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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES |
1-3Galß1-4GlcNAc-R (-Gal) Epitope
Department of Gene Function and Development, Roslin Institute, Roslin, Midlothian, United Kingdom
Key Words. Regenerative medicine • Selective ablation • Human embryonic stem cells • Stem cell therapy Complement-mediated lysis • Gal 
1,3-galactosyltransferase
Correspondence: Jim McWhir, Ph.D., Roslin Institute, Roslin, Midlothian, EH25 9PS, United Kingdom. Telephone: +44 131 527 4334; Fax: +44 131440 0434; e-mail: jim.mcwhir{at}bbsrc.ac.uk
Received September 30, 2005;
accepted for publication August 29, 2006.
First published online in STEM CELLS EXPRESS September 7, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Gal1-3Galß1-4GlcNAc-R (-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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The organs of nonprimate mammals transplanted into humans are rejected within a few hours or minutes because of the presence of -gal epitopes. Human serum normally contains high levels of circulating antibody to these epitopes [3, 4], due to a fixed stop mutation at the human 1,3-galactosyltransferase (GalT) locus (the product of which catalyzes -gal presentation), rendering us immunoreactive to dietary and gut flora sources of the epitope. We have engineered hESCs that express a functional copy of the GalT gene under the transcriptional control of the human telomerase reverse transcriptase (hTERT) promoter (Fig. 1A), normally active only in blastomeres, undifferentiated ESCs, and germ cells [5]. We show that exposure of undifferentiated cells to active human serum leads to cell death in vitro. However, when exposure of the same cell line follows a period of differentiation, the majority of hESC-derived cells are resistant. These data demonstrate an efficient method for removal of undifferentiated cells prior to engraftment and suggest that ongoing in vivo surveillance against residual undifferentiated cells may be effective in their continued elimination.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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Cell Culture and Transfection
H9 hESCs were a generous gift from Geron Corporation. hESCs were cultured as described previously by Xu et al. [7] on growth factor-reduced Matrigel substrate (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) in mouse embryonic fibroblast-conditioned hESC medium (knockout Dulbecco's modified Eagle's medium [DMEM] and 20% knockout serum replacement supplemented with 2 mM L-glutamine, 1x nonessential amino acids, and 100 µM ß-mercaptoethanol; Invitrogen) with 4 ng/ml recombinant human basic fibroblast growth factor (bFGF) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The cells were passaged using TEG (92.7 mM NaCl, 0.845 mM Na2HPO4, 1.58 mM KH2PO4, 4.46 mM KCl, 5 mM D-glucose, 22.28 mM Tris-HCl, 0.0009% phenol red, 0.25% trypsin, 1.05 mM EGTA, and 0.000105% polyvinylalcohol).
Exponentially growing cells were used for transfection as previously described [8]. They were rendered into a single-cell suspension by treatment with TEG and swollen in hypoosmolar buffer (Eppendorf, Hamburg, Germany, http://www.eppendorf.com) at room temperature for 20 minutes. One x 106 cells were then electroporated with 50 µg of linearized mGT or mfGT expression vector in a volume of 800 µl of hypoosmolar buffer in a 0.4-cm gap cuvette (Bio-Rad, Hercules, CA, http://www.bio-rad.com) and pulsed at 300 V for 100 µS in a Multiporator (Eppendorf). Cells were left at room temperature for 10 minutes and then plated onto a matrigelled 15-cm plate in conditioned hESC medium. After 48 hours, 100 µg/ml G418 was applied and maintained until colonies appeared after 10 days of selection. Colonies were physically picked up with a micropipetter into 48-well plates and expanded.
Fluorescence Microscopy for the Cell Surface -Gal Epitope
Unfixed cells in a tissue culture well were stained with fluorescein isothiocyanate (FITC)-conjugated Bandeiraea simplicifolia Isolectin B4 (BS-IB4) (Sigma-Aldrich), which specifically binds the -gal epitope [9]. Cells were washed with phosphate-buffered saline (PBS) (with Ca2+ and Mg2+) and incubated at 4°C for 90 minutes in 5 µg/ml BS-IB4-FITC in PBS (with Ca2+ and Mg2+) with 10% newborn calf serum. Cells were then washed twice for 15 minutes with PBS at room temperature and analyzed in PBS in the tissue culture well with a fluorescence microscope.
