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Prion protein (PrPC), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrPC in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline-regulated lentiviral vectors that up-regulate or suppresses PrPC expression. Here, we show that expression of PrPC in pluripotent hESCs cultured under self-renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrPC in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over-expression of PrPC in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrPC is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self-renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrPC is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self-renewal state, control cell proliferation activity, and define stem cell fate.
Misfolding and aggregation of a prion protein (PrP) underlies a key pathological event leading to several devastating transmissible neurodegenerative diseases in mammals including Creutzfeldt–Jakob disease and bovine spongiform encephalopathy (Prusiner 1997). The normal, cellular isoform of the prion protein, PrPC, is a glycoprotein that is expressed on the cell surface and is attached to the cell membrane via a C-terminal glycosylphosphatidyl-inositol anchor (Stahl et al. 1987). PrPC is expressed at high levels in cells of the central nervous system and at lower levels in various peripheral tissues (Manson et al. 1992).
A diverse range of activities have been proposed as candidates for the biological function of PrPC. In previous studies, PrPC was postulated to be involved in signal transduction (Mouillet-Richard et al. 2000), neuroprotection (Bounhar et al. 2001; Chiarini et al. 2002; Lopes et al. 2005; Roucou et al. 2005; Lima et al. 2007), neurotrophic activities (Chen et al. 2003; Santuccione et al. 2005; Lima et al. 2007), cell adhesion (Schmitt-Ulms et al. 2001; Santuccione et al. 2005; Viegas et al. 2006; Malaga-Trillo et al. 2009), cell proliferation and differentiation (Mouillet-Richard et al. 1999; Steele et al. 2006; Zhang et al. 2006; Lima et al. 2007; Lee and Baskakov 2010; Panigaj et al. 2011; Santos et al. 2011), or regulation of the cell cycle (Liang et al. 2007). Consistent with the hypothesis that PrPC is involved in differentiation of neural precursor cells, PrPC was found to localize to the surface of growing axons during development and along fiber bundles that contain elongating axons in the adult brain (Sales et al. 2002; Chen et al. 2003). Axonal transport of PrPC was found to increase significantly during post-traumatic axon regeneration (Moya et al. 2005). PrPC was also shown to induce polarization, synapse development, and neuritogenesis in embryonic neuron cultures (Kanaani et al. 2005; Lopes et al. 2005). Although the role of PrPC in neuronal differentiation has been well recognized, it remains unclear whether PrPC is involved in early embryogenesis.
To examine the role of PrPC in early embryogenesis, this study employed human embryonic stem cells (hESCs). hESCs are pluripotent cells with high self-renewal and proliferation activities that can be differentiated into any cell type of the three germ layers and subsequently any tissue (Thompson et al. 1998). In the past decade, hESCs have become an active venue of research because of their impressive potential as a tool for cell therapy in regenerative medicine. Moreover, because the developmental sequence of human embryoid bodies during differentiation of hESCs mimics the process of human embryogenesis (Nishikawa et al. 2007), hESCs offer an alternative to fetal tissues for examining molecular mechanisms involved in early human embryogenesis.
In previous work, we showed that treatment of hESCs with recombinant PrP folded into an α-helical conformation delayed spontaneous differentiation and helped to maintain the high proliferation activity of hESCs (Lee and Baskakov 2010). To examine the role of PrPC in human embryogenesis in detail, a panel of lentiviral vectors that up-regulates or suppresses PrPC expression in hESCs was generated. This work illustrates that PrPC is involved in key cellular activities that determine the status of hESCs: (i) it regulates the dynamics of the cell cycle, (ii) controls the cellular switch between self-renewal and differentiation, and (iii) contributes to determining the fate of cell differentiation.
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- Materials and methods
- Supporting Information
An increasing number of studies suggests that PrPC is involved in regulating stem cell self-renewal and proliferation (Lee and Baskakov 2010; Miranda et al. 2011; Peralta et al. 2011; Santos et al. 2011; Lopes and Santos 2012). To examine the role of PrPC in early embryogenesis, we employed a panel of lentiviral vectors that up-regulates or suppresses PrPC expression in hESCs. Human embryonic stem cells are pluripotent cells characterized by high proliferation rates, a self-renewal capacity and the ability to differentiate into a cell of any of the three germ layers. Because the developmental sequence of human embryonic bodies during spontaneous differentiation of hESCs mimics the process of human embryonic development (Nishikawa et al. 2007), hESCs are considered to be a valuable in vitro model of early embryogenesis.
