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发表于 2009-3-5 10:50 |只看该作者 |倒序浏览 |打印

a Department of Obstetrics and Gynecology, Rambam Medical Center, Faculty of Medicine, The Technion, Haifa, Israel;0 {! q& l' R6 p5 [7 {
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b Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany;
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c Robert Koch Institute, Berlin, Germany
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Key Words. Embryonic stem cells ? Murine leukemia virus ? Feeder cells

Correspondence: Ulrich Martin, Ph.D., Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Podbielskistr. 380, 30659 Hannover, Germany. Telephone: 49-511-906-3533; Fax: 49-511-906-3569; e-mail: martin.ulrich@mh-hannover.de

ABSTRACT
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Novel technologies of cell transplantation and tissue engineering will help to overcome current therapeutic limits and may enable improved treatment of life-threatening diseases such as Hunting-ton’s disease or myocardial infarction. For those upcoming technologies, one major prerequisite is suitable cell sources such as human embryonic stem cells (hESCs).
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Until recently, it was not possible to isolate and culture hESCs without support of so-called feeder cells, usually mouse embryonic fibroblasts (MEFs). These mouse-derived embryonic cells are necessary to supply factors that support undifferentiated growth and expansion of hESCs. Although it is now known that certain human cell types, including fetal and neonatal fibroblasts, support undifferentiated proliferation of hESCs  and activation of the canonical Wnt-pathway has been proposed to maintain self-renewal of hESCs , most hESC lines that have been registered (http://stemcells.nih.gov/registry/index.asp) have been in long-term contact with murine feeder cells.
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The continuing close contact of hESCs to MEFs poses the risk of transmission of pathogens from one species to another, and clinical application of such cells may result in xenozoonoses. Mice harbor a variety of parasites, bacteria, and viruses potentially pathogenic for humans. Special attention should be paid to endogenous retroviruses in the mouse genome. Similar to all other vertebrates, mice contain a variety of retroviral elements in their genome. Most of these retroviral elements are defective and do not lead to release of infectious retroviral particles. Nevertheless, it is well known that a variety of mouse strains contain functional intact viruses. These viruses cannot be eliminated by simple specified pathogen-free animal housing or gnotobiotic breeding.

Some endogenous retroviruses are able to infect foreign species, and some cause diseases, including leukemias, neuropathological effects, and immunodeficiencies, in mice and immunosuppressed primates . Murine endogenous retroviruses, especially mouse mammary tumor virus–related B-type retroviruses and C-type retroviruses of the murine leukemia virus (MuLV) group, are the most extensively investigated endogenous viruses.
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Because of the host range and potential pathogenicity of MuLVs, we focused on the analysis of potential MuLV transmission to hESCs. The dissemination of retroviral elements of the MuLV type in mice has been investigated extensively, in particular in a variety of inbreed strains. Most inbreed strains contain 40 to 60 copies per genome . Based on the receptor and host specificity, MuLVs can be arranged within at least six groups : MDEV (Mus dunni endogenous virus), 10A1, ecotropic MuLV (not able to infect other cells than murine), amphotropic MuLV (capable of infecting many species, including mice), xenotropic MuLV (able to infect foreign species but not mice), and polytropic MuLV (able to infect a limited number of species only). Potentially humantropic and therefore relevant for this study are xenotropic, polytropic, and amphotropic MuLVs. Moreover, one has to keep in mind that other endogenous retrovirus-like elements, which are not humantropic or not completely functional, can be transmitted to human cells via humantropic MuLV virions .: }. R; g/ v+ b" x
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Xenotropic MuLVs are present in different mouse strains . Xenotropic proviruses have been detected in all strains of Swiss mice analyzed so far . Swiss mice also include ICR (Institute of Cancer Research) mice, which are commonly used to isolate feeder cells. Whether this strain releases infectious xenotropic MuLVs has not been analyzed. At least in some animals of an ICR/Ha colony, an ecotropic, leukemia-inducing MuLV clone designated 334C leukemia virus has been isolated .) ?/ \% @/ [& t, E

