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Keywords:

  • embryonic stem cells;
  • neural differentiation;
  • prion protein;
  • proliferation;
  • self-renewal

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

J. Neurochem. (2010) 114, 362–373.

Abstract

The normal cellular form of prion protein (PrPC) has been shown to exhibit a diverse range of biological activities. Several recent studies highlighted potential involvement of PrPC in embryogenesis or in regulating stem cell self-renewal and proliferation. In the current study, we employed human embryonic stem cells (hESCs) for assessing the potential role of prion protein in early human development. Here, we showed that treatment of hESCs with full-length recombinant PrP folded into an α-helical conformation similar to that of PrPC delayed the spontaneous differentiation of hESCs and helped to maintain their high proliferation activity during spontaneous differentiation. Considering that administration of α-rPrP was also found to down-regulate the expression of endogenous PrPC, the effects of α-rPrP were likely to be indirect, i.e. executed by endogenous PrPC. Together with previous observations, these work support the hypothesis that PrPC is involved in regulating self-renewal/differentiation status of stem cells including hESCs.

Abbreviations used:
AP

alkaline phosphatase

bFGF

basic fibroblast growth factor

GAP43

growth associated protein 43

GPI

glycosyl-phosphatidylinositol

hESCs

human embryonic stem cells

PrPC

normal cellular isoform of the prion protein

α-rPrP

recombinant prion protein folded into an α-helical conformation

TH

tyrosine hydroxylase

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).

In the last decade, a diverse range of activities has been proposed as the possible biological function of PrPC. Some of these functions related to the Cu2+-binding properties of PrPC such as supplying cellular enzymes with Cu2+, acting as a Cu2+-sensor, acting as a superoxide dismutase and scavenging reactive oxygen species, or acting as a transmembrane Cu2+-transporter that operates through endocytosis (Pauly and Harris 1998; Brown et al. 1999; Vassallo and Herms 2003; Millhauser 2004). Other proposed roles of PrPC were not directly related to its Cu2+-binding ability but to its involvement in several signal transduction cascades that presumably act through a tyrosine kinase Fyn, a cAMP/Protein kinase A (PKA) pathway, or by binding to neural cell adhesion molecule (NCAM) (Mouillet-Richard et al. 2000; Chiarini et al. 2002; Santuccione et al. 2005). A number of studies indicated that PrPC has neuroprotective functions via regulating apoptosis (Bounhar et al. 2001; Roucou and LeBlanc 2005; Roucou et al. 2005). Other studies suggested that PrPC might be involved in cell adhesion (Schmitt-Ulms et al. 2001; Santuccione et al. 2005; Viegas et al. 2006; Malaga-Trillo et al. 2009) presumably by interactions with neural cell adhesion molecule (NCAM) (Schmitt-Ulms et al. 2001; Santuccione et al. 2005) and glycosaminoglycans (Gonzalez-Iglesias et al. 2002).

While some of the proposed functions are not mutually exclusive, the physiological role of PrPC remains poorly defined. A growing number of studies indicate that PrPC plays an important role in regulating self-renewal of adult stem cells (Zhang et al. 2006) and/or proliferation and differentiation of neural precursor cells (Steele et al. 2006). Consistent with these functions, PrPC was shown to localize on 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 is significantly increased during post-traumatic axon regeneration (Sales et al. 2002; Moya et al. 2005). PrPC was also shown to induce polarization in synapse development and neuritogenesis in embryonic hippocampal neuron cultures (Kanaani et al. 2005; Lopes et al. 2005). Neuritogenesis appears to be mediated via interaction with the extracellular matrix proteins, laminin and vitronectin (Graner et al. 2000a,b; Coitinho et al. 2006; Hajj et al. 2007), and stress-inducible protein 1 (Graner et al. 2000a; Zanata et al. 2002; Lopes et al. 2005).

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

See Appendix S1 for details.

