applied sciences Review Pluripotent Stem Cells for Transgenesis in the Rabbit: A Utopia? Worawalan Samruan, Nathalie Beaujean and Marielle Afanassieff * Stem Cell and Brain Research Institute U 1208, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, INRAE, USC 1361, F-69500 Bron, France; [email protected] (W.S.); [email protected] (N.B.) * Correspondence: marielle.afanassieff@inserm.fr; Tel.: +33-472-913-458 Received: 18 November 2020; Accepted: 5 December 2020; Published: 11 December 2020 Featured Application: Rabbit pluripotent stem cells represent valuable tools for creating human disease models in vivo via the production of transgenic animals and in vitro by generating organoids. They are also of interest for the creation of bioreactors, i.e., transgenic rabbits producing pharmacological molecules in their milk. Abstract: Pluripotent stem cells (PSCs) possess the following two main properties: self-renewal and pluripotency. Self-renewal is defined as the ability to proliferate in an undifferentiated state and pluripotency as the capacity to differentiate into cells of the three germ layers, i.e., ectoderm, mesoderm, and endoderm. PSCs are derived from early embryos as embryonic stem cells (ESCs) or are produced by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In mice, PSCs can be stabilized into two states of pluripotency, namely naive and primed. Naive and primed PSCs notably differ by their ability to colonize a host blastocyst to produce germline-competent chimeras; hence, naive PSCs are valuable for transgenesis, whereas primed PSCs are not. Thanks to its physiological and developmental peculiarities similar to those of primates, the rabbit is an interesting animal model for studying human diseases and early embryonic development. Both ESCs and iPSCs have been described in rabbits. They self-renew in the primed state of pluripotency and, therefore, cannot be used for transgenesis. This review presents the available data on the pluripotent state and the chimeric ability of these rabbit PSCs. It also examines the potential barriers that compromise their intended use as producers of germline-competent chimeras and proposes possible alternatives to exploit them for transgenesis. Keywords: embryonic stem cell; induced pluripotent stem cell; chimera; transgenesis; rabbit 1. Introduction Pluripotent stem cells (PSCs) were first derived from the inner cell mass (ICM) of mouse embryos in 1981 [1,2]. These cells, named embryonic stem cells (ESCs), were shown to be able to colonize the epiblast of host blastocysts and, consequently, to produce chimeric mice in 1984 [3]. This ability of mouse ESCs (mESCs) has made it possible to develop transgenesis techniques in this species, facilitating substantial progress in functional genetics. Later on, PSCs were derived from the late epiblast of mouse post-implantation embryos [4,5]. These cells, named epiblast stem cells (EpiSCs), display several different features from mESCs, especially with respect to their transcriptome and epigenome, and are notably not able to colonize host blastocysts. At the same time, mouse PSCs were obtained by reprogramming differentiated somatic cells by overexpression of four pluripotency factors, namely Oct4, Sox2, Klf4, and cMyc [6]. These cells, named induced pluripotent stem cells (iPSCs), can exhibit features of mESCs or EpiSCs depending on the medium used during their reprogramming [7,8]. Therefore, mESCs and EpiSCs epitomize two states of pluripotency existing Appl. Sci. 2020, 10, 8861; doi:10.3390/app10248861 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 8861 2 of 18 in vivo during early embryonic development—the naive state, corresponding to early epiblast cells from preimplantation embryos, and the primed state, corresponding to late epiblast cells from post-implantation embryos, respectively [9]. In vitro naive PSCs are sustained by the leukemia inhibitory factor (LIF)/gp130/STAT3 and bone morphogenetic protein 4 (BMP4)/ALK/ SMAD1-5-8 signaling pathways, whereas primed PSCs are supported by the fibroblast growth factor 2 (FGF2) and activin A/transforming growth factor beta (TGFβ)/SMAD2-3 signaling pathways [10]. To date, all PSC lines, ESCs and iPSCs, obtained in non-rodent mammalian species display features of primed pluripotency [11,12]. Therefore, it seems that non-rodent PSCs are only able to stabilize in culture in the primed state of pluripotency [13]. For example, primate PSCs present a transcriptome very different from that of human early blastocyst epiblast cells [14], but is rather closer to that of post-implantation late blastocyst epiblast cells according to an analysis in monkeys [15]. The actual research challenge in the PSC field is to reprogram primed cells toward the naive state of pluripotency, in order