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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: J Neurochem. 2012 Sep 3;124(3):310–322. doi: 10.1111/j.1471-4159.2012.07913.x

The cellular form of the prion protein is involved in controlling cell cycle dynamics, self-renewal and the fate of human embryonic stem cell differentiation

Young Jin Lee 1, Ilia V Baskakov 1,*
PMCID: PMC3505810  NIHMSID: NIHMS398906  PMID: 22860629

Abstract

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 upregulate 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, overexpression 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. The current study suggests that PrPC is at the cross-roads 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.

Keywords: human embryonic stem cells, prion protein, self-renewal, stem cell differentiation, stem cell fate

Introduction

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 has 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 (Roucou et al. 2005, Bounhar et al. 2001, Chiarini et al. 2002, Lopes 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 (Steele et al. 2006, Zhang et al. 2006, Lee & Baskakov 2010, Panigaj et al. 2011, Santos et al. 2011, Mouillet-Richard et al. 1999, Lima et al. 2007), 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). While 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, the current 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 due to 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 offers 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 & Baskakov 2010). To examine the role of PrPC in human embryogenesis in detail, a panel of lentiviral vectors that upregulates or suppresses PrPC expression in hESCs was generated. The current work illustrates that PrPC is involved in key cellular activities that determine the status of hESCs: (1) it regulates the dynamics of the cell cycle, (2) controls the cellular switch between self-renewal and differentiation, and (3) contributes to determining the fate of cell differentiation.

Materials and Methods

hESCs culturing and spontaneous differentiation

The protocol for using hESCs (H9, National Stem Cell Bank, Madison, WI, USA) was reviewed and approve by the University of Maryland, Baltimore Embryonic Stem Cell Research Oversight Committee and Institutional Review Board of the University of Maryland. H9 hESCs were maintained on mitomycin C (Sigma, St. Louis, MO, USA)-treated mouse embryonic fibroblasts (MEFs)(American Type Culture Collection, Manassas, VA, USA) feeder layers in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 20% knockout serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), 0.1 mM non-essential amino acids (Invitrogen), 50 U/ml penicillin G (Invitrogen), 50 µg/ml streptomycin (Invitrogen) and 4 ng/ml human recombinant basic fibroblast growth factor (bFGF, Invitrogen) at 37°C in an atmosphere of 5% CO2. hESC colonies were subcultured on new feeder cells every 5–7 days.

For inducing spontaneous differentiation, mechanically dissociated and harvested hESCs were grown in suspension culture without MEFs for 5 days using the same medium but in the absence of bFGF, during which they formed embryonic bodies and attached onto 0.1% gelatin-coated culture plates. Then hESCs were cultured for 14 days for further differentiation. On day 14, cells were immunostained with the antibodies specific for markers of the three germ layers.

Construction of lentiviral-derived vectors carrying shRNA-PrPC and human PrPC

Target sequences were derived from the 3’ UTR region of human Prnp gene (GenBank BC022532). CAATAGGGAGACAATCTAA (1899-1917, sequence #1) was selected as the target sequence for silencing the expression of PrPC; the scrambled sequence GAATGCAATAACGAGAGTA was used as a negative control for testing the effects of non-specific shRNA. To avoid off-target effects, a homology search was performed using BLAST (http://blast.ncbi.nlm.nih.gov) to ensure that only the PrPC mRNA sequence was targeted. Two complementary oligonucleotides necessary to create the hairpin insert for pENTR-H1/TO vectors (Invitrogen) were designed using SiRNA Scales software (Matveeva et al. 2007). The following single stranded oligonucleotides were synthesized:

  • shRNA-PrPC#1-Top, 5’-CACCGAATAGGGAGACAATCTAACGAATTAGATTGTCTCCCTATTC-3’

  • shRNA-PrPC#1-Bot., 5’-AAAAGAATAGGGAGACAATCTAATTCGTTAGATTGTCTCCCTATTC-3’

  • shRNA-Scrmbl-Top, 5’-CACCGAATGCAATAACGAGAGTACGAATACTCTCGTTATTGCATTC-3’

  • shRNA-Scrmbl-Bot., 5’-AAAAGAATGCAATAACGAGAGTATTCGTACTCTCGTTATTGCATTC-3’

