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Journal of Virology, November 2008, p. 10959-10962, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.01085-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Persistent Propagation of Variant Creutzfeldt-Jakob Disease Agent in Murine Spleen Stromal Cell Culture with Features of Mesenchymal Stem Cells

Sergey Akimov, Oksana Yakovleva, Irina Vasilyeva, Carroll McKenzie, and Larisa Cervenakova^*

Transmissible Diseases Department, American Red Cross Holland Laboratory, Rockville, Maryland

Received 22 May 2008/ Accepted 31 July 2008

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The transmission of variant Creutzfeldt-Jakob disease (vCJD) through blood transfusions has created new concerns about the iatrogenic spread of transmissible spongiform encephalopathies (TSEs)/prion diseases through blood and plasma-derived products and has increased the need to develop efficient methods for detection of the agent in biologics. Here, we report the first successful generation of spleen-derived murine stromal cell cultures that persistently propagate two mouse-adapted isolates of human TSE agents, mouse-adapted vCJD, and Fukuoka 1. These new cell cultures can be used efficiently for studies of the pathogenesis of the disease, for development of diagnostics and therapeutics, and as a rapid ex vivo assay for TSE inactivation/removal procedures.

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Efforts to infect cell cultures with infectious agents of transmissible spongiform encephalopathies (TSE)/prion diseases date back nearly 50 years. The earliest studies were oriented toward generating cell cultures from the brain tissues of scrapie-infected sheep and mice (7, 12), but none achieved high levels of prion multiplication and stable propagation of the agent until mouse neuroblastoma cell lines were explored (23). In general, the most successful results have been achieved with cell cultures developed from neuronal tissues, but infections of fibroblasts and microglial tissue and skeletal myoblast cell lines have recently been reported (8, 10, 16), and genetically engineered cell lines of rabbit epithelial tissue origin and neuronal tissue origin from transgenic mice carrying the ovine prion protein gene have been added to the list (9, 27).

Most of these cell cultures are permissive to one or more isolates of the mouse-adapted scrapie agent (28), and a few are susceptible to Fukuoka 1 (Fu) (1, 2, 4), a mouse-adapted isolate from the brain of a Japanese patient with Gerstmann-Sträussler-Scheinker disease (26). Two cell lines of epithelial and microglial tissue origin were reported to propagate mouse-adapted and vole-adapted bovine spongiform encephalopathy (BSE) agents, respectively (9, 16), and a fibroblast-like transformed cell line generated from the brain of a mule deer has been shown to propagate the agent of chronic wasting disease (24).

The outbreak of variant Creutzfeldt-Jakob disease (vCJD) consequent to infections with BSE, together with the occurrence of four secondary transmissions from transfusions of nonleukoreduced red blood cells (11, 13), has raised concerns about the safety of blood and its derivative products and, despite negative epidemiological evidence, about the possibility of similar transmissions from other forms of CJD.

The accumulation of PrP^TSE, the disease-associated, pathologically modified form of cellular prion protein (PrP^C), and evidence of its infectivity in the lymphoreticular tissues of vCJD patients have been documented (3, 14, 15). Transmission studies have also shown that vCJD readily infects various strains of conventional mice, unlike sporadic CJD, which rarely causes infection (30), and that the lymphoreticular tissues of vCJD agent-infected mice accumulate both PrP^TSE and infectivity (5). We have previously reported that SJL/OlaHsd (Ola) mice, closely related to SJL/J strain mice, which develop spontaneous B-cell lymphomas at an older age (22, 25), accumulate significant amounts of PrP^TSE in their spleens following infection with the mouse-adapted vCJD (mo-vCJD) agent (6). We now report success in establishing murine spleen-derived stromal cell cultures that persistently propagate mo-vCJD and Fu agents.