to obtain and culture more genetically stable cells, which are easier to handle by single-cell dissociation [16,17] and are more useful for cellular therapies or the production of disease models [18,19]. The possibility to revert primed PSCs into naive PSCs was first demonstrated in mice by overexpressing pluripotency genes such as Klf4, Nanog, Stat3, Tfcp2l1, or Prmd14, either alone or in combination [20–24], and by strengthening the signaling pathways sustaining the naive state using inhibitors of mitogen-activated extracellular regulated kinase (MEK) and glycogen synthase kinase 3 beta (GSK3β) in a medium called 2iLIF [25,26]. Such strategies for reprogramming primed PSCs have been extensively studied for human PSCs and have produced naive-like cells with a heterogenous reconfiguration of their transcriptome and epigenome [27–30] as well as a variable capacity to produce interspecies chimeras after microinjection into mouse blastocysts [31,32]. These variations in the molecular and functional characteristics of primate PSCs show that embryonic cells could be stabilized in vitro at different stages along a continuum of pluripotency, the ends of which are epitomized by the naive and primed states, respectively [33]. Lagomorphs and primates share many similarities in their embryonic development [34], in particular in the timing of the embryonic genome activation at the 8/16-cell stages [35], the timing of the waves of DNA demethylation and methylation [36,37], and the timing of the random inactivation of the second X chromosome [38]. Like their human counterparts, rabbit embryos develop as a flat disc on the surface of the conceptus [39], and present the advantage of implanting very late (at E6.75) due to a mechanism similar to that of human embryos [40]. Above all, the gastrulation of rabbit embryos begins before implantation (at E6.0) so that the epiblast remains more easily accessible for experimentation than in rodents [41]. These similarities and particularities make rabbits an interesting model not only for the study of the biology of PSCs, but also to be used to create transgenic animal models of human development and diseases and to improve interspecies chimerism tests. In this review, we provide a state-of-the-art discussion of the pluripotent state and chimeric ability of rabbit PSCs. We discuss the potential barriers to their use in the formation of chimeras and propose possible alternatives to exploit them for transgenesis in rabbit. 2. Rabbit Pluripotent Stem Cells (rbPSCs) 2.1. Rabbit Embryonic Stem Cells (rbESCs) Rabbit embryonic cell cultures were described by American teams in 1993 [42,43], but the first lines of rbESCs were not published until 15 years later by two teams from China and Japan [44,45]. RbESCs are derived from ICM cells of early blastocysts (E3.5–E4.0). They are cultured on feeder cells and form flat colonies (Figure1). The self-renewal of rbESCs depends on the activin A /TGFβ/SMAD2-3 and FGF2 pathways [46,47]. FGF2 appears indispensable by inducing the PI3K/AKT and MAPK pathways [48,49], while the WNT/β-catenin pathway may also be indirectly activated by FGF2 [50]. It is possible to derive cells without any growth factor in the medium if they are cultured on feeder cells, showing that LIF is not necessary for the maintenance of rbESCs [51]. However, the addition of Appl. Sci. 2020, 10, 8861 3 of 18 LIF to the culture medium of rbESCs has often been used [48,49,52], and several studies have described the effAppl.ect Sci. of 2020 LIF, 10 on, x FOR the PEER derivation REVIEW of rbESCs and the induction of LIF-receptor expression3 of [ 5218 ,53]. Unlike the derivation of rodent ESCs stabilized in the naive state [54], MEK and GSK3β inhibitors do not[52,53]. enhance Unlike epiblast the derivation cell differentiation of rodent ESCsin vivo stabilized[55] or rbESCin the naive derivation state [54],in vitro MEK[51 and]. InGSK3 addition,β the maintenanceinhibitors do ofnot dome-shaped enhance epiblast naive-like cell differentiation rbESC colonies in vivo [55] in theor rbESC presence derivation of these in vitro two [51]. inhibitors In requiresaddition, both FGF2the maintenance and feeder of cells dome-shaped [56]. naive-like rbESC colonies in the presence of these two inhibitors requires both FGF2 and feeder cells [56]. Scale of primed pluripotency Blastocyst E3.5 Passage ICM by clumps CKF CKL Derivation AKF AKSL AKSF AKSgff rbESC lines Enzymatic single cell dissociation hOct4 hSox2 hKlf4 hc-Myc rbiPSC-B19 KOSR +FGF2 Lentiviral transduction Reprogramming Adult fibroblasts Reversion rbiPSC lines Piggybac transfection + medium change rbEKA FBS + LIF Toward the naive state FigureFigure 1. Schematic 1. Schematic representation representation of of the the rabbit rabbit pluripotentpluripotent