http://orcid.org/0000-0003-1138-9712
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ABSTRACT
Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig.
Abbreviations
| AFP | = | α feta protein |
| AP | = | alkaline phosphatase |
| bFGF | = | basic fibroblast growth factor |
| CFU | = | colony forming unit |
| DMEM | = | Dulbecco's modified eagle medium |
| dpt | = | days post transfection |
| EB | = | embryoid body |
| ESC | = | embryonic stem cell |
| FACS | = | fluorescent-activated cell sorting |
| FBS | = | fetal bovine serum |
| GO | = | gene ontology |
| iPSC | = | induced pluripotent stem cell |
| LPR | = | liquid permanent red |
| MACS | = | magnetic-activated cell sorting |
| MSC | = | mesenchymal stem cell |
| Muse | = | multilineage-differentiating stress enduring |
| pEF | = | porcine embryonic fibroblast |
| pen/strep | = | penicillin and steptomycin |
| piPSC | = | porcine induced pluripotent stem cell |
| RMA | = | robust multiarray average |
| rt | = | room temperature |
| SSEA-1 | = | stage-specific embryonic antigen-1 |
| SEER | = | SSEA-1 expressing enhanced reprogramming |
| SMA | = | smooth muscle actin |
Disclosure of potential conflicts of interest
In the case of each author, there is no commercial association or conflict of interest with the submitted manuscript.
Acknowledgments
We would like to thank Tina Christoffersen for cell culture support, Anita Pacht for assistance with qRT-PCR. We also thank Ellegaard Göttingen Minipigs A/S for supplying us with Göttingen embryos that were used to produce the porcine embryonic fibroblasts in this study.
Funding
Funding for this research was provided by: The Danish Council for Independent Research, Natural Sciences (FNU), grant number: HEALTH-2007-B-223485; US Department of Agriculture (USDA) grant number: 2011–67015–30688 and personal PhD fellowship to Dong Li sponsored by China Scholarship Council (CSC).