In this study, we showed that (i) ectopic expression of PrPC in pluripotent hESCs cultured under self-renewal conditions induced cell differentiation; (ii) silencing of PrPC in hESCs undergoing spontaneous differentiation changed the balance between lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed; (iii) silencing of PrPC in differentiating hESCs altered the dynamics of the cell cycle and suppressed the G1 to S phase transition; (iv) over-expression of PrPC in hESCs under spontaneous differentiation conditions suppressed differentiation toward all three germ layers and helped to preserve high proliferation activity. These results provide strong evidence that PrPC is involved in key activities that dictate the status of hESCs: (i) it regulates cell cycle progression, (ii) controls the cellular switch between self-maintenance and differentiation, and (iii) determines the fate of cell differentiation.
Previous studies established that PrPC was undetectable in pluripotent hESCs or at the initial stage of spontaneous differentiation. However, PrPC expression increased gradually together with other markers of neuronal lineages during spontaneous hESC differentiation (Lee and Baskakov 2010). Studies using mouse and human ESCs showed that PrPC modulates neural commitment during early ESC differentiation (Lee and Baskakov 2010; Peralta et al. 2011). Consistent with the previous results, here we observed that the expression of PrPC was not detectable in pluripotent hESCs. Surprisingly, induction of ectopic PrPC expression was sufficient to trigger differentiation of hESCs cultured under self-renewal conditions, as was evident by changes in cell morphology, dynamics of the cell cycle, a decrease in pluripotency marker Oct-3/4 expression and appearance of ectodermal, endodermal, and mesodermal markers. Several alternative mechanisms have to be considered for explaining these effects. First, PrPC could be directly involved in regulating the cell cycle G1 to S phase transition. The ESC cell cycle is significantly shorter than that of somatic cells, which is largely because of an abbreviated G1 phase (Becker et al. 2006). Inhibition of the G1 to S transition is expected to suppress hESC self-renewal activity and stimulate their differentiation. Second, PrPC could be directly involved in regulating the self-renewal activity and differentiation status, whereas changes in cell cycle dynamics occur as a result of changes in self-renewal status. It might be difficult to distinguish between the possibilities above because of the close connections between the cell cycle, self-renewal, and differentiation and because the same signaling pathways appear to be involved in the maintenance of the self-renewal activity and controlling the cell cycle (Burdon et al. 2002; Liang and Slingerland 2003; Ruiz et al. 2011). Nevertheless, recent studies reported that PrPC modulates mRNA expression level of Nanog, a transcription factor involved in self-renewal (Miranda et al. 2011). Moreover, PrPC was also found to stimulate self-renewal and proliferation of neurosphere-derived stem cells via interaction with stress-inducible protein I (Santos et al. 2011).
Previous studies indicated that PrPC was mostly involved at a relatively late stage of neuronal development (Mouillet-Richard et al. 1999) and was important for adult morphogenesis (Steele et al. 2006). In the mouse nervous system, Prnp gene expression was found to begin in post-mitotic neural cells that have undergone neuronal differentiation (Tremblay et al. 2007). Furthermore, in mouse, PrPC levels were shown to correlate with differentiation of multipotent neural precursor cells during developmental and adult neurogenesis (Steele et al. 2006) and to be involved in self-renewal activity of hematopoietic stem cells (Zhang et al. 2006). Furthermore, PrPC was found to promote regeneration of adult skeletal muscle tissue (Stella et al. 2010). In cattle, PrPC is differentially expressed in the neuroepithelium, the “stem cells” of the nervous system that differentiate into neurons, astrocytes, and other glial cells (Peralta et al. 2011). Notably, PrPC was found predominantly at the intermediate and marginal layers where more differentiated neuroepithelial cells were located (Peralta et al. 2011). In fetal human forebrain, PrPC immunoreactivity was observed in axonal tracts and fascicles from the 11th week to the end of gestation (Adle-Biassette et al. 2006). Increasing levels of PrPC expression was found throughout synaptogenesis (Adle-Biassette et al. 2006). Silencing of PrPC upon induction of differentiation of a neuroectodermal cell line impaired neuritogenesis (Loubet et al. 2012). PrPC was shown to be involved in neuritogenesis via interaction with Stress-Inducible Protein 1 (Lopes et al. 2005). Moreover, PrPC supported axonal growth (Hajj et al. 2007) and was important for astrocyte development (Arantes et al. 2009). On the other hand, several studies suggested that PrPC might be involved in regulating early embryogenesis and, possibly, the very early stages of ESC differentiation (Malaga-Trillo et al. 2009; Khalifé et al. 2011; Syed et al. 2011). Knocking down one of the two PrP genes in zebrafish embryos caused a disruption in morphogenetic cell movement, loss of embryonic cell adhesion, and ultimately developmental arrest (Malaga-Trillo et al. 2009; Syed et al. 2011). PrPC and its paralog Shadoo were required for early mouse embryogenesis, as lethality was observed at E10.5 in Sprn-knockdown, Prnp-knockout embryos (Young et al. 2009).
