Neural Stem Cell Model for Prion Propagation - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:01 |只看该作者 |倒序浏览 |打印

作者：Ollivier Milhaveta, Danielle Casanovaa, Nathalie Chevalliera, Ronald D. G. McKayb, Sylvain Lehmanna作者单位：aInstitut de Gntique Humaine, CNRS-UPR, Montpellier, France;bLaboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland, USA
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      【摘要】
   Correspondence: Sylvain Lehmann, M.D., Ph.D., Institut de G¨¦n¨¦tique Humaine, CNRS-UPR1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France. Telephone:  33-499619931; Fax:  33-499619901; e-mail: Sylvain.Lehmann@igh.cnrs.fr; or Ollivier Milhavet, Ph.D., Institut de G¨¦n¨¦tique Humaine, CNRS-UPR1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France. Telephone:  33-499619930; Fax:  33-499619901; e-mail: Ollivier.Milhavet@igh.cnrs.fr
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The study of prion transmission and targeting is a major scientific issue with important consequences for public health. Only a few cell culture systems that are able to convert the cellular isoform of the prion protein into the pathologic scrapie isoform of the prion protein (PrPSc) have been described. We hypothesized that central nervous system neural stem cells (NSCs) could be the basis of a new cell culture model permissive to prion infection. Here, we report that monolayers of differentiated fetal NSCs and adult multipotent progenitor cells isolated from mice were able to propagate prions. We also demonstrated the large influence of neural cell fate on the production of PrPSc, allowing the molecular study of prion neuronal targeting in relation with strain differences. This new stem cell-based model, which is applicable to different species and to transgenic mice, will allow thoughtful investigations of the molecular basis of prion diseases, and will open new avenues for diagnostic and therapeutic research. ) N4 _7 m( ~5 b8 E$ G0 p
      【关键词】 Neural stem cells Prions Neural differentiation Diagnosis
   INTRODUCTION

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal transmissible neurodegenerative diseases that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in elk, and scrapie in sheep , the detection of this particular strain among the other scrapie strains is both a challenge and a necessity.  }/ t3 z' I; m: P3 _
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Historically, propagation of TSE infectious agents was tested in cultured neuronal cells as early as 1970 . However, this model suffers from major limitations, such as restriction to some strains of scrapie and limited sensitivity, and also from difficulties of use, since it takes time to make a new culture from mice, which renders the model inadequate for any large scale or screening usage. Also, this model does not accurately reflect the cellular environment found in the brain. Thus, we hypothesized that neural stem cells (NSCs) might be permissive to infection by prions and would be a versatile and powerful model for studying prions.7 I6 |0 P9 ~5 l5 h9 d0 Y  j

NSCs are the self-renewing, multipotent cells that generate neurons, astrocytes, and oligodendrocytes in the nervous system. Cultured CNS stem cells have proved useful in defining the pathways that lead to generation of neurons and glia . They have been the object of increasing attention because of their potential use in cell replacement or gene therapy. We hypothesized that a model based on NSCs would be permissive to prion infection because of their neural origins and their ability to differentiate into different cell types from the brain. Moreover, the possibilities of manipulating cell fate by various growth factors would allow the accurate determination of the conditions propitious to production of PrPSc for a particular strain. Ultimately, the availability of these cells and the possibility of obtaining large number of cells opened the opportunity to establish powerful new diagnostic and therapeutic approaches for prion disease.
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Here, we report the generation of a new and versatile cellular model for prion propagation using differentiated mouse fetal NSCs and adult multipotent progenitor cells. This model can be adapted to transgenic mouse and to other species, and it therefore represents a major asset to study prion propagation and transmission.