An Ola female mouse developed multiple spontaneous tumors involving the lymph nodes, Peyer's patches, and spleen. The mouse was euthanized by exsanguination while under anesthesia at 540 days of age, and the spleen was aseptically removed and used for the preparation of a primary cell culture. A homogeneous cell suspension was prepared by the dissociation of cells from the tissue. Continuous growth of adherent spleen stromal cells (SP-SC) was established by a 1:3 split twice per week for nearly 8 weeks, and the cells became spontaneously immortalized within the 8 weeks. All experiments were performed using IMDM with HEPES and L-glutamine (Lonza Walkersville) supplemented with 10% fetal bovine serum, 5% recombinant mouse interleukin-3-conditioned medium from X63Ag8-653 myeloma cells (17), 10% BIT9500 (Stemcell Technologies), 50 µM 2-mercaptoethanol (Invitrogen), 5 ng/ml sodium selenite (Sigma-Aldrich), and penicillin-streptomycin. The immortalized cells displayed a fibroblast-like appearance, but with some distinctive features, such as less-well-developed stress fibers, a less-polygonal shape, and a large number of well-developed filopodia. Based on these observations, we concluded that the morphological features of SP-SC resemble the typical morphology of bone marrow-derived mouse mesenchymal stem cells (21). To test the survival of immortalized SP-SC in the presence of brain homogenate, the culture was inoculated with a 0.1% brain suspension prepared from a healthy mouse and was propagated as described above. At passage ten, the SP-SC culture became transformed (tSP-SC). The tSP-SC displayed less spread (10-fold) on plastic (Fig. 1A) and lost contact inhibition. The rate of cell division increased, and the tSP-SC required splitting 1:20 twice per week. A soft-agar transformation assay confirmed the transformed status of the culture (data not shown). All further work was performed using the tSP-SC culture.

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FIG. 1. Phenotypic characterization of stromal cell culture derived from spleen of an SJL/Ola mouse. (A) Phase-contrast image of cell morphology in spontaneously transformed culture. Scale bar, 50 µm. (B) Expression of PrPC and Sca1 on transformed cell culture as shown by flow cytometry using FITC-labeled 6D11 and Ly-6A/E antibodies, respectively. FITC-labeled mouse immunoglobulin G2a (IgG2A) and rat IgG2a represent isotype controls for 6D11 and Sca1, respectively. (C) Expression of PrPC by transformed cell culture before and after cryopreservation, as shown by an immunoblot of cell extracts developed by using an 6D11 antibody. Di-, mono-, and unglycosylated isoforms are indicated by arrows. The molecular mass standard in kilodaltons is shown on the left.

Stromal cell cultures from murine lymphoid organs are thought to constitute a heterogeneous cell population consisting of fibroblasts, endothelial cells, macrophages, and dendritic cells, present in various proportions, and a small population of hematopoietic stem cells (HSC) (19). We performed an immunophenotypic characterization of a tSP-SC culture by using a FACSCanto flow cytometer and FACSDiva software (BD Biosciences) with either fluorescein isothiocyanate (FITC)- or phycoerythrin-conjugated antibodies against the cell surface antigens listed in Table 1. The analysis revealed some variations in the cell subpopulations due to the heterogeneity of the tSP-SC culture (Table 1), but in repeated experiments, the major proportion of the cells was always positive for PrP^C (Fig. 1B, left panel), and a significant number of cells was steadily positive for stem cell antigen 1 (Sca1) (Fig. 1B, right panel), a marker of multipotent HSC, and CD13, which is expressed on a variety of cells, including mast cells, monocytes, macrophages, immature dendritic cells, endothelia, and brain cells. All cells in the culture exhibited high levels of CD44 glycoprotein, one of the most frequently identified markers of stromal cells. The greatest fluctuation was observed in the distribution of cell populations expressing HSC/mesenchymal stem cell marker CD90.1 (18) and the HSC/progenitor cell marker CD34. Constantly present was a small population of cells positive for the B-cell lineage marker CD45R/B220. A negligible proportion of cells was positive for the myeloid cell markers CD11b and Gr-1 or dendritic cell marker CD11c, and no cells tested positive for the endothelial cell markers CD31, CD106, and Flk1 or the cell marker of mature dendritic cells, CD86. Our data on the characterization of tSP-SC subpopulations are consistent with those of Peister et al. (21), who reported on the variability of hematopoietic CD34, Sca1, and CD106 cell populations in adult stem cells from the bone marrows of various strains of inbred mice.

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TABLE 1. Expression of cell surface markers on murine spleen stromal cells as determined by FACS analysis

We have shown that PrP^C is expressed on the surfaces of tSP-SC, a requirement for the support of TSE replication in vitro. In order to confirm that tSP-SC express steady-state levels of PrP^C through multiple passages and to explore the effect of cryopreservation, cells that were either continuously propagated from the original culture or recovered after cryopreservation were collected at various passages after growing to a confluent monolayer. The amount of total PrP^C in tSP-SC remained at high levels during cell propagation through more than 50 passages following immortalization, and cryopreservation had no effect on its levels, as determined by Western blotting (Fig. 1C). The PrP^C extracted from the cells showed a characteristic three-glycoform pattern in which the diglycosylated form of the protein was dominantly represented over the monoglycosylated and unglycosylated forms. We observed no change in the molecular weight of the PrP^C nor in the glycosylation profile during multiple passages of the tSP-SC culture.