Results

Depending on in vitro culture conditions, hESCs could be maintained in either an undifferentiated state with high self-renewal potential or differentiated into wide variety of somatic cell types. When cultured on mitotic-inactivated mouse embryonic fibroblast in the presence of basic fibroblast growth factor (bFGF), colonies of hESCs showed positive staining for oct-3/4, the pluripotency marker which is known to be expressed in undifferentiated hESCs (these conditions will be referred to as self-renewal conditions) (Fig. 1a). Under conditions for spontaneous differentiation (without mouse embryonic fibroblast and bFGF in culture medium; these conditions will be referred to as differentiation conditions), hESCs were found to form embryoid bodies by day 5 of differentiation (Fig. 1b). On the day 14 of differentiation, immunostaining revealed the expression of markers specific for three germ-layers: an endoderm marker, alpha-fetoprotein (AFP) (Fig. 1c); a mesoderm marker, brachyury (Fig. 1d); and the following ectoderm and neural markers: nestin, glial fibrillary acidic protein (GFAP), tau-1, microtubule-associated protein 2 (MAP2), tyrosine hydroxylase (TH), and growth associated protein 43 (GAP43) (Fig. 1e–g). The immunostaining confirmed that the two culturing conditions employed in the current studies either maintain hESCs in the undifferentiated state or induce hESCs to differentiate into several cellular lineages.

image

Figure 1.  Spontaneous differentiation of hESCs. (a) hESCs maintain undifferentiated state when cultured in the presence of basic fibroblast growth factor: staining for oct-3/4 (red) and Hoechst 33342 (blue). The boundary between hESCs colonies and feeder cells is indicated by yellow line in phase contrast image. (b) When cultured in the absence of basic fibroblast growth factor, hESCs formed embryoid bodies by day 5 of culture and showed expression of markers specific for multiple cell lineages (c–g). Double immunostaining for the following markers on day 14 of spontaneous differentiation: (c) alpha-fetoprotein (AFP) (red), (d) brachyury (red), (e) nestin (red) and glial fibrillary acidic protein (GFAP) (green), tau-1 (red), (f) microtubule-associated protein 2 (MAP2) (green), (g) tyrosine hydroxylase (TH) (red) and growth associated protein 43 (GAP43) (green). Figure parts (c and d) shows Hoechst 33342 staining of nuclei (blue). Scale bars = 50 μm.

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PrPC expression is increased during spontaneous differentiation of hESCs

As judged from immunocytochemistry and immunoblotting, PrPC was not detectable at the initial stages of spontaneous differentiation, at which a pluripotency marker oct-3/4 was clearly visible (Fig. 2a and b). In the course of spontaneous differentiation, however, the oct-3/4 expression decreased to undetectable level, whereas the expression of PrPC was found to be gradually increased (Fig. 2b). These data were consistent with previous observations that oct-3/4 is expressed at a high level in undifferentiated hESCs but down-regulated upon differentiation (Rao and Stice 2004), whereas expression of PrPC was shown to be increased with neuronal differentiation (Novitskaya et al. 2007). The glycosylation pattern of PrPC was similar to that observed in normal hamster brain, where diglycosylated PrPC is known to be the predominant species (Nishina et al. 2006).

image

Figure 2.  Expression of PrPC in undifferentiated and differentiated hESCs. (a) Expression of PrPC (green) and Oct-3/4 (red) as detected by immunostaining in undifferentiated hESCs (33 passages on feeder cells, U) and in hESCs during spontaneous differentiation for 30 days. Hoechst 33342 was used for staining of nuclei (blue). Under undifferentiating conditions (U), PrPC was detected only in feeder cells (green arrow). Scale bar = 100 μm. (b) Expression of PrPC in hESCs during the time course of spontaneous differentiation for 30 days as determined by western blotting using 3F4 antibody. ES, undifferentiated hESCs; NBH – 10% normal brain homogenate from hamster is included as reference. Oct-3/4 was used for confirmation of a differentiation status; β-actin was used as a loading control.

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α-rPrP delays spontaneous differentiation of hESCs

To test whether normal prion protein exhibits any negative or positive trans-effect on hESCs differentiation, full-length rPrP was folded in vitro into a monomeric α-helical conformation (α-rPrP) as previously described (Bocharova et al. 2005) and supplemented in the culture medium. In contract to PrPC, rPrP is unglycosylated and lacks GPI anchor. The effect of α-rPrP was tested for both undifferentiated cells and cells undergoing spontaneous differentiation. Upon cultivation for 7 days, hESCs cultured under self-renewal conditions either in the absence or presence of 1 μM α-rPrP [referred to as U (undifferentiated) or U+1, respectively] or under differentiation conditions in the presence of 1 μM α-rPrP (referred to as D+1) consisted mainly of cells with the morphology of undifferentiated stem cells (Fig. 3a). hESCs were assembled into large, compact, multicellular clusters with a distinguishable border between the stem cells and the feeder cells (Fig. 3a). The cells cultured under those conditions (U, U + 1, and D + 1) showed expression of the pluripotency marker protein oct-3/4 (Fig. 3a). The cells cultured under differentiation conditions in the absence of α-rPrP (D) did not maintain their embryonic stem cell-like morphology and showed clear signs of differentiation (Fig. 3a). The expression of oct-3/4 in these cells was markedly reduced compared with that in cells cultured in the presence of α-rPrP (Fig. 3a). The results of immunostaining were further confirmed by immunoblotting that showed reduced amounts of oct-3/4 expression in differentiating cells cultivated in the absence of α-rPrP (Fig 3b).