After annealing, each double-stranded oligonucleotide (5nM) was cloned into pENTR/H1/TO vector (Invitrogen). To construct shRNA-expression vectors, recombination reactions of pENTR/H1/TO vectors that included specific target sequences with pLenti4/BLOCK-iT-DEST vector (Invitrogen) were performed. To construct lentiviral PrPC expressing vector, human PrPC cDNA (ID: HsCD00043335) was obtained from DF/HCC DNA Resource Core (http://plasmid.med.harvard.edu/PLASMID/); DNA sequencing was conducted to confirm its cDNA sequence. In addition, single strand oligonucleotides for the tetracycline operator (TetO2) sequence, (5’-TCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGA-3’ and 5’-TCTCTATCACTGATGGGAGATCTCTATCACTGATAGGGA-3’), were synthesized and annealed for cloning into TR-removed pLenti6/TR vector (Invitrogen) via Spe I and Pst I restriction enzyme sites. PrPC cDNA, amplified by primers with Bspe I or EcoR I restriction enzyme sites, was inserted into pLenti6/TR with TetO2 (Fig. 1A).

Figure 1. Lentiviral vectors and cell establishing procedures.

Figure 1

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(A) Schematic representation of the four lentiviral vectors expressing tetracycline (Tet) repressor (Lenti-TetR), short hairpin RNA against PrPC (Lenti-ShPrPC), scrambled shRNA (Lenti-ShScram) and PrPC (Lenti-HuPrPC). RSV, Rous Sarcoma Virus enhancer/promoter; LTR, long terminal repeats; 5’, 5’ splice donor; Ψ, HIV-1 packaging signal; RRE, HIV-1 Rev (regulator of virion expression) response element; CMV, cytomegalovirus promoter; SV40, simian virus early promoter; EM7, synthetic prokaryotic promoter; ΔU3/3’UTR, a deletion in the 3’LTR; TO, two tetracyclin operator sequences; PH1/TO, hybrid promoter consisting of the human H1 promoter and TO. (B) Experimental procedure for the establishment of inducible shRNA- or PrPC-expressing hESC lines. Bla, Blasticidin; Zeo, Zeocin.

For producing lentiviruses, pLenti4/BLOCK-iT-DEST that expresses shRNA, pLenti6/TetO2/PrPC that expresses PrPC, or pLenti6/TR that expresses tetracycline repressor were mixed with ViraPower Packaging Mix (Invitrogen) and transfected into 293FT cells (6 × 106). Virus-containing supernatants were harvested 72 hours post-transfection. The lentiviruses were added to hESCs cultured in media conditioned by MEFs on dishes coated with Matrigel (BD Biosciences, San Jose, CA, USA) at a multiplicity of infection (MOI) of 2–10 with 5 µg/ml Polybrene (American Bioanalytical, Akron, OH, USA). Cells were cultured using a complete medium containing 5 µg/ml Zeocin (Invitrogen) and/or 5 µg/ml Blasticidin (Invitrogen) for 6 weeks to establish stable cell lines (Fig. 1B).

Induction of shRNAs or PrPC expression

To induce expression of shRNAs or PrPC in hESCs under self-renewal conditions, cells were cultured on the Matrigel-coated plates using culture medium supplemented with 1 µg/ml tetracycline (Tet) (Invitrogen). The cell culture medium supplemented with 1 µg/ml Tet was changed every day. To induce expression of shRNAs or PrPC in hESCs under spontaneous differentiation conditions, spontaneous differentiation was induced as described above, and cells were cultured using the medium (without bFGF) supplemented with 1 µg/ml Tet. The culture medium supplemented with Tet was changed every 2 days.

Results

Generation of hESCs that conditionally express PrPC or shRNA against PrPC

To examine the role of PrPC in hESC self-renewal and differentiation, three lentiviral vectors were constructed: (1) ShPrPC vector that expresses shRNA against PrPC; (2) ShScram vector that expresses scrambled shRNA, and (3) HuPrPC vector that expresses the human PrPC gene (Fig. 1A). In addition, lentivirus with a pLenti6/TR lentiviral vector that expresses tetracycline repressor (TetR) protein was constructed (Fig. 1A). After producing lentivirus with helper plasmids in 293FT cells, the titer of each lentiviral stock was determined using a HeLa cell line. Each lentivirus was added to hESCs at a multiplicity of infection (MOI) of 2–10 and cells were cultured with antibiotics for 6 weeks to select for stably transduced cells (Fig. 1B). To test whether modified hESC lines maintain the basic characteristics of pluripotent cells, the expression of a pluripotency marker Oct-3/4 was examined by immunostaining of hESCs cultured under self-renewal conditions. All modified hESC lines grew as colonies and expressed Oct-3/4 at a level similar to that observed in unmodified hESCs (Fig. 2A). As expected, the modified hESCs stably expressed TetR protein (Fig. 2A, B).

Figure 2. Characterization of hESC lines with inducible expression systems.

