Prion protein plays a key role in transmissible neurodegenerative diseases in mammals such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (Prusiner 1997). The cellular isoform of prion protein (PrPC) is a glycoprotein that is expressed early in embryogenesis, present at highest levels in the neurons of the brain and spinal cord and at lower levels in various peripheral tissues in the adult (Manson et al. 1992; Harris 1999; Bosque et al. 2002). PrPC is localized on the cell surface and is attached to the cell membrane via a C-terminal glycosyl-phosphatidylinositol (GPI) anchor (Stahl et al. 1987).
While several recent studies highlight the involvement of PrPC in embryogenesis or stem cell self-renewal and proliferation (Steele et al. 2006; Zhang et al. 2006; Malaga-Trillo et al. 2009), the previous work on PrPC function was limited to animal models or animal-derived cell lines. In this current study, we employed human embryonic stem cells (hESCs) for assessing the role of PrPC in human stem cell differentiation and proliferation. hESCs are pluripotent cells that are directly derived from the inner cell mass of blastocyst stage embryos (Thompson et al. 1998). The basic characteristics of hESCs include the ability for self-renewal, for multi-lineage differentiation in vitro and in vivo, cologenicity, a normal karyotype, and extensive proliferation (Brivanlou et al. 2003). The injection of hESCs into immune-deficient mice produced teratomas consisting of many cell types (Thompson et al. 1998). In vitro, hESCs are capable of differentiating into a broad range of cell types including neural cells (Schulz et al. 2003), keratinocytes (Green et al. 2003), cardiomyocytes (Mummery et al. 2002), hepatocytes (Lavon et al. 2004), pancreatic and β-cells (Segev et al. 2004), and blood cells (Chadwick et al. 2003). In recent years, hESCs have received enormous attention mainly because of their ability to repair normal tissues or organs after in vitro differentiation and great promise for cell therapy in regenerative medicine.
Considering that PrPC is likely to be involved in interaction and/or communications between cells during neural differentiation, we were interested in assessing PrPC biological effects when it is provided as an extracellular factor. While PrPC is known to be bound to the plasma membrane via a GPI anchor, several secreted forms of PrPC were identified in cell cultures and biological fluids including blood and cerebrospinal fluid (Borchelt et al. 1993; Harris et al. 1993; Simak et al. 2002; Robertson et al. 2006; Vella et al. 2007; Taylor et al. 2009). Most recent studies revealed that a disintegrin and metalloproteinases (ADAM) are involved in shedding of PrPC from the cell surface via cleaving PrPC at the C-terminus near GPI anchor (Taylor et al. 2009). To mimic secreted form of PrPC, we used human full-length recombinant prion protein (residues 23–231, unglycosylated) converted into its native α-helical conformation (α-rPrP). We found that supplementing α-rPrP to cell culture media delayed spontaneous differentiation of hESCs and helped to maintain their high proliferation activity. Considering that treatment with α-rPrP also down-regulated expression of endogenous PrPC, it is likely that the effect of α-rPrP was indirect, i.e. mediated via suppression of PrPC expression. This study suggests that PrPC is involved in controlling the self-renewal/differentiation status of hESCs.
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- Materials and methods
- Supporting Information
The PrPC sequence has been highly conserved throughout evolution (Rivera-Milla et al. 2006) and its expression has been observed in many cell types starting from early embryogenesis suggesting that PrPC plays an important physiological function (Manson et al. 1992; Harris 1999; Bosque et al. 2002). Several recent studies illustrated the involvement of PrPC in embryogenesis and in regulating the self-renewal activity of stem cells (Steele et al. 2006; Zhang et al. 2006). In Xenopus, PrP transcripts were detected throughout embryonic development starting from the neurulation stage (van Rosmalen et al. 2006). Recently, Malaga-Trillo and coauthors reported that 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). While these results highlight an essential role for PrPC in cell adhesion and communication during zebrafish embryogenesis, it is unclear whether PrPC plays any role in human embryogenesis. Considering that the developmental sequence of human embryoid bodies during spontaneous differentiation of hESCs mimics the process of human embryonic development (Nishikawa et al. 2007), hESCs offers a valuable model for assessing the role of PrP.