MATERIALS AND METHODS

Mouse CNS Stem Cell Cultures
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For mouse fetal CNS stem cells called NSCs, the conditions described by Kim et al.  and incubated at 37¡ãC in 95% air/5% CO2. Basic fibroblast growth factor (bFGF) (Abcys, Paris, http://www.abcysonline.com) was added daily at 25 ng/ml to expand the population of proliferative precursors, and the medium was changed every 2 days at the time of bFGF addition. Cells at 80% confluence were subcultured in N2 medium in the presence of bFGF. The neural progenitors were induced to differentiate by withdrawing bFGF and kept in differentiation medium (N2 medium).
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For serial transmission of prions, the culture protocol was changed to have cultures consisting of a mixture of NSCs, progenitor cells, and more mature cells. At day 0 (D0), instead of splitting the culture, cells were kept for 1 additional day in bFGF-containing medium. At D1 cells were split and allowed to expand for a second passage in bFGF-containing medium. The same procedure was applied for the subsequent passages.6 R, ?- N7 @) T4 ?+ V
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Figure 1. Model of mouse fetal NSCs. (A): Scheme depicting the general monolayer cell culture protocol. After dissection, cells are plated in coated dishes and referenced as P0. On an average basis, 6 days are needed to reach 80%¨C100% confluence after expansion in N2 medium supplemented with bFGF. At this stage, either cells are passaged or differentiation is induced by bFGF withdrawal (D0). Infection of cells was made either on the day before differentiation (D-1) or the same day (D0). Differentiation can be maintained for several days before analysis (PnD6 and PnD12). (B, C): Immunostaining of cells during expansion and differentiation. b, undifferentiated cells are revealed by nestin immunostaining (red), and nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). ß-III tubulin, a neuronal marker, and glial fibrillary acidic protein (GFAP), an astrocytic marker, could not be detected in these cultures. Scale bar = 10 µm. (C): After 6 days of differentiation, cells expressed ß-III tubulin (red) and GFAP (green), as revealed by immunofluorescence. Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. (D): Expression profile of PrPC in mouse cortical NSCs at the undifferentiated state and during differentiation for 20 days. PrPC from equal amount of proteins extracted at various time of culture was revealed by Western blot using SAF32 antibody. Molecular mass markers (in kilodaltons) are indicated on the right. Abbreviations: bFGF, basic fibroblast growth factor; D, differentiation day; P, passage; Pn, number of passages after initial plating.
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For mouse adult multipotent progenitor cells, culture conditions were adapted from Song et al. . Briefly, intact hippocampal formations were dissected from female adult CD1 mice. Tissues from three mice were diced into small fragments and then digested for 20 minutes at 37¡ãC in 14 ml of HBSS containing a mixture of 1 mg/ml papain, 0.2 mg/ml cystein, 0.2 mg/ml EDTA, and 0.01% DNase I (Sigma-Aldrich). Digestion was stopped with HBSS containing 0.7 mg/ml ovomucoid inhibitor (Sigma-Aldrich). After mechanical dissociation, tissues were plated on polyornithine- and fibronectin-coated plates in N2 medium containing 10% fetal bovine serum. The next day, the medium was replaced with N2 medium and bFGF (25 ng/ml). The cells grew as attached cultures and were cultured under the same conditions as fetal stem cells.- {- c& O  {; A3 A

Prion Infection of NSCs3 ?; ~7 ]. q# U3 C4 u+ P# C
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The material used for infection was prepared from the brain of terminally ill mice inoculated with the various prion strains. Ten percent brain homogenates were prepared in phosphate-buffered saline (PBS)/5% glucose and stored at ¨C80¡ãC until use. To infect cells, partial purification of the homogenate was completed to avoid sticking of tissues on cells by first treating the homogenate with low amount of Triton-deoxycholate (DOC) lysis buffer for 20 minutes (150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl ) before centrifugation at 10,000g for 10 minutes; supernatant was then resuspended in culture medium and filtrated through 0.22-µm membranes. Cells were exposed to the indicated dilution of brain homogenates for 24 hours. Culture medium was then changed every other day.
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For cell-to-cell infection, cells corresponding to 12-day-differentiated infected cells were resuspended in PBS with a low concentration of Triton-DOC lysis buffer. The solution obtained was then diluted and used as infectious material after filtration. Cells were exposed to infectious material at the time of differentiation (D0) for 24 hours, and culture medium was then changed every other day.
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Proteinase K Resistance Assay and Immunoblotting. w3 ?9 s' C5 Z- @: T+ E

