Transcriptome diversity at multi- and single-cell levels in rabbit pluripotent stem cells

Pluripotent stem cells (PSCs) can exist in at least two morphologically, molecularly and functionally distinct pluripotent states, designated as the naive and primed states; PSC exist in both states in rodents, whereas only the primed stated has been described in primates so far. Not much is known about the pluripotent state of PSCs in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts, and 4 lines of embryonic stem cells (rbESCs). All cell lines required FGF2 for their growth and proliferation. We purified the SSEA subpopulations by FACS sorting in each line, and performed a global analysis of their expression profiles. For this, we used a rabbit-specific gene expression microarray containing approximately 13,000 independent genes. Hierarchical clustering showed that all subpopulations of rbESC lines were clustered on one side and all subpopulations of rbiPSC lines were clustered together on the other. This indicates that the variability is mainly associated with the cell status (rbESC vs rbiPSC) and not with the SSEA expression status. We next performed a qPCR analysis of Blimp1, Cdx2, Cdh1 (E-cadherin), Cdh2 (N-cadherin), Cldn6, Dax1 (NrOb1), Dazl, Essrb, Fbxo15, Fgf4, Gbx2, Klf4, Lefty2, Nanog, Oct4 (Pou5f1), Otx2, Pecam1, Pitx2, Piwil2, Rex1 (Zfp42), Tbx3, and Tcfcp2l1, which have been used to evaluate stemness and demarcate the naive and primed pluripotent states in rodents. In agreement with the microarray data, correlation clustering of the Delta Ct values showed that the rbiPSC subpopulations exhibited fewer differences between them than with the rbESC subpopulations. Moreover, when compared to the rabbit inner cell mass (ICM), the rbiPSCs were the closest; all rbESC subpopulations, irrespective of their SSEA1 status, were more distantly related to the ICM than the SSEA1+ rbiPSCs. Of note, both rbiPSCs and ICM cells expressed the naive markers Cdh1, Essrb, Cldn6, Klf4, Dazl, Piwil2, and Pecam1. However, rbiPSCs did not express all the molecular markers of naive pluripotency, including Rex1, Tbx3, Gbx2, Fgf4, and Dax1. Based on these observations, we propose that rabbit iPSCs self-renew in an intermediate state between naive and primed pluripotency. To gain further insight into cell diversity within the rabbit PSC populations, we started examining the expression of pluripotency markers by single-cell qRT-PCR. Preliminary results showed that rabbit ESC populations contained some rare cells, which co-expressed the naive markers Essrb and Tbx3. Our ongoing experiments aim to identify these cells both in iPS cell populations and in the preimplantation embryo. This work should help decipher the molecular signature of naive pluripotency in rabbits and, ultimately, will lead to generate PSC lines suitable for blastocyst colonization and chimaera production.