Figure 2

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(A) Expression of TetR (green) and Oct-3/4 (red) in undifferentiated hESCs (37 passages on feeder cells) analyzed by immunostaining. Hoechst 33342 was used for staining of nuclei (blue). Scale bar, 100 µm. (B) hESCs stably transfected with Tet-inducible PrPC were incubated without (−) or with (+) 1 µg/ml Tet for up to 72 hrs, and PrPC and TetR expression were analyzed by Western blotting in two independently established cell lines as a function of induction time. β-actin was used as a loading control.

To establish a time frame for inducing PrPC expression, hES+TetR+HuPrPC cells were treated with Tet, and the time course of PrPC expression was examined by Western blotting. Consistent with the previous study (Lee & Baskakov 2010), hES+TetR+HuPrPC cells cultured under self-renewal conditions lacked any detectible PrPC in the absence of Tet treatment. However, PrPC was detected upon 24 hours of treatment with Tet and its expression level increased gradually afterwards (Fig. 2B). TetR protein was stably expressed in Tet-treated and untreated hESCs (Fig. 2B).

Expression of PrPC in hESCs under self-renewal conditions induces cell differentiation and changes the dynamics of the cell cycle

To examine the role of PrPC in hESC differentiation, hES+TetR+HuPrPC cells cultured under self-renewal conditions were treated with Tet. Three days after treatment, hES+TetR+HuPrPC cells showed notable signs of differentiation including changes in shape of the hESCs colonies and morphology of individual cells (Fig. 3A,B,C,D) and a decrease in Oct-3/4 expression level (Fig. 3E). No signs of differentiation were observed in the control cell lines including hES, hES+TetR, or hES+TetR+HuPrPC in the absence of Tet treatment (Fig. 3A,B,C,E). After five days of Tet treatment, expression of markers for three germ layers (ectodermal, endodermal and mesodermal), including TH, GAP43, AFP and Bra, was detected by Western blotting and immunofluorescence in the hES+TetR+HuPrPC line, but not in a control line hES+TetR (Fig. 4A,B). At the same time, Oct-3/4 expression in Tet-treated hES+TetR+HuPrPC was lower than in Tet-treated hES+TetR or untreated hES+TetR+HuPrPC (Fig. 4 A,B). These data suggest that ectopic expression of PrPC was sufficient to trigger differentiation of hESCs cultured under self-renewal conditions and that PrPC-induced differentiation does not seem to favor any particular germ layer within this time frame.

Figure 3. PrPC induces differentiation of hESCs cultured under self-renewal conditions.

Figure 3

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Phase-contrast images of hES (A), hES+TetR (B), or hES+TetR+HuPrPC lines cultured in the absence (C) or presence of Tet (D) for 72 h. hES+TetR+HuPrPC with Tet did not maintain morphology typical for embryonic stem cells. Scale bar = 15 µm. (E) Analysis of PrPC and Oct-3/4 expression in hES, hES+TetR or hES+TetR+HuPrPC lines by Western blotting. Induction of PrPC in hES+TetR+HuPrPC for 72 h using Tet treatment (+) was accompanied by a decrease in Oct-3/4 expression. β-actin was used as a loading control. (F) Analysis of cell cycle structure in hES, hES+TetR, or hES+TetR+HuPrPC lines cultured in the absence (−) or presence of Tet (+) for 72 hrs. Cells were stained with propidium iodide and analyzed by flow cytometry. In the hES+TetR+HuPrPC(+) line, a higher percentage of cells were found in G1 phase in comparison to the control lines. The data represent a mean±SD from three independent experiments. Statistical significance was determined by Student’s t-test: *, p<0.05; ***, p<0.0005.

Figure 4. PrPC-induced differentiation of hESCs.

Figure 4

Figure 4

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(A) hES+TetR or hES+TetR+HuPrPC lines were cultured under self-renewal conditions in the absence (−) or presence of 1 µg/ml Tet (+) for 5 days. Cells were stained with antibodies to Oct-3/4 and PrPC (top panels), TH and GAP43 (middle panels), or AFP and Bra (bottom panels). Nuclei were stained with Hoechst 33342. Scale bars = 100 µm or 15 µm (for high magnification images). (B) Expression of PrPC, Oct-3/4, TH, GAP43, AFP, Bra and TetR in hES+TetR or hES+TetR+HuPrPC cell lines cultured for 5 days under self-renewal conditions in the presence (+) or absence (−) of Tet, as analyzed by Western blotting. β-actin is used as a loading control. Expression levels of marker proteins were normalized to that of β-actin. The data represent a mean±SD from three independent experiments. Statistical significance was determined by Student’s t-test: *, p<0.05 (Student’s t-test).