image

Figure 3.  Treatment with α-rPrP helps to preserve pluripotent status of hESCs. (a) hESCs were cultured under self-renewal conditions in the absence or presence of 1 μM α-rPrP (U and U + 1, respectively), or under differentiation conditions in the absence or presence of 1 μM α-rPrP (D and D + 1, respectively). After culturing for 7 days, hESCs were immunostained for oct-3/4 (red) and PrP using P antibody (green). Hoechst 33342 was used for staining of nuclei (blue). Scale bar = 100 μm. (b) Western blot (upper panel) of oct-3/4 and PrPC expression (with 3F4 antibody) in hESCs cultured under four different conditions for 7 days: U, U + 1, D, and D + 1. NBH – 10% normal hamster brain homogenate; β-actin was used as a loading control. rPrP runs faster than PrPC in PAGE, because it lacks glycosylation. Densitometric analysis (lower panel) of oct-3/4 expression normalized to actin loading control. (c) Quantitative assay of alkaline phosphatase in each hESC group (top panel) and standard calibration curve showing OD dependence on concentration of recombinant alkaline phosphatase (bottom panel). For each group, the experiments were performed in triplicate with approximately 20 000 cells assayed for each repeat.

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In addition to oct-3/4, undifferentiated hESCs are known to display high activity of alkaline phosphatase (AP) (Thompson et al. 1998; Reubinoff et al. 2000). As judged from the enzyme activity assay, hESCs supplied with α-rPrP maintained their high AP expression level regardless of whether they were cultured under differentiation or self-renewal conditions, whereas hESCs cultured under differentiation conditions in the absence of α-rPrP showed substantial reduction in AP activity (Fig. 3c). These data support the previous results that treatment with α-rPrP inhibited spontaneous differentiation.

To test whether the effect of α-rPrP on hESCs differentiation was attributed to its native α-helical conformation, hESCs were treated with two non-native rPrP isoforms, the β-oligomers and amyloid fibrils that were prepared in vitro as previously described (Bocharova et al. 2005; Novitskaya et al. 2006; Makarava et al. 2007). We did not observe any noticeable effects on hESCs status upon treatment with non-native rPrP isoforms regardless of whether the treatment was performed under self-renewal or differentiation conditions (Fig. S1a and b). Consistent with our previous work (Novitskaya et al. 2006, 2007), amyloid fibrils caused some apoptotic cell death in hESCs as was evident from DNA-fragmentation (Fig. S1a and b).

To determine whether the treatment with α-rPrP delays or arrests hESCs differentiation, hESCs were cultured in the presence of 0.1 μM or 1 μM α-rPrP under differentiation conditions for 20 days and the course of differentiation was monitored using several markers specific for neuronal lineages (Fig. 4a). On the day 14 of culture, the expression of GAP43 and TH were down-regulated in the cells treated with 1 μM α-rPrP when compared with the control cells or cells treated with 0.1 μM α-rPrP. The immunocytochemistry confirmed that the control cells displayed more advanced stages of differentiation on the day 14 than the cells treated with 1 μm α-rPrP (Fig. 4b). Consistent with the previous results, the expression of pluripotency marker oct-3/4 was substantially higher in the cells treated with 1 μM α-rPrP relative to that in control cells (Fig. 4a). On the day 20 of culturing, the expression levels of GAP43 or TH appeared to be similar in all three groups. However, the expression of synaptophysin, a marker of advanced differentiation into neuronal lineages, was substantially lower in cells treated with 1 μM α-rPrP than that in control cells or cells treated with 0.1 μM α-rPrP (Fig. 4a). Oct-3/4 was not detectable in all three groups on the day 20 of differentiation. Consistent with the previous results, the expression of PrPC increased throughout the time course of spontaneous differentiation in all three groups (Fig. 4a; lane 1, 2 and 5). However, when compared with the non-treated control cells, the cell treated with α-rPrP (0.1 μM or 1 μM) showed a dose-dependent decrease of PrPC expression on days 14 and 20 of differentiation (Fig. 4a, lane 2–7). Therefore, treatment with α-rPrP was found to have a negative feedback on expression of endogenous PrPC that presumably highlights a self-regulalting mechanism of PrPC expression during differentiation and/or a negative effect of α-rPrP on cell differentiation. Taken together, these results illustrate that treatment with α-rPrP delays the differentiation of hESCs. In opposite to α-rPrP, the β-oligomers or amyloid fibrils produced very minor accelerating effect if any at all on differentiation course of hESCs, as judged by expression levels of Oct-3/4, PrPC, and GAP43 at the day 14 (Fig. S1c).

