Derivation and characterization of pluripotent porcine cells
Reproductive Medicine, Urology
Final Report Abstract
The objectives to produce fully pluripotent porcine iPSC using different cell types as starting material (pMSCs) and by optimization of the culture conditions through testing small molecule library were not fulfilled. Similarly, the attempts to generate transgene-free iPSC and to test the pluripotency by chimera production were not successful. Testing of small molecule library did not lead to discovery of compounds that provided better conditions for generation and maintenance of porcine iPSC. Surprisingly, HDAC inhibitors were able to maintain high expression of multiple pluripotency-related genes in porcine iPSC-like cells in feeder-free conditions in absence of any other supplements, but the cells did not show any differentiation abilities. The studies were performed in an objective and unbiased manner. One reason for the lack of success in generating true porcine iPSC is the fact that the culture conditions that have been proven optimal for mouse and human pluripotent cells have only limited efficacy for porcine cells. This is evidenced by the current absence of fully pluripotent porcine stem cell lines despite the number of allegedly successful reports published to date, the majority of which are incomplete (lacking empirical evidence of pluripotency such as chimera production) or not reproducible (there is only one report of chimera production that has not been reproduced). Moreover, the pluripotency genes used to identify true pluripotent cells in the mouse and human species did not prove to be reliable markers for porcine cells, since our iPSC-like cells (selected for expression of OCT4-EGFP reporter) express high levels of endogenous OCT4 as well as multiple pluripotency markers characteristic for naïve mouse ESC/iPSC but still did not show empirical evidence of pluripotency (ability to differentiate in vitro and in vivo). This paradox may be explained by: a) lack of optimal culture conditions and b) lack of reliable pluripotency markers to identify the truly pluripotent porcine cells. The optimization of culture conditions could be eventually achieved by further trial-anderror experiments which are expensive and time-consuming. To answer the question regarding the reliable pluripotency markers, more basic research into the pluripotency networks and differentiation pathways of proven porcine pluripotent cells (blastocyst inner cell mass cells), including genetic manipulation experiments (e.g. pluripotency gene knock out) would be necessary in the future. In the meantime, it remains unclear how to establish truly (germ line competent) pluripotent porcine stem cells.
Publications
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(2013). The choice of expression vector promoter is an important factor in the reprogramming of porcine fibroblasts into induced pluripotent cells. Cellular Reprogramming 15(1):1-8
Petkov SG, Hyttel P, and Niemann H
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(2014). The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells. Cellular Reprogramming 16(4): 235-240
Petkov SG, Hyttel P, and Niemann H
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(2016). Long-term culture of porcine iPSC-like cells under feeder-free conditions in the presence of histone deacetylase inhibitors. Stem Cells and Development Jan 29 (Epub ahead of print)
Petkov SG, Glage S, Nowak-Imialek M, Niemann H
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Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency. J Stem Cells Regen Med. 2017; 13(1): 20–28
Petkov SG, Glage S, Niemann H