Polytropic MuLVs have been isolated and described somewhat later as their xenotropic relatives ; their dissemination has been analyzed in less detail, as in the case of xenotropic MuLVs.
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Amphotropic strains have been isolated initially from wild Californian mice  and are not naturally found in common laboratorystrains. However, exogenous infections of the mice strains used to prepare feeder cells cannot be excluded completely. Modern retroviral vectors are based on amphotropic MuLVs.+ M8 y. B: W; q% S. u
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Whereas MuLV usually replicate efficiently in differentiated somatic cells, there is some evidence that MuLV expression in embryonic cells is depressed . So far, it has not been investigated whether MuLV expression is inhibited in embryonic/fetal fibroblasts.
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The MuLV types described above differ particularly in the sequence of the envelope protein env, which is responsible for binding to the specific cellular receptor . Human receptors for amphotropic MuLVs  and xenotropic/polytropic  MuLVs recently have been cloned from HL-60 cells and T lymphocytes. Many adult and fetal tissues express the receptor for xenotropic/polytropic MuLV, at least at the mRNA level , whereas high levels of the amphotropic receptor can be found on different hematopoietic stem cells . Whether these receptors are expressed on hESCs has not been analyzed hitherto.

The aim of this study was to determine whether embryonic fibroblasts of mouse strains, which are usually used to prepare feeder cells, release xenotropic, polytropic, or amphotropic MuLVs and whether hESCs, which have been grown for extended time on MEFs, have been infected by humantropic MuLV.  w" i3 E; j& A: T) q

MATERIALS AND METHODS  B8 _5 q; ~1 i5 ~. b

MEF Cells Express Xenotropic and Polytropic MuLVs

RT-PCRs specific for MuLV pol as well as for xenotropic, poly-tropic, and amphotropic env genes of MuLVs were performed using total RNA of MEFs isolated from different mouse strains. Analysis of ecotropic MuLV expression has not been included, because those viruses are not able to infect human cells. Because most groups working with hESCs use feeder cells isolated from ICR mice or during genetic modification of embryonic stem (ES) cells, also from ICR mice crossbred with neomycin-resistant 129neo or fourfold-resistant DR4 mice, we analyzed those strains for MuLV expression. As depicted in Figure 2, strong RNA expression of xenotropic and polytropic MuLV was detected in MEFs, whereas no expression of amphotropic MuLV could be demonstrated. As expected, also no proviral sequences for amphotropic MuLV could be detected in the tested MEF cultures by PCR (data not shown).
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Figure 2. Xenotropic and polytropic MuLVs are expressed on the mRNA level in MEFs. Reverse transcription–polymerase chain reaction specific for xenotropic (xeno.) and polytropic (poly.) MuLV was performed on RNA of embryonic fibroblasts of different mouse strains frequently used to produce MEFs for human embryonic stem cell culture. pBlueAmpho, DG75 cells, and MCF13 cells were positive controls. Internal controls without RT excluded false-positive results due to contaminating genomic DNA. Abbreviations: ampho., amphotropic; MEF, mouse embryonic fibroblast; MuLV, murine leukemia virus; RT, reverse transcriptase.: G) U: E+ _- ^) P$ S, x3 a9 y- v
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Because cellular MuLV RNA expression does not necessarily imply release of viral particles, RNA was also prepared from cell culture supernatants. RT-PCR performed on these samples confirmed the results of RT-PCRs performed on cellular RNA (Fig. 3). Further evidence for release of MuLV particles was obtained by an enzymatic assay capable of detecting RT activity as a retroviral marker in cell culture supernatants. In all tested cell supernatants of cultured MEFs, RT activity was detected (Fig. 4).  v! b' Y  m$ |7 D) j; r. t( D