image

Figure 4.  Treatment with α-rPrP delays spontaneous differentiation of hESCs. hESCs were cultured under differentiation conditions in the absence or presence of 0.1 μM or 1 μM α-rPrP for 14 or 20 days and analyzed by western blotting (a) or immunofluorescence (b). Growth associated protein 43 (GAP43), TH, tyrosine hydroxylase; Snp, synaptophysin; ES, undifferentiated hESCs; NBH, normal hamster brain homogenate; β-actin was used as a loading control. (b) Immunostaining for GAP43 (green) and PrP (red) on day 14 of differentiation. Scale bar = 50 μm.

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α-rPrP helps to maintain proliferation activity during spontaneous differentiation of hESCs

As high proliferation activity is considered to be one of the key characteristics of undifferentiated pluripotent cells, we were interested in evaluating the effect of α-rPrP on the rate of cell proliferation during hESCs differentiation. To assess the rate of cell proliferation, cells collected at different time-points of differentiation were analyzed using a 5-bromo-2-deoxyuridine (BrdU) incorporation assay followed by flow cytometry. Three experimental formats were employed, where α-rPrP was supplemented in the culture media starting on the days 1, 7, or 14 of differentiation (Fig. 5a). If α-rPrP was supplemented starting on day 1, the proliferation activity first peaked (on day 7 of cultivation) and then decreased in both α-rPrP-treated and non-treated cells (Fig. 5b and c). The proliferation activities, however, were consistently higher in the α-rPrP-treated cells compared with those in the non-treated controls. For instance, on day 7 of differentiation, the percentage of BrdU-positive cells in α-rPrP-treated samples was 67.9 ± 1.95%, comparing with 53.6 ± 2.4% observed for non-treated samples. If α-rPrP was supplied to culture medium starting on day 7 of differentiation, the percentage of BrdU-positive cells was maintained at the relatively high level (46.7 ± 1.1%) even on day 30 of differentiation, when the proliferation activity in the control group dropped to 22.7 ± 1.2%. If the treatment with α-rPrP started on day 14, however, the cells did not maintain their high proliferating activity. The rate of proliferation dropped to 16.6 ± 0.5% in α-rPrP-treated cells by day 30 of differentiation, which was similar to 22.7 ± 1.2% observed for the control group. This experiment revealed that α-rPrP helped to maintain stem cell proliferation activity if α-rPrP-treatment started at the early stages of hESCs differentiation. While α-rPrP was not able to prevent differentiation completely, it helped to maintain the undifferentiated status with high proliferation activity in substantial portion of the cell population.

image

Figure 5.  α-rPrP-treated hESCs display high proliferation activity. (a) Experimental set up for assessing proliferation activity of hESCs. hESCs were cultured under differentiating conditions either in the absence (blue arrows) or in the presence of 1 μM α-rPrP (red arrows) and their proliferation activity was assayed on days 7, 14, 20, or 30 of culture. 7d + 1, 20d + 1 and 30d + 1 refer to hESCs cultures that were supplied with α-rPrP starting from the beginning of differentiation, whereas 7d [RIGHTWARDS ARROW] 30d + 1 and 14d [RIGHTWARDS ARROW] 30d + 1 refer to hESCs cultures that were supplied with α-rPrP starting from day 7 or day 14 of differentiation, respectively. (b) Analysis of cell proliferation using BrdU incorporation assay and flow cytometry. Cells were treated with 10 μM BrdU for 24 h, then trypsinized and labeled with antibody against BrdU. (c) The rate of proliferation in hESCs cultures measured by BrdU incorporation assay as a function of differentiation time. The data points represent an average from two independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

We thank Regina Savtchenko for purifying rPrP and Pamela Wright for editing the manuscript. This work was supported by a Maryland Stem Cell Commission grant (to IVB).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Appendix  S1. Materials and methods.

Figure S1. Treatment of hESCs with &bgr;-oligomers or amyloid fibrils.

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