSCs can be derived from any somatic cell and can self-renew and differentiate into all types of somatic cells.Therefore,they can be used in biomedicine to establish disease models to study disease development,drug detection,and disease treatment.In terms of agriculture,it can also be used to develop genetically modified animals or protect excellent genetic resources.But so far,although many teams have tried to establish the horse iPSs,there are no authentic horse iPSCs has been reported and also no try has been reported in Mongolian horse at home and abroad.Therefore,in order to establish Mongolian horse induced pluripotent stem cells,in this research we used a polycistronic lentiviral vector to introduce the four foreign genes OCT4,SOX2,KLF4 and c-MYC into Mongolian horse embryonic fibroblasts for reprogramming.Our research laid a strong foundation for establishing a further wide-range of biological and medical cell models of Mongolian horse.In Summary,our research outcomes are as follows:1.The 34-day embryonic fibroblasts of Mongolian horse and the13.5-day embryonic fibroblasts of mouse were separated and cultured by the digestion method.Both cell morphologies are in line with the morphological characteristics of fibroblasts,can be digested and passaged normally,and can proliferate normally without abnormal morphology after freezing and resuscitation.These two kinds of cells could be successfully used as fonder cells for reprogramming and feeder layers for follow-up horse iPSCs research.2.To optimize the polycistronic lentivirus packaging system,we transfected 293 T cells with liposome 3000 and liposome LTX transfection reagents,and compared the transfection efficiency of the lentiviral plasmid encoding the green fluorescent protein gene and the ability of the virus to infect cells.The result showed that liposomal 3000 is better in terms of transfection efficiency of virus DNA and infection efficiency of the packaged virus,which were 79.4±6.3% and 74±3.6%,respectively.3.With the method ofinfection of polycistronic lentivirus encoding Oct4,Sox2,Klf4 and c-Myc genes,we successfully obtained the Mongolian horse iPS-like cells(E-iPS):(1)Mongolian horse iPS-like cells exhibit clear edges and grow as cell colonies;(2)Alkaline phosphatase(AP)staining showed that our Mongolian horse iPS-like cells are AP-positive,implicating the poluripotency of it;(3)The RNA of Mongolian horse iPS-like cells was extracted,and real-time PCR was conducted to detect the m RNA expression level of pluripotency-related genes in the Mongolian horse iPS-like cells.The results showed that in the Mongolian horse iPS-like cells highly expressed Oct4,Sox2,Nanog,Bmp4,Rex1;(4)The obtained Mongolian horse iPS-like cells were subjected to immunofluorescence staining experiments.The results showed that the Mongolian horse iPS-like cells express the pluripotent markers Oct4,Sox2,Nanog,Lin28,SSEA-1 and SSEA-4;(5)The culture medium components 2i,LIF and BFGF were removed,and the morphological changes of Mongolian horse iPS cells were observed.The results showed that E-iPS lost its clone morphology when 2i and BFGF were removed from the culture medium,indicating that Mongolian horse iPS cells may maintain pluripotency through the BFGF pathway.