Figure 3. RNA of xenotropic and polytropic MuLV as a marker for MuLV particles can be detected in cell culture supernatants of MEFs. Reverse transcription–polymerase chain reaction specific for xenotropic (xeno.) and polytropic (poly.) MuLV was performed on RNA isolated from cell culture supernatants of embryonic fibroblasts of different mouse strains. pBlueAmpho, DG75 cells, and MCF13 cells were positive controls. Internal controls without RT excluded false-positive results due to contaminating genomic DNA. Abbreviations: ampho., amphotropic; MEF, mouse embryonic fibroblast; MuLV, murine leukemia virus; RT, reverse transcriptase.5 }# {3 y# J) S% s5 x/ m6 k
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Figure 4. Detection of reverse transcriptase activity in cell culture supernatants of mouse embryonic fibroblasts (MEFs) as a marker for retroviral particles. Detection of reverse transcriptase activity was performed using a polymerase chain reaction–based reverse transcriptase assay. Avian myeloblastosis virus (AMV) reverse transcriptase was used as positive control; HEK293 cells were used as a negative control.
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Expression of Receptors for Xenotropic/Polytropic MuLVs on hESCs

Besides release of infectious virions, expression of specific receptors is a second prerequisite for productive MuLV infection of human cells in coculture settings. We have therefore analyzed mRNA expression of human receptors for xenotropic and polytropic MuLVs on human HEK293T cells as positive control and on several hESC lines. Human HEK293T cells expressed mRNA for the receptor for xenotropic/polytropic MuLV. A strong mRNA expression of this receptor was also found on all tested hESC lines (H9, I4, and I6) (Fig. 5).

Figure 5. The receptor for xenotropic and polytropic MuLV is expressed on human HEK293T cells and hESCs. Total RNA of human HEK293 cells and different hESC lines was tested by reverse transcription–polymerase chain reaction specific for the human receptor for xenotropic and polytropic MuLV. Internal controls without RT excluded false-positive results due to contaminating genomic DNA. Abbreviations: hESC, human embryonic stem cell; MuLV, murine leukemia virus; RT, reverse transcriptase.: g0 Q4 b4 Y; Z

MuLV Infection of HEK293T Cells and hESCs After Coculture with MLV-X; Y1 `5 w0 L; i8 n" q

Because receptor expression is not ample evidence for susceptibility, infection experiments using the mink cell line MLV-X, which releases infectious xenotropic MuLV, were performed. To facilitate analysis, MLV-X was pretreated with mitomycin C. After initial culture with mitomycin-treated MLV-X, HEK293 cells were cultivated for an additional eight passages to eliminate MLV-X cells and DNA. In cases of hESCs, human cells were cultured on a 1:1 mixture of mitotically inactivated MEFs and MLV-X before being cultured for eight passages on human feeder cells, which was necessary to exclude false-positive results based on MEF/MLV-X contaminations. Eight passages after contact with MLV-X cells, MuLV proviral sequences could be demonstrated in HEK293 cells and in hESCs (Fig. 6). Moreover, robust MuLV mRNA expression was detected in hESCs after coculture with MLV-X, suggesting that in contrast to murine embryonic carcinoma cells , MuLV expression is not significantly repressed in hESCs on the transcription level (Fig. 6).8 H; o5 L" k9 U' k2 u  b! o8 d
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Figure 6. Productive infection of human HEK293 cells and hESCs after coculture with a mink cell line releasing infectious xenotropic MuLV. HEK293/hESCs, which had been cocultured for one passage with mitomycin-treated MLV-X, a mink cell line releasing infectious xenotropic MuLV, have been cultured for at least seven passages without further contact to mouse embryonic fibroblasts or MLV-X before preparation of genomic DNA or total RNA. Results of MuLV pol–specific PCR and, in case of hESCs, RT-PCR are depicted. Internal controls without RT excluded false-positive results due to contaminating genomic DNA. Abbreviations: hESC, human embryonic stem cell; MuLV, murine leukemia virus; RT-PCR, reverse transcriptase–polymerase chain reaction.