One of the key characteristics of hESCs is their high self-renewal potential and proliferation activity. According to an emerging view, the switch between self-renewal and differentiation states is controlled by the dynamics of the cell cycle (Becker et al. 2006, Menchon et al. 2011). To test whether PrPC affects the structure of the cell cycle, unsynchronized hESCs were analyzed using flow cytometry. Consistent with a previous study that hESCs exhibit a very short G1 phase under self-renewal conditions (Becker et al. 2006), only 15.9±1.3% of hESCs were found to reside in G1 phase, whereas 51.7±3.5% cells were in S phase (Fig. 3F). The cell cycle structure of hES+TetR or hES+TetR+HuPrPC in the absence of Tet treatment was very similar to that of hESCs. In contrast, in hES+TetR+HuPrPC cells treated with Tet for three days, the percentage of cells in G1 phase was substantially higher (32.9±1.9%) than that in control groups (Fig. 3F). This change occurred entirely at the expenses of the S phase (Fig. 3F). This result illustrates that PrPC has a negative effect on the G1 to S phase transition. It is not clear whether PrPC triggered differentiation first, while the cell cycle structure transformed as a consequence of the changes in hESC status, or alternatively, whether PrPC delayed the G1 to S phase transition, a process that can cause differentiation. Nevertheless, these results suggest that PrPC should not be considered only as a marker of cell commitment to differentiation according to neuronal lineages, but can play an active role in inducing differentiation.

PrPC regulates hESCs differentiation

To test whether PrPC affects the fate of hESC differentiation, four cell lines, hES+TetR, hES+TetR+ShPrPC, hES+TetR+ShScram and hES+TetR+HuPrPC, were cultured in the presence of Tet under spontaneous differentiation conditions for 14 days; and the expression of three germ-layer marker proteins was examined by Western blotting (Fig. 5A) and immunocytochmistry (Fig. 5B,C). In the hES+TetR+ShPrPC line, where PrPC was suppressed, the expression of ectodermal neuronal markers (GAP43, synaptophysin and TH) was down-regulated, but no notable changes in the endodermal or mesodermal markers, AFP and Brachyury, respectively, were observed when compared to the control lines, hES+TetR or hES+TetR+ShScram (Fig. 5A). Remarkably, in the hES+TetR+HuPrPC line that overexpressed PrPC, the expression of markers of all three germ layers was down-regulated (Fig. 5A). Both control lines hES+TetR and hES+TetR+ShScram displayed more advanced stages of neuronal differentiation than hESC lines, in which PrPC was silenced or overexpressed. In addition, endodermal and mesodermal differentiation was suppressed in the PrPC-overexpressed line hES+TetR+HuPrPC (Fig. 5A to C). These data suggest that PrPC is actively involved in determining hESC fate and that proper levels of PrPC are required for neuronal differentiation. At the same time, these results also suggest that PrPC upregulation acts as a repressor of spontaneous hESC differentiation into the cells of all three germ layers.

Figure 5. PrPC modulates the cell cycle transition and fate of hESCs cultured under spontaneous differentiation conditions.

Figure 5

Figure 5

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hES+TetR or hES+TetR transfected with ShPrPC, ShScram or HuPrPC vectors were cultured under spontaneous differentiation conditions for 14 days in the presence of Tet (+) and analyzed by Western blotting (A), immunostaining (B, C) of flow cytometry (D). (A) Analysis of expression of PrPC and the following markers: GAP43, TH, synaptophysin (Syn), AFP, Bra; and TetR. β-actin was used as a loading control. Expression level of each protein was normalized to those observed in a control line (hES+TetR). The data represent a mean±SD of three independent experiments; statistical significance was determined by Student’s t-test: *, p<0.05, **, p<0.005, ***, p<0.0005. Immunostaining for PrPC (red) and GAP43 (green) (B), or AFP (red) and Brachyury (green) (C). Nuclei were stained with Hoechst 33342. Scale bar, 50 µm. (D) Analysis of the cell cycle distribution by flow cytometry. In hES+TetR+ShPrPC(+) line where PrPC was silenced, a higher percentage of cells were found in G1 phase in comparison to the lines with normal or high PrPC expression level. The data represent a mean±SD from three independent experiments. Statistical significance was determined by Student’s t-test: *, p<0.05; **, p<0.005.