Cells were lysed in Triton-DOC lysis buffer (150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl ), and protein concentration in cell lysates was measured by the BCA protein assay (Perbio Science, Brebi¨¨res, France, http://www.perbio.com). The same amount of protein (100¨C200 µg) was treated with proteinase K or left untreated (12 µg/mg protein; Roche Diagnostics, Meylan, France, http://www.roche-applied-science.com) for 30 minutes at 37¡ãC. All samples were then supplemented with 2 mM Pefabloc for 5 minutes at 4¡ãC and centrifuged at 20,000g for 45 minutes. Pellets were resuspended in loading buffer, boiled, subjected to SDS-polyacrylamide gel electrophoresis on precast 10% bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bis-Tris) gels (Invitrogen), and transferred onto polyvinylidene difluoride membranes. PrPC was detected by using SAF32 monoclonal antibody, and PrPSc was detected by using a mixture of three monoclonal antibodies, SAF 60, SAF 69, and SAF 70. Glyceraldehyde-3-phosphate dehydrogenase was detected using a mouse monoclonal antibody (clone 6C5; Ambion, Huntingdon, U.K., http://www.ambion.com). Western blots were revealed with an enhanced chemiluminescence detection system (GE Healthcare, Saclay, France, http://www.amershambiosciences.com).
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Immunohistochemistry
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Cells were fixed in 4% paraformaldehyde plus 0.15% picric acid in PBS, and standard immunohistochemical protocols were followed. The following primary antibodies were used: for stem cell progenitors characterization, nestin (rat-401) monoclonal antibody at 1:500 (Chemicon, Temecula, CA, http://www.chemicon.com); for stem cell differentiation, ß-tubulin type III (Tuj1) monoclonal antibody at 1:1,000 (Covance, Princeton, NJ, http://www.covance.com) and rabbit glial fibrillary acidic protein (GFAP) at 1:1,000 (DakoCytomation, Trappes, France, http://www.dakocytomation.com). Appropriate fluorescence-tagged secondary antibodies (AlexaFluor 488 and 555; Invitrogen) were used for visualization. 4,6-Diamidino-2-phenylindole was used for nuclear counterstaining.
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RESULTS7 q6 [- H3 A; p6 D" s' X. u8 y

The culture protocol for mouse cortical neural stem cells and the description of the model is summarized in Figure 1A. Undifferentiated cells expressed primarily nestin (Fig. 1B), an intermediate filament protein expressed in neural stem cells and progenitors , whereas ß-III-tubulin, a neuronal marker, and GFAP, an astrocytic marker, were not. At 80% confluence, these cells were subcultured or differentiated by removal of bFGF. After 6 days, cells could be identified as neurons (ß-III tubulin-positive cells) or astrocytes (GFAP-positive cells) (Fig. 1C). We also checked for PrPC expression by Western blot (Fig. 1D). Undifferentiated NSCs expressed low but detectable amounts of PrPC (Fig. 1D, day ¨C2). During the course of differentiation, the levels of PrPC increased massively (Fig. 1D).# N8 W+ d7 n( q! {3 g/ m! S
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Figure 2. Infection of mouse fetal NSCs with mouse prions. (A): Cells were exposed to 0.1% 22L crude brain homogenate at differentiation day 0 and then lysed at various time of differentiation. Cell lysates were digested with proteinase K, and PrPSc was revealed using a mixture of SAF 60, SAF 69, and SAF 70 (SAFmix) antibody. (B): Cells were exposed to semipurified 0.1% 22L brain homogenate at D0 and then lysed at various times after starting the differentiation. Cells from Prnp¨C/¨C were used to control residual signal from the brain homogenate. Cell lysates were digested with proteinase K, and PrPSc was revealed using SAFmix antibody. (C): Detection of PrPSc in mouse NSCs 6 days after differentiation and infection with serial dilutions of semipurified 22L brain homogenate. Molecular mass markers (in kilodaltons) are indicated on the right. All results are representative of three independent experiments. Abbreviations: KO, knockout; NSC, neural stem cell; WT, wild-type.* r; k7 P6 n: ~" T4 G9 c