To determine the susceptibility of tSP-SC to infection, we inoculated the cells with a clarified 1% brain suspension diluted with Opti-MEM, which was prepared from the brains of healthy mice (negative controls) or terminally ill mice infected intracerebrally with either the mo-vCJD agent or Fu, as described elsewhere (29), and monitored the presence of PrP^TSE at 72 h and at various passages following inoculation. In three independent experiments, we were able to infect tSP-SC with Fu, and in one experiment, with the mo-vCJD agent, and the infected cell cultures continued to propagate PrP^TSE through over 50 (Fu) and 30 (mo-vCJD) passages, as demonstrated by Western blotting (Fig. 2) using anti-PrP-specific monoclonal 6D11 antibodies (20). It is well established that the misfolded proteins of various TSE strains are characterized by distinct patterns of three glycosylated isoforms after being digested proteolytically with proteinase K (PK). Our data confirmed the difference in migration profiles of core fragments of PrP^TSE resistant to PK (PrPres) in the Fu and mo-vCJD strains. Although the PrPres from mo-vCJD agent-infected cells had a lower molecular weight than the PrPres from cells infected with Fu, the ratios of glycoforms remained similar and consistent with our previous observation for PrPres extracted from the brains of infected mice (5). Cryopreservation of tSP-SC had no effect on the ability of cells to produce PrP^TSE. Transmission studies using experimental mice are under way to confirm our expectation that the formation of PrP^TSE in tSP-SC cultures correlates with the presence of infectivity. Multiple studies showing the formation of PrP^TSE in persistently infected cell cultures in close association with the level of infectivity argue that our cell cultures should be infectious as well.

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FIG. 2. Immunoblot of murine tSP-SC cultures uninfected (Control) or persistently infected with 1% Fu or 1% mo-vCJD. The presence of PrPTSE in cell lysates at various passages was confirmed by the treatment of samples with 5 µg/ml of PK. The PK-treated samples (+) loaded into the gel are fivefold more concentrated than samples not treated with PK (–). The membranes were developed by using 6D11 antibodies (0.2 µg/ml). Three glycoforms of PrP are present as bands corresponding to di-, mono-, and unglycosylated isoforms. Note the differences in the glycoform profiles of PrPres in the Fu- and mo-vCJD-infected samples. The molecular mass standard in kilodaltons is shown on the left.

Although it remains to be seen whether a developed tSP-SC culture is susceptible to a broad range of TSE agents, including the scrapie, BSE, and chronic wasting disease agents and various human CJD isolates, our data provide clear evidence that tSP-SC with features of mesenchymal stem cells are susceptible to TSE infection and can persistently propagate the mo-vCJD agent and Fu through multiple passages. In future studies, we aim to determine more precisely the identity and distribution of the cell population(s) responsible for propagating the infectious agents, with special attention to any involvement of Sca1- and Thy1.1-positive cells, and to examine whether interactions between different cell types are necessary for efficient propagation.

We propose that the tSP-SC culture, because of its splenic origin, represents a particularly appropriate ex vivo model to study the early events of infection at the cellular level. In addition, tSP-SC cultures persistently infected with mo-vCJD and Fu agents might serve as better models than previously developed cell cultures for both diagnostic and therapeutic assays and for validation studies of the methods used to remove vCJD from blood, plasma-derived products, and other biologics. Finally, our results should alert the clinical research community to the potential for transmitting CJD infection in the course of stem cell therapy.

 
We are grateful to Gordon Keller for providing the interleukin-3-producing X63Ag8-653 myeloma cells, Paul Brown, Suzette Priola, and Robert Hawley for critical readings of the manuscript, and Donna Sobieski for editorial corrections.

 
* Corresponding author. Mailing address: Transmissible Diseases Department, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Phone: (301) 738-0765. Fax: (301) 738-0495. E-mail: cervenakl{at}usa.redcross.org

Published ahead of print on 20 August 2008.

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Journal of Virology, November 2008, p. 10959-10962, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.01085-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Akimov, S. Articles by Cervenakova, L. Search for Related Content PubMed PubMed Citation Articles by Akimov, S. Articles by Cervenakova, L.