Abstract
Concerted efforts have been expended in deriving porcine induced pluripotent stem cells (piPSC) which are envisaged to more faithfully mimic human physiology than existing rodent-derived iPSC lines. While initial piPSC lines, first generated in 2009, exhibit the majority of hallmarks displayed by iPSCs derived from other mammalian species, this is not without some caveats. Firstly, all existing piPSC-like cells are afflicted by insufficient activation of endogenous pluripotency genes. Secondly and associated with this, lack of silencing of exogenous pluripotency genes is a general drawback: in contrast, human and murine episomal reprogramming approaches lead to integration of such transgenes. Thirdly, current culturing conditions fail to support the maintenance of either porcine embryonic stem cells (pESC) or piPSC. Lastly, piPSC are unable to reproducibly contribute to chimeric embryos as demonstrated for mouse and rat iPSC. Acquiring insight into these caveats will greatly benefit the generation of bona fide piPSC lines.
Here, we sought to examine these caveats through: 1) comprehensive characterization of the pluripotency state of piPSC-like cells and 2) comparative transcriptome analyses between the original neonatal fibroblasts, piPSC-like cells, and porcine embryos at embryonic day 6/7 and day 10/11. The inner cell mass (ICM) of day 6/7 porcine embryos represents the naïve state of pluripotency, whilst at day 10/11 the blastocysts have hatched and the ICM has given rise to the hypoblast and the epithelial epiblast constituting the embryonic disc. In the epiblast, we expected to detect a primed state of pluripotency.
To generate piPSC, neonatal fibroblasts were transduced with a Tet-ON lentiviral construct expressing porcine Oct4, Sox2, c-Myc, and Klf4. A comparative study was then conducted on these piPSC-like cells using the 2i approach with either FGF or LIF to establish and maintain them in a näive (LIF) or primed (FGF) condition. Subsequently, the potency of piPSC-like cells was comprehensively assessed, including through chimeric embryo contribution assays. Finally, extensive RNAseq analysis was performed to compare iPSC-like cells with the original fibroblasts and cells derived from porcine embryos at days 6/7 and 10/11.
In summary, we were able to show that piPSC-like cells derived and maintained on 2i LIF exhibit a more naïve state than those derived with 2i FGF. Moreover, we identify a unique gene expression profile indicating the missing links in regards to pluripotency factors and growth factor receptors. These will be incorporated in our future efforts to derive and maintain bona fide stable naïve pluripotent piPSC.
Here, we sought to examine these caveats through: 1) comprehensive characterization of the pluripotency state of piPSC-like cells and 2) comparative transcriptome analyses between the original neonatal fibroblasts, piPSC-like cells, and porcine embryos at embryonic day 6/7 and day 10/11. The inner cell mass (ICM) of day 6/7 porcine embryos represents the naïve state of pluripotency, whilst at day 10/11 the blastocysts have hatched and the ICM has given rise to the hypoblast and the epithelial epiblast constituting the embryonic disc. In the epiblast, we expected to detect a primed state of pluripotency.
To generate piPSC, neonatal fibroblasts were transduced with a Tet-ON lentiviral construct expressing porcine Oct4, Sox2, c-Myc, and Klf4. A comparative study was then conducted on these piPSC-like cells using the 2i approach with either FGF or LIF to establish and maintain them in a näive (LIF) or primed (FGF) condition. Subsequently, the potency of piPSC-like cells was comprehensively assessed, including through chimeric embryo contribution assays. Finally, extensive RNAseq analysis was performed to compare iPSC-like cells with the original fibroblasts and cells derived from porcine embryos at days 6/7 and 10/11.
In summary, we were able to show that piPSC-like cells derived and maintained on 2i LIF exhibit a more naïve state than those derived with 2i FGF. Moreover, we identify a unique gene expression profile indicating the missing links in regards to pluripotency factors and growth factor receptors. These will be incorporated in our future efforts to derive and maintain bona fide stable naïve pluripotent piPSC.
| Original language | English |
|---|---|
| Publication date | 1 Dec 2014 |
| Number of pages | 1 |
| Publication status | Published - 1 Dec 2014 |
| Event | International Embryo Transfer Society: 41th Annual Confernce - Versailles, France Duration: 10 Jan 2015 → 13 Jan 2015 Conference number: 41 |
Conference
| Conference | International Embryo Transfer Society: 41th Annual Confernce |
|---|---|
| Number | 41 |
| Country/Territory | France |
| City | Versailles |
| Period | 10/01/2015 → 13/01/2015 |