Differentiated Mouse NSCs Can Efficiently and Persistently Produce PrPSc. K9 p4 L5 U% X( F" U, t- e5 u
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In a first attempt to infect NSCs with prions, cells were exposed to crude 22L prion strain brain homogenate at D0 of the differentiation induced by bFGF removal. PrPSc was assayed by Western blot every 2 days until day 12 of differentiation (Fig. 2A). PrPSc amounts increased with time, indicating that cells were able to produce de novo PrPSc. However, since PrPSc from the homogenate could not easily be distinguished from conversion of endogenous PrPC, the signal observed at early days could be due partly to detection of PrPSc from residual brain homogenate. To address this, we established a protocol to semipurify prions from brain homogenate and used as a control fetal NSC cultures established from mice deleted for the Prnp gene . Cells from wild-type mice produced increasing amount of PrPSc with time, whereas in Prnp¨C/¨C cells, no PrPSc was detected in the early days of infection, indicating that PrPSc from the inoculum did not remain in detectable amounts and that PrPSc accumulation in wild-type NSCs was most likely the result of conversion of endogenous PrPC (Fig. 2B). We also could propagate other mouse-adapted prion strains, in particular RML and C506M3 (data not shown). In an attempt to evaluate the sensitivity of the model, we infected NSCs with serial dilutions of 22L brain homogenate (Fig. 2C). Positive signals for PrPSc were obtained in cells inoculated with 1 ml of a 0.001% dilution, meaning that infectivity could be detected in the equivalent of 10 µg of brain tissue from terminally ill mice.6 K9 `$ V8 }0 E1 ^: A8 U# \- r2 S

Figure 3. Infection of adult multipotent progenitor cells and serial transmission. (A): Adult multipotent progenitor cells from wild-type and Prnp¨C/¨C mice were exposed to 0.1% 22L semipurified brain homogenate at D0 and then lysed after 6 days of differentiation for detection of PrPSc using a mixture of SAF 60, SAF 69, and SAF 70 antibody after proteinase K digestion. (B): Cells were infected with 0.1% 22L semipurified brain homogenate at D0 and kept for 1 more day in bFGF-containing medium. At D1, cells were split and allowed to expand for a second passage. At P2D1 cells were either split or allowed to differentiate until D6 after removal of bFGF. The same procedure was applied for P3. Differentiated cells from P2D6, P3D6, and P4D6 were then lysed and digested with proteinase K to detect PrPSc. (C): WT NSCs were exposed to cell homogenates from P0D12 infected WT NSCs (lane 1) or P0D12 infected Prnp¨C/¨C (KO) NSCs (lane 2). In lane 3, KO NSCs were exposed to cell homogenates from P0D12 infected WT NSCs. The amount of lysed cell used for infection corresponded to a 1:2 dilution of the infected cells. After 6 days of differentiation, samples were analyzed for presence of PrPSc. Molecular mass markers (in kilodaltons) are indicated on the right. All results are representative of three independent experiments. Abbreviations: D, differentiation day; KO, knockout; NSC, neural stem cell; P, passage; WT, wild-type.