Coculture of HEK293T Cells with MEFs Does Not Result in MuLV Infection

Although productive MuLV infection of hESC could be demonstrated after coculture with MLV-X, and although in the culture supernatant of MEFs, MuLV-RNA and RT activity as markers for virion release could be demonstrated, only coculture with susceptible human cells can prove the release of significant titers of replication-competent humantropic MuLV. To investigate potential productive infection of susceptible human cells, mitomycin-treated MEFs were cocultured with human HEK293T (Fig. 7) as well as with HEK293 cells (data not shown). Cocultures were continued until no murine DNA could be detected (passage 7). Infection of the human cells by MEF-derived MuLV was not observed: MuLV proviral sequences and MuLV RNA were not detected at passage 7 or 8 (Fig. 7).
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Figure 7. No infection of human HEK293T cells after coculture with MEFs. Mitomycin-treated MEFs were cocultured with human HEK293T cells. Cocultures were tested after seven or eight passages by MuLV pol–specific PCR/RT-PCR, which did not result in any evidence for MuLV infection. False-positive results due to contaminating murine DNA/RNA were excluded by murine mtDNA (CyO II)–specific PCR. Internal controls without RT excluded false-positive results due to contaminating genomic DNA. GAPDH was an internal positive control. MEFs served as positive control for all PCRs. Noninfected HEK293T cells were negative control. Faint bands visible in the upper left panel have a size below 100 bp and most likely represent surplus primers. PCR sensitivity: MuLV pol, detection of one MEF cell within 105 to 106 human cells; murine mtDNA: detection of one MEF cell within 106 to 107 human cells. Abbreviations: CyO, cytochrome oxidase; MEF, mouse embryonic fibroblast; MuLV, murine leukemia virus; RT-PCR, reverse tran-scriptase–polymerase chain reaction.; j6 O% O  z% H% C

No Evidence for MuLV Infection of hESC Lines After Sustained Contact with MEFs. [: ^, x% e" {; K* ?: U4 s# X
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Four hESC lines were analyzed for proviral MuLV sequences by quantitative real-time PCR. To minimize contaminations with murine cells or murine DNA, not only hESCs on MEFs but also hESCs, which had been cultivated for at least five passages in the absence of MEFs, were analyzed. As demonstrated in Table 1, cultivation on MEF matrix, and even more on matrigel or on human feeders, indeed strongly reduced contaminating murine DNA, resulting in significantly increased threshold cycles during real-time PCR. All samples tested resulted in ratios of  of approximately 1. For several samples, no ratio was obtained because the amount of MuLV pol template was below the detection limit. Provided that the actual CTpol was at least 31.84 for these samples, the obtained ratios would still be in a range of 1 or greater than 1. Because ratios of approximately 1 or greater than 1 exclude productive MuLV infection, our data clearly demonstrate that none of the tested hESC lines has been productively infected by MEF-derived MuLV. Further evidence for the absence of a productive MuLV infection has been obtained by analyzing MuLV expression and release in hESC cultures. Neither cellular MuLV RNA (Fig. 8) nor RT activity in culture supernatants (Fig. 9) was detected in the hESC lines I3, I6, I8, and H9 after several passages on human feeder cells.

Figure 8. No evidence for productive MuLV infection of hESCs after sustained culture on murine feeder cells by MuLV pol–specific RT-PCR. RT-PCR specific for MuLV pol was performed on RNA of hESCs cultured for at least seven passages on human foreskin fibro-blasts. The obtained results confirm the results of the quantitative real-time PCR assay. GAPDH was used as positive control. In parallel, all samples were spiked with MEF DNA for internal positive control. Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast; MuLV, murine leukemia virus; RT-PCR, reverse transcriptase–polymerase chain reaction.
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Figure 9. No release of RT activity in culture supernatants of hESCs. C-type RT activity in supernatants of hESCs, cultured for at least seven passages on human foreskin fibroblasts (huF10), has been determined using a commercial enzyme-linked immunoabsorbent assay–based assay. Culture supernatants of MEFs, HEK293T, and huF10 have been used as controls. Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast; RT, reverse transcriptase.

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We wish to thank Victoria Margulets and Yael Miropolski for technical assistance. DG75, MCF13, and MLV-X cells and pBlueAmpho were kindly provided by Dr. Y. Takeuchi, Wohl Virion Center, Windeyer Institute, University College London. ICRx129neo- and ICRxDR4-derived feeder cells were a gift from N. Benvenisty, Hebrew University, Jerusalem. This research was partly supported by NIH grant 1R24RR018405-01 (J.I.). In accordance with the German Stem Cell Act, no hESCs were cultured by the German researchers; instead, supernatants, DNA, and RNA of the analyzed hESCs were provided by the Israeli collaborators./ A  m# J7 q2 [/ k$ I

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