Generation Of Rat Lung In Mouse By Interspecific Blastocyst Injection Of Rat Pluripotent Stem Cells

Objective:To verify feasibility of interspecific lung regeneration by using rat iPSCs and blastocyst complementation in the TTF-1 knockout mice model whose lung development was completely blocked. This study will pave the way to achieve the ultimate goal of regenerating humanized lung organs in large animals including pigs, monkeys.Methods:(1) EGFP labeled rat iPSCs were cultured and maintained using N2B27+2i (PD0325901, CHIR99021) culture system. (2) Alkaline phosphatase staining, RT-PCR, and immunofluorescent staining, teratoma formation and embryoid body (EB) formation were performed to evaluated the sternness of rat iPSCs. (3) TTF-1 heterozygous mice were bred and mated for collecting embryos. Rat iPSCs as donor cells were used to perform interspecific blastocyst complementation using micromanipulation technique. (4) The TTF-1 genotypes of chimeric mice were determined by PCR analysis; the lung morphology of chimeric mice was checked by H/E staining; immunohistochemical analysis of EGFP and TTF-1 were used to reveal distribution of rat iPSC-derived cells in the chimeric mice lung and other organs; the extent of chimerism of chimeras by FACS analysis; the development and differentiation of regenerated lung were assessed by immunohistochemical detection of CCSP, SP-C and Foxj 1.Results:(1) Rat iPSCs were positive by alkaline phosphatase staining; the expression of pluripotent stem cell markers could be detected by RT-PCR and immunofluorescence analysis; teratoma and embryoid body (EB) formation experiments indicated that rat iPSCs could differentiate into three germ layers. (2) Three blastocyst injection experiments were successfully conducted by using micromanipulation technology, totally,191 blastocysts were injected,44 mice were produced, among these mice,5 were chimeric mice. (3) PCR genotyping of 5 chimeric mice using primers specific for mouse TTF-1 gene indicated that one of the chimeric mice was TTF-1 double knockout. Lung regeneration was observed in the TTF-1 double knock out mice by H/E staining; immunohistochemical analysis of EGFP and TTF-1 demonstrated that the regenerated lung was composed of rat iPSC-derived cells; FACS analysis demonstrated that chimeric rate of chimeric TTF-1’-mice was 3.37%; CCSP, SP-C and Foxj1 immunohistochemical staining revealed the differentiation of clara cells, pulmonary alveolar type II cells, terminal bronchiolar epithelium cells in the regenerated lung.Conclusion:The major accomplishment of this study is generation almost entirely rat iPSC-derived lung in TTF-1-/- mice by combination of rat iPSCs and blastocyst complementation. These data validate the feasibility of interspecific lung organ generation in vivo, which will pave a way for generation humanized lungs in large animals including pigs.