This study strongly supports the hypothesis that PrPC is actively involved in determining ESC fate. Indeed, perhaps the most surprising finding was that in PrPC-silenced hESCs, early neuronal (ectodermal) differentiation was suppressed, whereas differentiation toward endodermal and mesodermal lineages was not affected. Unexpectedly, over-expression of PrPC suppressed hESC differentiation toward lineages of all three germ layers and helped to maintain high proliferation activity, one of the characteristics of non-differentiated stem cells. This observation has remarkable parallels with our previous observation that treatment of hESC with recombinant PrP that mimicked the PrPC α-helical conformation delayed spontaneous differentiation and helped to maintain hESC high proliferation activity (Lee and Baskakov 2010). Taken together, the current results suggest that PrPC might balance cell differentiation between lineages of the three germ layers and contribute to switching between self-maintenance and differentiation.
The hypothesis that PrPC is involved in the control of stem cell fate appears to contradict previous studies where PrPC was not required for neuronal development/differentiation in mice as judged from PrPC knockout experiments (Bueler et al. 1993; Tobler et al. 1996). These discrepancies could be attributed to differences in the signaling involved in early embryogenesis of mouse and human, and/or to the possibility that a loss of PrPC function in mice is compensated by other proteins such as Shadoo or Dopple (Young et al. 2009). Interestingly, transgenic mice that express PrPC with a deletion in the conservative region 105–125 were found to develop neonatal lethality (Li et al. 2007).
A cell cycle consists of four phases: G1 (Gap 1), S (Synthesis), G2 (Gap 2), and M (Mitosis) phase. Fast proliferation of ESCs is achieved via an unusual structure in the cell cycle, which is characterized by a much shorter G1 phase in comparison to that of other cell types (Becker et al. 2006; Fluckiger et al. 2006). Cyclins and cyclin-dependent kinases are required for the cell cycle G1 to S phase transition (Neganova et al. 2009). It has been proposed that the length of the G1 phase controls an important switch between self-renewal and differentiation (Ruiz et al. 2011). In this study, we showed that in pluripotent hESCs, ectopic PrPC expression inhibited the cell cycle G1 to S phase transition. In contrast, in hESCs that were undergoing spontaneous differentiation, it was down-regulation of PrPC expression that inhibited the G1 to S transition. Such apparently contrasting effects of PrPC on the G1 to S phase transition could be attributed in part to the fact that ectopic expression of PrPC in pluripotent hESCs stimulated their differentiation, i.e., changed their status, and that cell cycle dynamics is known to change dramatically as a function of hESC status (White and Dalton 2005). These results can also indicate that the precise role of PrPC in the G1 to S phase transition depends on the status of hESCs and that PrPC might reverse its role at different stages of hESC differentiation. Nevertheless, to our knowledge, this is the first report illustrating that PrPC is involved in the cell cycle G1 to S transition in hESCs. Interestingly, PrPC over-expression in differentiating hSECs had no notable impact on cell cycle structure, but was associated with high proliferation rates. In fact, a nice correlation between PrPC expression level and proliferation activity was observed in hESCs undergoing differentiation.
In previous studies, PrPC was linked to cell cycle transitions in cancer cell models. Ectopic expression of PrPC in human gastric cancer cells was shown to promote the cell cycle G1 to S transition and stimulate their proliferation and metastatic activity (Liang et al. 2007). In cancer cells, PrPC effects were mediated via the PI3K/Akt pathway with subsequent transcriptional activation of cyclin D1, which is known to regulate the G1 to S phase transition (Liang et al. 2007). Furthermore, the expression level of PrPC in cancer cell lines and tissues was found to correlate with their proliferation activity and tumor aggressiveness (Liang et al. 2006; Erlich et al. 2007; Meslin et al. 2007; Antonacopoulou et al. 2008). Antibodies against PrPC were found to be effective in suppressing proliferating activity and inhibiting tumor growth (McEwan et al. 2009). Overall, the current findings of the effects of PrPC on hESC proliferation and cell cycle dynamics have parallels with those previously described for cancer cells. Together, the current and previous results illustrate that PrPC function in regulating the cell cycle and proliferation appears to be preserved across cells of various types.
While the range of PrPC biological activities appears to be very diverse, this study strongly supports the hypothesis that PrPC is at the cross-roads of several signaling pathways that regulate stem cell switches to preserve or depart from the self-renewal state, control cell proliferation activity, and contribute to defining stem cell fate. Because these activities are ultimately linked and, possibly, controlled by cell cycle dynamics, it is not surprising that PrPC was also found involved in the G1 to S phase transition. It is reasonable to speculate that PrPC couples extracellular signals, including those generated by cell–cell contacts or the extracellular matrix, to the cell cycle. Depending on the physiological environment, PrPC acts to provide negative or positive cues for cell self-renewal, proliferation, or differentiation.