Generation Of Bat Induced Pluripotent Stem Cells And Their Preliminary Applications

In 2006, Japanese scientist Shinya Yamanaka used four pluripotent factors Oct4, Sox2, cMyc, and Klf4 to convert mouse embryoic fibroblasts (MEFs) into mouse induced pluripotent stem cells (iPSCs)via retroviral infection. Using these pluripotent factors, scientists around the world generated iPSCs from human, monkeys, rats, pigs, rabbits, bovines, canines, sheep, equines and other species during the following years. As an important anti-viral and longevity models,bats have been a hot research area in recent years. Unlike common animal models such as rodents and livestock, bats are not suitable for isolating EFs or even somatic cells conveniently and safely. Moreover, iPSCs have the ability of infinite proliferation and can be good substitute for embryoic stem cells (ESCs) to perform gene targeting and generate chimeras so as to discover gene functions. As a result, bat iPSCs would have important application values.We successfully established bat embryoic fibroblasts (BEFs) cell lines from Myotis lucifugus that could be stably passaged. We constructed bat specific inducing vector pStem-h103. It contains eight pluripotency factors Oct4, Sox2, cMyc, Klf4, Nanog, Lin28, Nr5a2 and miRNA302/367 which are mediated by piggyBac (PB) system. The induction efficiency of this vector can reach 1% which is much higher than vectors consisting of only four or six pluripotency factors. The generated iPSCs of bat have similar colony morphology to mouse embryoic stem cells (mESCs) which are compact, shiny, with clear edges and 3D structure. Our bat iPSCs were passaged for over 45 times in 3i medium. After acquiring the optimal transfection conditions, we tried to establish iPSC lines without exogenous insertional sequences by using PB transposase vector. The generated bat iPSCs retained normal karyotype of 42+XY and expressed alkaline phosphatase (AP). Various amounts of bat iPSCs were subcutaneously injected into SCID mice, and tumors (teratomas) of different sizes were observed after approximately 4-5 weeks. The teratomas contained tissue cells of all three germ layers confirmed by hematoxylin-eosin(HE) staining. Embryoid bodies (EBs) could be formed by culturing bat iPSCs using ESC medium without LIF.Teratomas and EBs formation demonstrated the bat iPSCs can differentiate both in vivo and in vitro. Real-time quantitative PCR was used to compare pluripotency gene expression differences between bat iPSCs and BEFs. Compared with BEFs, gene expression of Oct4 and Sox2 increased dramatically for bat iPSCs. However, gene expression of cMyc and Klf4 decreased.Immunofluorescence staining and western blot were used to detect the protein expression of bat iPSCs pluripotency genes. The results showed that pluripotency markers OCT4, SOX2, NANOG, TBX3 and TRA-1-60 were positive. Protein expression levels of OCT4 and SOX2 in bat iPSCs were similar to those in mESCs. Bat iPSCs could form interspecific chimeras with pig blastocysts, and they were able to hatch out from the zona pellucida together with pig blastocysts as well. It laid a good foundation for future to obtain heterologous chimeric pigs and produce chimeras using bat iPSCs. All the results above confirmed the generated bat iPSCs have similar pluripotent characteristics to mESCs.One of the most important applications for iPSCs is to perform gene targeting. We used conventional homologous recombination method to perform gene targeting in pig embryoic fibroblasts(PEFs), the iPSCs of pig and bat. Gene targeting in PEFs was successful, but failed in iPSCs, which indicated that gene targeting on iPSCs was more difficult than that of somatic cells. Advanced technologies for gene targeting should be applied to improve the efficiency. We then created CRISPR-Cas9 targeting vector of the bat Oct4 gene and tried in bat iPSCs. The results showed that positive clones of bat iPSCs could be acquired by the Cas9 gene targeting system.