Karyotyping
Exponentially growing cultures were arrested in metaphase using Karyomax colcemid solution (Invitrogen) at 100 ng/ml for 2 hours at 37°C. Cells were disaggregated by treatment with TEG, pelleted at 200g, and resuspended in 0.56% potassium chloride for 10 minutes at room temperature. Cells were pelleted and resuspended in a fixative of three parts methanol, one part acetic acid. Cells were then washed twice with fixative by pelleting and resuspending. Cells in fixative were dropped onto a slide and dried for 3 days. Resulting chromosome spreads were washed in x2 SSC at 60°C for 2 hours and partially digested with 0.01% Bacto trypsin (Difco Labs, Oxford, U.K., http://www.bdbiosciences.com) before being stained with 5% Gurr's improved Giemsa R66 (BDH, Lutterworth, U.K., http://www.vwr.com) in Gurr's phosphate buffer.
In Vitro Differentiation of hESCs
hESCs were disaggregated with TEG and seeded at high density into a nonadherent bacterial Petri dish in conditioned hESC medium. After 2 days, the medium was changed to differentiation medium (knockout DMEM [Invitrogen], 10% FBS [Globepharm, Surrey, U.K.], 2 mM L-glutamine [Invitrogen], 1x nonessential amino acids [Invitrogen], and 100 µM ß-mercaptoethanol [Invitrogen]). After a further 5 days of suspension culture, the resulting aggregates (embryoid bodies) were plated onto gelatin in differentiation medium and allowed to reattach to the culture surface. Differentiation was allowed to proceed for a further 2 weeks. Alternatively, hESCs were spontaneously differentiated as monolayers by removing conditioned hESC medium and bFGF and replacing it with differentiation medium, as described above, for a specified time scale (0–22 days).
Neural Differentiation of hESCs
hESCs were differentiated into neural progenitors in accordance with the protocol of Gerrard et al. [10]. Briefly, hESCs at near confluence were passaged with 0.5 mM EDTA/PBS at a ratio of 1:5 onto tissue culture plastic coated with poly(L-lysine)/laminin (Sigma-Aldrich). These cells were then cultured in N2B27 medium (1:1 mix of DMEM/F-12 supplemented with N2 and Neurobasal medium supplemented with B27 all from Invitrogen) in the presence of 100 ng/ml mouse recombinant noggin (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) for 3 weeks [10].
Immunofluorescence
In all cases, differentiated cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes. When intracellular staining was performed, cells were washed a further two times with PBS and permeabilized with absolute ethanol at room temperature for 2 minutes, followed by a further two PBS washes. Nonspecific protein binding was blocked with 10% normal goat serum for 1 hour at room temperature. Primary antibodies were bound to their antigens in PBS with 1% normal goat serum plus 0.02% Triton-X for 1–2 hours at room temperature. Antibody dilutions were as follows: monoclonal anti-ß-tubulin III (Sigma-Aldrich) at a 1:200 dilution, monoclonal muscle-specific actin (Dako Cytomation, Glostrup, Denmark, http://www.dakocytomation.com) at a 1:50 dilution, monoclonal anti--fetoprotein (Sigma-Aldrich) at a 1:500 dilution, monoclonal anti-human specific nestin (Chemicon International, Temecula, CA, http://www.chemicon.com) at a 1:200 dilution, and polyclonal anti-human musashi (Chemicon International) at a 1:200 dilution. Unbound antibody was removed by three 5-minute room temperature washes with PBS. FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich), Alexa Fluor 568-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen) were used as secondary antibodies at dilutions of 1:200 (Sigma-Aldrich) and 1:400 (Invitrogen) in PBS with 1% normal goat serum incubated at room temperature for 30 minutes, and unbound antibody was washed away as before. Coverslips were mounted in Vectashield with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and viewed with a fluorescence microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).
Formation of Tumors in SCID Mice and Histological Analysis
SCID mice were obtained from Harlan UK Limited (Bicester, Oxon, U.K., http://www.harlanseralab.co.uk) and maintained in a sterile environment. hESCs for injection into SCID mice were disaggregated by treatment with TEG, washed once in PBS, and resuspended in PBS at 1 x 108 cells per milliliter. A 100-µl aliquot was injected into the leg muscle of each of three SCID mice.