Since prion disease is mainly an adult disease, one can argue that a fetal model is not representative of the disease. We therefore performed infection of adult multipotent progenitor cells isolated from the hippocampus of CD1 mice with 0.1% 22L semipurified brain homogenate. After 6 days of differentiation, PrPSc was also detected in adult NSCs from wild-type animals (Fig. 3A). However, the amount of PrPSc produced seemed lower than that in fetal cells, possibly because of a less efficient neuronal differentiation.
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Figure 4. Modulation of mouse fetal NSC infection by growth factors. (A): Immunostaining of cells exposed to different growth factors after 6 days of differentiation. Neurons are revealed with ß-III tubulin antibody (red), and glial cells were detected using glial fibrillary acidic protein antibody (green). Cells were counterstained with 4,6-diamidino-2-phenylindole (blue). Shown are FCS (1%), NGF (20 ng/ml), BDNF (20 ng/ml), RA (5 µM), and CNTF (20 ng/ml). Scale bars = 20 µm. (B, C): Mouse cortical NSCs were exposed to different growth factors and exposed to 0.1% 22L semipurified brain homogenates and then lysed after 6 days of differentiation for analysis of PrPSc and PrPC. (B): After proteinase K digestion, PrPSc was detected using a mixture of SAF 60, SAF 69, and SAF 70 antibody. (C): Detection of PrPC into cell lysates by Western blot using SAF32 antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as loading control. Molecular mass markers (in kilodaltons) are indicated on the right. All results are representative of three independent experiments. Abbreviations: BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; Ctrl, control (untreated cells); FCS, fetal calf serum; NGF, nerve growth factor; RA, retinoic acid.7 j5 ^  y* [+ a
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Serial Transmission of Prions in NSCs
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Importantly, we did not succeed in infecting highly purified populations of undifferentiated NSCs (data not shown). It is possible that in these cells, conversion occurred at very low rate and was not detectable by our methods. This is unlikely, however, since even long-term culture of highly purified undifferentiated CNS stem cells did not consistently accumulate PrPSc. To carry the infectivity in dividing cells, we thus modified our culture conditions and could eventually infect NSCs as a mixture of NSC, progenitor cells, and more mature cells (Fig. 3B). This protocol allows a low amplification of PrPSc, as demonstrated by the slight increase of PrPSc signal when cells were diluted and differentiated between each passage.

To confirm infection and to address whether prions produced in our cell system could be transmitted, we used a simplified transmission protocol based on cell-to-cell infection. After preparation of cell homogenates from P0D12 wild-type and Prnp¨C/¨C infected cells, the solutions obtained were used to infect cultures of wild-type NSCs at D0 (cell lysates used for infection were used at a ratio of 1:2). Cell homogenates from infected wild-type NSCs were able to induce production of PrPSc after 6 days of differentiation but not cell lysates from Prnp¨C/¨C infected NSCs (Fig. 3C, lanes 1 and 2). As an additional control, we verified that no PrPSc was produced in NSC cultures established from Prnp¨C/¨C animals and exposed to the lysate of infected wild-type NSCs (Fig. 3C, lane 3). Similar results were obtained using a higher dilution (1:100) of the homogenate (not shown).5 @! W$ y1 r$ E8 |& |" F
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Modulation of Infection Following Various Differentiating Conditions1 K# ~8 ]4 \! g% T/ Y
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Treatment with different growth factors at the time of differentiation can alter cell fate, as demonstrated previously .; t6 _% G" m# }+ Q9 E* k

DISCUSSION' T$ u) p- ]/ `6 K8 r) B" ~+ j$ D

Our primary objective was to develop a new and relevant experimental model able to propagate prions in vitro. Such a model is critical for both basic and applied research, including screening of drugs and detection of prion infectivity.

Using a well-established model system of mouse NSC culture, we demonstrated that differentiated NSCs could propagate prions. Indeed, following inoculation with low concentrations of brain homogenate, a progressive increase of PrPSc signal in wild-type but not in control Prnp¨C/¨C cells was observed. This PrPSc is not that of the inocula, since the low concentration of infectious material used, as well as the optimized procedure of infection (as described in Materials and Methods), resulted in a consistent absence of PrPSc signal at day 2 or 3 following inoculation. Incidentally, we are confident that the production of PrPSc by the cells is not the result of an acute conversion process that occurs in the first 72 h of contact with the inocula and does not generate infectivity . Indeed, we believe that PrPSc produced by NSCs is associated with the production of infectivity, as seen in all cell culture models producing PrPSc. We could, in fact, propagate the infection in serial transmission experiments. However, we will have a better idea of the exact amount of infectivity generated by the cells by studying the incubation time in mice inoculated with dilution of cell lysates (experiments in progress).

