Comparative Study Of Reprogramming Efficiency And Pluripotency Of Horse,Donkey And Mule Induced Pluripotent Stem Cells

As the interspecies hybrid of horse and donkey,mule has better muscular endurance,disease resistance,and longevity than its parents.However,there are few studies on the characteristics of mule cells,and no studies have compared the differences between horse,donkey and mule at the cellular and molecular levels.Fibroblasts reprogramming to induced pluripotent stem cells(iPSCs)has still been inefficient,and it is still a challenge to establish iPSCs of large livestock species,such as horse,donkey and mule.To explore whether mule could reflect the advantages than horse and donkey at cellular and molecular levels,we examined adult fibroblasts of mule(MAFs)compared with the donkey adult fibroblasts(DAFs)and horse adult fibroblasts(HAFs)in cell proliferation,apoptosis,and glycolysis.Subsequently,Dox-independent iPSCs of horse,donkey and mule were successfully established after reprogramming.Finally,we characterized the pluripotency and differentiation potential of these iPSCs.The detailed results were shown below:1.The cellular characteristics and reprogramming efficiency of MAFs compared with HAFs and DAFs.Firstly,different cell lines of HAFs,DAFs and MAFs were cultured and compared under the same condition,and MAFs were found to proliferate faster than DAFs and HAFs.Cell cycle analysis revealed that MAFs had a higher percentage of S phase compared to DAFs and HAFs.The enrichment analysis of upregulated differentially expressed genes in MAFs revealed that biological processes and pathways such as DNA replication and cell cycle were enriched,compared with DAFs and HAFs.RT-qPCR and Western blot results indicated that pro-apoptotic genes(Dffa,Bax and Caspase-3)were all highly expressed in DAFs and HAFs,compared with MAFs.Furthermore,the results of RT-qPCR and RNA-seq data revealed glycolysis-related genes had higher expression levels in MAFs than DAFs.The cell proliferation,apoptosis and glycolysis could impact reprogramming efficiency.Then,we expressed Dox-inducible exogenous reprogramming factors in HAFs,DAFs and MAFs,delivered via piggy Bac transposition.Our results showed that MAFs had higher reprogramming efficiency and stem cells establishment efficiency than HAFs and DAFs.2.Establishment of Dox-independent iPSCs of horse,donkey and muleAfter reprogramming,the horse,donkey and mule iPSC colonies were passaged in single-cell suspension in M15 medium supplemented with Dox.Upon Dox removal,mule iPSCs differentiated rapidly within 6 days,concomitant with the increased expression of three germ layers genes and the loss of endogenous pluripotency genes expression.We screened several other culture conditions and successfully derived horse,donkey and mule Dox-independent iPSCs under SERUM/LIF culture condition,named as hiPSCs,diPSCs and miPSCs.These hiPSCs,diPSCs and miPSCs had high nuclear:cytoplasmic ratios,formed compact colonies with smooth colony edges,were AP-positive and could be passaged for more than 50 passages with high expression levels of key endogenous pluripotency genes.hiPSCs,diPSCs and miPSCs had a normal karyotype after long-term passages and the leaky expression of the exogenous reprogramming factors was not readily detected in RT-PCR.These results indicated that we successfully established Dox-independent iPSCs of horse,donkey and mule.3.Transcriptome profiling of hiPSCs,diPSCs and miPSCs and their respective adult fibroblastsTo further gain molecular insights into hiPSCs,diPSCs and miPSCs and their respective adult fibroblasts,we determined global gene expression profiles using RNA-seq analysis.Compared with their respective adult fibroblasts,the pluripotency genes,such as Pou5f1,Nanog and Esrrb,in hiPSCs,diPSCs and miPSCs were all significantly increased,whereas the somatic cell genes,including Zeb1 and Thy1,were significantly decreased.Furthermore,pearson correlation analysis revealed miPSCs clustered closer to diPSCs than to hiPSCs.4.The differentiation potential of miPSCs compared with diPSCs and hiPSCs in vivo and in vitroTo functionally evaluate the differentiation potential of hiPSCs,diPSCs and miPSCs in vivo and in vitro,EB formation in vitro was assessed.RT-qPCR analysis and immunostaining results showed that hiPSCs-,diPSCs-and miPSCs-EBs differentiated into cells expressing all three germ layer,such as ectoderm genes:Noggin and Nestin;mesoderm genes:Hand1 and Gata2;endoderm genes:Gata4 and Afp.Furthermore,teratoma formation assays in vivo showed that miPSCs have faster teratoma formation and higher formation efficiency.Next,the differentiation potential of miPSCs and diPSCs in interspecies chimeras were tested.The td Tomato-labeled miPSCs and diPSCs were injected into gastrula-stage mouse embryos.After 48 h culture in vitro,miPSCs proliferated more extensively than diPSCs.Immunostaining analysis revealed that td Tomato~+cells of miPSCs expressed markers of embryonic cell lineages and PGC.Interestingly,diPSCs could not differentiate into endoderm,as endoderm marker were not detected in chimeric embryos.In conclusion,this study compared hiPSCs,diPSCs and miPSCs and their respective adult fibroblasts at cellular and molecular levels and found significant differences.We found MAFs have advantages in reprogramming efficiency than HAFs and DAFs,and miPSCs have significant advantages in pluripotency and differentiation potential than diPSCs and hiPSCs.The establishment of hiPSCs,diPSCs and miPSCs provide unique research materials for the investigation of heterosis and perhaps will be more significant to study hybrid gamete formation.