Three to five months later, the mice were sacrificed, and the tumors were removed. Tumors were fixed whole or, if very large, cut into segments. Fixation was by treatment with 4% PFA for 20 minutes at room temperature. Tumors were embedded in paraffin wax in an automated system using a Shandon Hypercenter XP processor (Shandon Scientific UK Ltd., http://www.thermo.com). Once embedded, tumors were cut into 10-µm sections using a Microm HM325 rotary microtome (MICROM International GmbH, Walldorf, Germany, http://www.microm-online.com) and stained using standard hematoxylin and eosin protocols. Tumor sections were analyzed and photographed by Dr. David Brownstein (University of Edinburgh, Scotland, U.K.).
Lysis of Cells with Human Serum
Human sera from healthy screened volunteers of blood group A was either purchased from Harlan Sera-Lab Limited (Loughborough, Leicestershire, U.K., http://www.harlanseralab.co.uk) as a pooled sample or collected fresh and pooled from blood group A healthy volunteers from the Roslin Institute. Blood group B serum was purchased from Harlan Sera-Lab Limited.
Where required, heat inactivation of complement in serum was achieved by heating to 56°C for 30 minutes. Cells were disaggregated with TEG, pelleted (200g), resuspended at 1 x 105 cells per 0.1 milliliter per replicate in undiluted human serum, and incubated for 1 hour at 37°C. Experiments were performed in triplicate. Disaggregation with TEG generated robust single-cell suspensions with, on average, 93% viability as determined by flow cytometry for propidium iodide (PI) inclusion (data not shown).
Calcein-Release Assay
A calcein-release assay was used to measure cell lysis, based upon the methods of Spiller [11] and Iwanowicz et al. [12]. Calcein AM (Invitrogen) is weakly fluorescent and nonpolar and is readily taken up into cells where the acetoxymethyl ester group is cleaved, leaving fluorescent, polar calcein trapped in the cell. Cells were incubated with Calcein AM in conditioned hESC medium at 40 µg/ml for 2 hours at 37°C to load the cells. For lysis in suspension, all pelleting of cells was performed at 260g to ensure a robust pellet and no carry-over of cells in the supernatant. One x 105 cells were loaded with Calcein AM in a volume of 50 µl, and cells were washed with knockout DMEM prior to lysis in 100 µl of active or heat-inactivated serum for 1 hour at 37°C. As controls, cells were also incubated with either 100 µl of conditioned hESC medium as a measure of spontaneous release or 0.1% Triton X-100 as a measure of total release. A 50-µl sample of the supernatant after pelleting of unlysed cells was transferred to a black 96-well assay plate containing 50 µl of 0.2% Triton X-100. Cells were then resuspended in 100 µl of 0.1% Triton X-100 and incubated for 15 minutes at 37°C to lyse intact cells. A 50-µl sample of lysate was transferred to an assay plate as before. Fluorescence was measured using a Victor [2] plate reader (PerkinElmer Wallac, Boston, http://www.perkinelmer.com) with an excitation filter for 485 nm and an emission filter for 535 nm. Calcein release was calculated as the fluorescence in the serum lysate as a percentage of the fluorescence in the sum of the fluorescence in the serum lysate and Triton lysate.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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-gal epitope [9]. The epitope was clearly detected on four of the mGT clones and eight of the mfGT clones. However, there was no obvious improvement in expression level associated with the use of the mfGT version of GalT, and all mfGT clones analyzed showed variegated patterns of expression.
One mGT clone, M2, was selected for further analysis due to its low level of variegation and was compared with a more variegated mfGT clone, F11. Near ubiquitous GalT expression in M2 cells and variegated expression in F11 cells is illustrated by lectin binding in Figure 1. GalT expression in M2 cells (Fig. 1Ce, 1Cf) was similar to that observed with a positive control ovine fetal fibroblast cell line (PDFF) (Fig. 1Cc, 1Cd), and consistent with their dysfunctional GalT genotype, H9 cells showed no detectable level of expression (Fig. 1Ca, 1Cb). Cytometric analysis of the same populations (Fig. 1B) confirmed that 95.40% of the cells in the M2 cell line expressed the -gal epitope (mean fluorescence intensity of 478.29 compared with 615.27 for PDFF), whereas only 84.74% of the cells expressed in the more variegated F11 population (mean fluorescence intensity 395.96).
