# Pluripot Stem Cells for Transgenesis in the Rabbit: A Utopia?
## 2 Rabbit Pluripotent Stem Cells (rbPSCs)
### Pluripotate State of rbPSCs  
Both rbESC and rbiPSC lines express the cardinal markers of PSCs [47]. They are positive for alkaline phosphatase activity [44]. They express the pluripotency-associated OCT4 and NANOG transcription factors, as well as the SSEA-1, SSEA-4, TRA1-60, and E-cadherin cell surface markers [52; 61]. They also display a normal karyotype (42 XX or 42 XY) [44; 47], produce embryoid bodies in vitro [45; 63], and form teratoma containing tissues of ectodermal, mesodermal and endodermal origin upon injection under the kidney capsule in severe combined immunodeficient (SCID) mice [61; 47]. However, rbPSCs can only be stabilized in the primed state of pluripotency. The suppression of FGF2 and/or culturing without feeder cells lead to their differentiation. Thus, these cells can be cultured only on a synthetic matrix, with either medium conditioned on mitomycin-treated MEF or addition of activin A [65; 51]. In the same way, rbPSCs differentiate in the presence of inhibitors of SMAD2-3, TGF\(\beta\)-receptor, or FGF-receptor [46; 50].  
Transcriptomic comparisons of rbPSCs with rabbit epiblast cells from E4- and E6-stage embryos show overexpression of 17 genes involved in the WNT/\(\beta\)-catenin signaling pathway, including _TCF4_, _LEF1_, and _WNT5A_[60]. This result is consistent with the decrease in the rate of rbESC multiplication after inhibition of the WNT pathway by a Frizzled-1 antagonist or an anti-Wnt3a antibody [50]. This effect is associated with an increase in phosphorylation of \(\beta\)-catenin and SMAD1-5-8, as well as a reduction in expression of SSEA-4 pluripotency marker, indicating cell commitment to differentiation. The WNT pathway is involved in maintaining the balance between self-renewal and differentiation of both mouse and human ESCs [66; 67; 68]. It is therefore also important in sustaining the pluripotency of rbPSCs, although the 2 iLIF medium allowing the self-renewal of rodent PSCs and containing the GSK3\(\beta\) inhibitor CHIR99021 did not allow the stabilization of rbESCs.  
RbPSCs form flat colonies characteristic of their primed pluripotent state (Figure 1). They express 173 genes involved in cytoskeletal organization and function compared to rabbit epiblast cells at the E4 and E6 stages [60]. Among them are gene-encoding filamins (FLNA and FNLC) and vinculin(VCL), which function to stabilize the actin cytoskeleton and its anchoring to the plasma membrane. These molecules promote cell-to-matrix interactions rather than cell-to-cell adhesions, and therefore may interfere with colony morphology. These proteins are also more expressed in EpiSCs than in mESCs [69; 70].  
Interestingly, a comparison of rbESC and rbiPSC lines produced from the same breed of New Zealand white rabbit showed different features that bring rbiPSC lines closer to the naive state of pluripotency than rbESC lines [47]. These characteristics mainly concern their proliferation rate, their resistance to unicellular dissociation, their global transcriptome, and their expression of markers specific to the primed and naive pluripotency states [71]. The variation of these characteristics allows the classification of rbPSC lines on a graduated scale of primed pluripotency with rbESCs at one end (most primed state) and rbiPSCs at the other end (closer to the naive state) (Figure 1).