In the current study, we assessed the possible effects that PrP might have on hESCs, when it is provided as an extracellular factor. In addition to its expression as a form bound to a membrane via GPI anchor, PrPC is also known to be secreted into various biological fluids (Borchelt et al. 1993; Harris et al. 1993; Simak et al. 2002; Robertson et al. 2006; Vella et al. 2007; Taylor et al. 2009). We found that treatment of hESCs with α-rPrP delays the spontaneous differentiation and helps to maintain high proliferation activity during spontaneous differentiation. Specifically, when cells were exposed to α-rPrP, the expression of endogenous PrPC and neural differentiating markers was delayed, whereas the expression of the pluripotency marker oct-3/4 was prolonged. α-rPrP-treated cells also showed a high activity for AP, an enzyme that is known to be active in undifferentiated cells. Furthermore, cells treated with α-rPrP during the early stages of differentiation displayed a substantially higher proliferation activity than non-treated controls and maintained similar high rate of proliferation as cells cultured under non-differentiating conditions. We do not know whether the effects of α-rPrP on hESCs were mediated through a negative feedback of α-rPrP on PrPC expression level or, alternatively, whether PrPC expression level was suppressed as a result of α-rPrP-induced delay in spontaneous differentiation. In other words, the causative relationship between the down-regulation of PrPC expression and the delay in spontaneous differentiation remains to be clarified.
The current observation that treatment with α-rPrP helps to maintain high proliferation activity while delaying spontaneous differentiation in embryonic stem cells has remarkable parallels with the recent findings on PrPC involvement in adult neurogenesis. Using wild-type, PrP knockout and over-expressing mice, Steele et al. (2006) showed that the PrPC expression level correlates well with the rates of cellular proliferation of neural precursors during adult neurogenesis that occurs constitutively in the olfactory bulb in the subventricular zone. The fact that PrPC-stimulated cell proliferation was observed without an increase in the net neurogenesis is consistent with our observation that treatment with α-rPrP helps to preserve pluripotent status and delay differentiation. Remarkably, in the subventricular zone, PrPC was found to be expressed immediately adjacent to the proliferating region but not in mitotic cells, a finding that highlights that PrPC is involved in trans-regulation of stem cell proliferation activity (Steele et al. 2006). The pattern of PrPC expression in placenta is consistent with possible involvement of PrPC in trans-regulation of the proliferation and differentiation during embryogenesis. In mouse embryos, the earliest expression of Prnp and PrPC were detected beginning from the post-implantation stage (E7.5 to E8.5 p.c.), at which PrPC was observed in the post-mitotic cells that undergo differentiation (Tremblay et al. 2007). In placenta (human), however, PrPC was found throughout pregnancy with the highest expression levels observed at 10–12 weeks during the first trimester of pregnancy (Donadio et al. 2007). PrPC localization in the placental villi suggests involvement of PrPC in uterine–embryo interactions and, possibly, trans-regulation of proliferating and differentiating activities of embryonic cells.
It has been well established that Prnp (prion protein gene) mRNA and PrPC are not only expressed in CNS but also in non-neuronal tissues. In fact, extremely high levels of Prnp mRNA expression were found in Sertoli cells (Johnston et al. 2008). The function of Sertoli cells is to support development and high proliferation activity of all spermatogenic cells including spermatogonial stem cells, a process that involves an intimate physical association between Sertoli and spermatogenic cells. In a manner similarly to adult neurogenesis, a fine balance must be maintained between differentiation and self-renewal of spermatogonia stem cells. It is reasonable to speculate that PrPC expressed in Sertoli cells is involved in trans-regulating the proliferation of spermatogonial stem cells while balancing their self-renewal and differentiating activities.