Importantly, we believe that these results could only be obtained because of the high susceptibility of the NSCs that allowed us to use low concentrations of homogenates, in combination with our improved infection protocol. The latter is actually the result of an extensive testing of different infection conditions to minimize the amount of residual inocula in the culture. However, it remains to be established whether this cell culture model can be used for all prion strains and thus will not suffer from limited sensitivity to some of them.
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One explanation for the high susceptibility of NSCs to prions may reside in the fact that these cells are primary cultures of brain origin. So far, with the exception of the model established by Cronier et al. , cells used for infection were not of brain origin or were immortalized cells. Moreover, the mixed nature of the NSC culture after differentiation, accurately recapitulating the brain environment in vitro, might provide the factors favoring, or essential for, the generation of PrPSc.
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To fully compare our model with the available models to date, direct comparison would be necessary, requiring precise parallel study and titration in vivo of infectivity produced by the different models. Based on the following remarks, we believe, however, that our NSC model has new potentials for studying prions. First of all, undifferentiated NSC cells can be passaged easily up to 10 times, with a fivefold increase at each passage, maintaining their differentiation and infection potential. This allows the building up of important frozen stocks of cells for various experiments and screening. We can, in fact, envision developing a "scrapie cell assay" that would allow prion infectivity titration as introduced by Klohn et al. , a preliminary observation that would need further investigation.

In this work, specific culture conditions had to be used to passage nondifferentiated infected cells, which were then able to propagate prions (Fig. 3B). Actually, in our hands, pure populations of undifferentiated NSCs were not able to sustain prion infection (data not shown). Our protocol for prion propagation did not let cells grow as true undifferentiated NSCs but rather as a mixture of NSCs, progenitor cells, and more mature cells, as can be seen in neurosphere cultures. This probably allowed for a low amplification of PrPSc waiting for appropriate conditions, such as differentiation, to produce large amounts of PrPSc. This NSC model could therefore help in the determination of the key cellular events and the precise time required for cells to become susceptible to infection.. f) K+ Q$ U( }; N1 |0 {- q- I

The possibility of infecting differentiating adult multipotent progenitor cells (Fig. 3A) also raises the question of the role of these cells in vivo during the course of the disease. In an adult brain, new neurons can be produced from NSCs, especially during cerebral insults . Therefore, it is possible that endogenous adult multipotent progenitor cells act as a reservoir for infection and that migration and differentiation of these cells participate in the development of the disease rather than providing brain repair. Further work is certainly needed to test this hypothesis.& }& K. s) N5 Q

In conclusion, we describe here a new cell culture model of TSEs based on the use of mouse NSCs. The model will be applicable to transgenic mice expressing PrP from different species, as well as to human and hamster neural stem cells. It will help to decipher the molecular mechanism of prion replication and targeting and will open new avenues for diagnostic and therapeutic research.$ I* q, S% C; ?' \! i, d& o% m
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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We thank R. Carp (New York State Institute for Basic Research, Staten Island, NY) for mouse brain homogenates. We thank M. Pastore and C. Crozet for helpful assistance. Special thanks go to M. Guentchev (National Institute of Neurological Disorders and Stroke, Bethesda, MD) for training NSC cultures. This work was supported by grants from the European Community (Brussels, Belgium) Network of Excellence "Neuroprion", the Department for Environment, Food and Rural Affairs (London) Grant SE2002, and the Centre National de la Recherche Scientifique (Paris).2 j$ V. i4 c! v. A
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