To examine the role of PrPC in regulating the cell cycle in differentiating hESCs, flow cytometry analyses were performed using the four hESC cell lines described above. When compared to the hESCs cultivated under self-renewal conditions (Fig 2F), all four hESC lines showed a lower percentage of cells in S phase at the 14th day of differentiation, consistent with a change in their status. In hESCs with silenced PrPC (hES+TetR+ShPrPC), the percentage of cells in G1 phase (45.9±4.2%) was significantly higher than those in other lines (hES+TetR: 22.5±2.1%; hES+TetR+ShScram: 24.7±2.9%; hES+TetR+HuPrPC: 18.5±2.1%) (Fig. 5D). Accordingly, the percentage of cells that resided in S phase in hES+TetR+ShPrPC (18.6±1.5%) was significantly lower than those in the three other lines (hES+TetR: 34.9±3.1%; hES+TetR+ShScram: 35.5±6.6%; hES+TetR+HuPrPC: 34.1±2.8%) (Fig. 5D). Remarkably, no significant effect of PrPC overexpression on cell cycle distribution was observed. The percentage of cells in G2/M phase or apoptotic cells was very similar in all four lines. This result illustrates that downregulation of PrPC slows down or blocks the transition from G1 to S phase, whereas overexpression of PrPC has no significant effect on cell cycle distribution.

Analysis of cell proliferation activity using the BrdU incorporation assay revealed a direct correlation of proliferation rate with the level of PrPC expression (Fig. 6A,B). The highest proliferation activity was observed in the hES+TetR+HuPrPC line with the highest level of PrPC expression, whereas the lowest proliferation activity was found in the hES+TetR+ShPrPC line with the lowest level of PrPC.

Figure 6. hESC proliferation activity correlates with PrPC expression level in cells cultured under spontaneous differentiation conditions.

Figure 6

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hES+TetR or hES+TetR transfected with ShPrPC, ShScram or HuPrPC vectors were cultured under spontaneous differentiation conditions for 14 days in the presence of Tet and analyzed by analyzed by flow cytometry using staining with allophycocyanin (APC)-conjugated anti-BrdU antibody and 7-amino-actinomycin D (7-AAD). (A) Analysis of cell distribution between sub-G1, G1, G2/M or S phase. (B) Histograms for BrdU labeling for four cell lines. Bracketed lines shows the gates used to identify BrdU-positive cells with an average percentage of BrdU positive cells from two independent experiments indicated.

Discussion

An increasing number of studies suggests that PrPC is involved in regulating stem cell self-renewal and proliferation (Miranda et al. 2011, Lee & Baskakov 2010, Peralta et al. 2011, Santos et al. 2011, Lopes & Santos 2012). To examine the role of PrPC in early embryogenesis, we employed a panel of lentiviral vectors that upregulates 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 the current 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) overexpression 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: (1) it regulates cell cycle progression, (2) controls the cellular switch between self-maintenance and differentiation, and (3) 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 & Baskakov 2010). Studies using mouse and human ESCs showed that PrPC modulates neural commitment during early ESC differentiation (Peralta et al. 2011, Lee & Baskakov 2010). 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 due to 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, while 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 & 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 neuroshpere-derived stem cells via interaction with stress inucible 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, Syed et al. 2011, Khalifé 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).

The current 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, overexpression 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 & 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 the current 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 & 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 overexpression 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, the current 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.

Supplementary Material

Supp Appendix S1

Acknowledgements

We thank Pamela Wright for editing the manuscript. This work was supported by a Postdoctoral Fellowship Grant of Maryland Stem Cell Commission to YJL and NIH grant NS045585 to IVB.

Abbreviations

PrPC

normal, cellular isoform of the prion protein

hESCs

human embryonic stem cells

bFGF

basic fibroblast growth factor

MEFs

mouse embryonic fibroblasts

Tet

tetracycline

TetR

tetracycline repressor

Lenti-TetR

lentiviral vector expressing tetracycline repressor

Lenti-ShPrPC

lentiviral vector expressing short hairpin RNA against PrPC

Lenti-ShScram

lentiviral vector expressing scrambled shRNA

Lenti-HuPrPC

lentiviral vector expressing PrPC

hES+TetR+ ShPrPC

hESCs transfected with Lenti-TetR and Lenti-ShPrPC

hES+TetR+ShScram

hESCs transfected with Lenti-TetR and Lenti-ShScram

hES+TetR+HuPrPC

hESCs transfected with Lenti-TetR and Lenti-HuPrPC

TH

tyrosine hydroxylase

GAP43

growth associate protein 43

AFP

alpha fetoprotein

Bra

brachyury

BrdU

5'-bromo-2'-deoxyuridine.

Footnotes

The authors declare no conflicts of interest.

Supporting information

Appendix S1. Materials and Methods including Flow cytometry, Immunostaining and Western blots.

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