Considering the striking parallels between stem cells and cancer cells it has been proposed that similar signaling pathways might regulate self-renewal, proliferation, and differentiation of stem cells and cancer cells (Reya et al. 2001). Remarkably, the level of PrPC expression in cancer cells was found to correlate with the tumor aggressiveness such as their metastatic potential for a number of cancer tissues and cell lines including colorectal carcinoma tissues (Antonacopoulou et al. 2008), breast carcinoma cell lines (Diarra-Mehrpour et al. 2004; Meslin et al. 2007), gastric cancer cell lines and tissues (Du et al. 2005; Liang et al. 2006; Pan et al. 2006), and glioma (Erlich et al. 2007). Antibodies against PrPC were found to be effective in suppressing the proliferating activity of tumor cells and inhibiting tumor growth in vitro and in vivo (McEwan et al. 2009). We speculate that activities of PrPC in regulating proliferation and self-renewal could be generic and extend well beyond that of embryonic or adult stem cells.
The self-renewal and differentiation activities of hESCs appear to be regulated by a coordinated interaction between internal and external factors including Oct-3/4, SRY (sex determining region Y)-box 2 (SOX2), NANOG, bFGF, transforming growth factor-β, and wnt (Thompson et al. 1998; Sato et al. 2004; Boyer et al. 2005). Based on the results from the previous and current studies, we speculate that PrPC could be a new factor involved in regulating the balance between self-renewal and differentiation of stem cells. Specifically, PrPC might help to maintain the undifferentiated pluripotent status for a small fraction of embryonic stem cells during human embryogenesis and adult life. While the role of PrPC in regulating proliferation and self-renewal does not appear to be essential for development under normal conditions, it might become very important for regeneration after exposure to lethal stress conditions. PrPC knockout mice develop and reproduce normally and show similar behavior to that of wild-type mice except for relatively minor abnormalities in circadian activity and sleep rhythms (Büeler et al. 1992; Tobler et al. 1996). Exposure to irradiation, however, was shown to be lethal for PrP-deficient mice, whereas reconstitution of PrPC expression in hematopoietic cells improved the rate of animal survival as PrPC was shown to be important for self-renewal of long-term hematopoietic stem cells (Zhang et al. 2006).
In the current study, the expression of PrPC was not detectable in undifferentiated hESCs but increased during spontaneous differentiation of hESCs. These results were consistent with previous observations where the levels of PrPC expression were found to increase during neuronal differentiation from NTERA cells (Novitskaya et al. 2007) or from multipotent neural precursors (Steele et al. 2006). While PrPC expression level correlates with neuronal differentiation on one hand, PrPC was also shown to be a surface marker of long-term hematopoietic stem cells and important for self-renewal activity of stem cells on the other hand (Zhang et al. 2006). How can we reconcile this apparent paradox that PrP accompanies neural differentiation but is also important for self-renewal activity and even capable of delaying the differentiation as was shown in the current study? We propose that the biological effects of PrP depend on whether it acts as a cis or trans factor and whether trans-interactions are homophilic by nature (interactions between two or more PrP molecules on adjacent cells) or heterophilic (interactions of PrP with a non-PrP molecule on adjacent cells). For instance, expression of PrP on the surface of neural cells that undergo differentiation during human embryogenesis might help to maintain a pluripotent status for a small fraction of undifferentiated hESCs cells via heterophilic trans-interactions. Considering that PrPC might be involved in multiple modes of interactions with several partner molecules including homophilic interactions, it is likely that PrPC performs a diverse range of biological functions that might be different in embryonic and adult cells. To further examine the role of PrPC in human embryogenesis, gain- and loss-of-functional study using hESCs during maintenance and differentiation might be required.
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- Materials and methods
- Supporting Information
Appendix S1. Materials and methods.
Figure S1. Treatment of hESCs with &bgr;-oligomers or amyloid fibrils.
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