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Chimeric Pigs Produced from Induced Pluripotent Stem Cells Demonstrate Germline Transmission and No Evidence of Tumor Formation in Young Pigs

From the October Edition of Stem Cells
By Stuart P. Atkinson

A brief report in the October edition of Stem Cells from the laboratory of Steven L. Stice at the University of Georgia, Athens, Georgia, USA describes the ability of pig induced pluripotent stem cells (piPSCs) to be transmitted through the germline and studies the effect of piPSC incorporation on offspring development and tumorigenicity, the first experiment of its kind outside the mouse model (West et al, 2011). Using pig as a model for such studies holds great interest due to the relative similarity of pigs to humans when compared to the more widely used mouse model.

In a previous study (West et al. 2010), the authors generated female piPSCs from pig mesenchymal cells using the human POU5F1, SOX2, NANOG, LIN28, KLF4, and C-MYC genes. These piPSCs were then used to generate chimeric piglets by cell injection into blastocyst-stage embryos. 29 piglets were produced and through the analysis of human POU5F1 expression, it was shown that the piPSCs contributed to organs arising from the ectoderm, endoderm, and mesoderm lineages and, interestingly, the gonads. Of the 132 samples tested from organs from all 29 piglets, 37% were hPOU5F1+ with ectoderm (45%), mesoderm (41%), and gonad (45%) showing similar piPSC tissue incorporation percentages, while the piPSC tissue incorporation percentage was lower for endoderm (29%). Encouragingly, gross analysis of organs showed no abnormalities and no evidence of tumors, while tissues found to be entirely hPOU5F1+ were normal, overall suggesting that iPSCs can safely incorporate into tissues without tumor formation. Further examination of hPOU5F1+ tissues found hPOU5F1 mRNA expression in 39% of the samples, but tumour-associated hC-MYC mRNA expression was not observed in any samples.

Next, the germline competence of piPSCs was tested by crossing chimeric females with non-chimeric mice and analysing the 43 offspring (F1), one of which was stillborn. Of these 43 piglets, two from the same litter were hPOU5F1+ and hNANOG+, so while there is clear germline contribution, the overall transmission is low. However, the two hPOU5F1+hNANOG+ piglets were unhealthy, with one being the stillborn piglet and the other living for three days, while the non-iPSC derived piglets thrived and showed normal development. Analysis of hPOU5F1+hNANOG+ piglets however revealed no overt abnormalities. Analysis of sperm from four male-chimeras indicated a lack of incorporation into the male germline, as expected given that the piPSCs were female (XX) and had been introduced into male (XY) embryos. Multiple studies have shown that XX cells fail to incorporate into the adult male germline and that specific Y chromosome-related factors are critical for normal spermatogenesis, overall suggesting that that XX piPSCs are likely fail to form sperm in chimeric pigs.

The data presented in this paper suggests that porcine models could be utilised to study the efficacy and safety of iPSC-mediated stem cell therapies outside of rodent systems. The research may also be of agricultural importance, allowing the generation of distinct pig strains. However, the fact that iPSC germline transmission only gave unhealthy pigs is of concern.

 

References

Franklin D. West, Elizabeth W. Uhl, Yubing Liu, et al
Brief Report: Chimeric Pigs Produced from Induced Pluripotent Stem Cells Demonstrate Germline Transmission and No Evidence of Tumor Formation in Young Pigs
Stem Cells Article first published online: 27 SEP 2011

West FD, Terlouw SL, Kwon DJ, et al
Porcine induced pluripotent stem cells produce chimeric offspring.
Stem Cells Dev. 2010 Aug;19(8):1211-20.