The specificity of isolectin BS-IB4 binding was confirmed by costaining with anti--gal antibody. Flow cytometry analysis for costaining confirmed that 93.8% of M2 cells both expressed GalT and stained with the isolectin BS-IB4 (supplemental online Fig. 1C). As expected, the variegated clone F11 showed two subpopulations, one costaining with both reagents and the other negative with both reagents. Few cells were stained for only one marker (supplemental online Fig. 1D), further confirming the specificity of the isolectin staining.
GalT-Expressing hESCs, but Not Nonexpressing hESCs, Are Lysed by Exposure to Human Serum
To investigate their sensitivity to human serum, M2, F11, positive control PDFF, and negative control parental hESCs (H9) were exposed to human serum for 1 hour. Cell death was quantified using a calcein-release assay [11, 12]. In initial experiments, we tested the capacity of serum from blood groups A and B to lyse M2 cells as monolayers (data not shown) and found that serum from blood group A led to higher levels of lysis than blood group B. This is consistent with previous reports that AB serum results in lower lysis of GalT-expressing cells [14, 15]. This is presumed to arise from cross-reactivity of anti-B and anti-GalT antibodies, due to homology between the B blood group antigen and the Gal epitope. We observed improved lysis when cells were exposed to serum as a suspension. This lysis was specific to active serum and did not result from the method of disaggregation. Flow cytometry for propidium iodide inclusion indicated that TEG generated a robust single-cell suspensions with, on average, 93% viability (data not shown). Therefore, in subsequent experiments, cells were exposed in suspension only to serum from blood group A.
Positive control ovine (PDFF) cells and M2 hESCs were both sensitive to lysis in active human serum, and for both cell types approximately 90% of total calcein was released in response to a 1-hour incubation with active human serum (Fig. 2A). H9 hESCs were insensitive to active human serum, as predicted by their lack of GalT activity, and F11 cells were intermediate in their level of lysis, which varied between experiments as indicated by the increase in standard deviation. This is consistent with their variegated pattern of transgene expression. Figure 2B shows H9, PDFF, M2, and F11 cells stained with Giemsa 24 hours after incubation with either active or heat-inactivated human serum. Both PDFF and M2 cultures exposed to active serum for 1 hour contained small residual numbers of surviving cells. However, there was an increase in the number of surviving cells observed by Giemsa staining in the F11 cultures after exposure to serum, confirming the results suggested by the calcein-release assay.
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Minority Populations of GalT-Expressing (M2) Cells Are Efficiently Lysed by Exposure to Human Serum in Mixed Populations
In the context of regenerative medicine, in which differentiated populations are engrafted, residual GalT-expressing cells would normally be small in number. To determine whether different proportions of GalT-expressing cells could be efficiently killed when cocultured with GalT-negative cells, we mixed varying numbers of M2 cells with HEK 293 cells that do not express GalT, exposed the mixture to active or inactive human serum, and then selected for surviving M2 cells in G418 for 7–10 days. A single G418-resistant colony was observed among three replicates when the proportion of M2 cells exposed to active human serum was less than 25%, although no colonies were observed at proportions less than 1.6% (Fig. 4). At higher concentrations of M2 cells, small numbers of colonies were occasionally observed after lysis. It was not possible to count the number of surviving colonies in heat-inactive controls at these concentrations and therefore the frequencies of surviving ESC colonies provided in Figure 4 are overestimations based on a count greater than 500 colonies. These data indicated again that, at high cell concentrations, a single 1-hour incubation killed most, but not all, GalT-expressing cells.
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-gal epitope on differentiated derivatives of M2 such that only undifferentiated cells are subject to complement-mediated lysis. To determine whether M2 cells downregulated GalT expression when they differentiated, we first cultured cells as monolayers in spontaneous differentiation conditions for 10 days and stained with BS-IB4 (Fig. 5A). After 10 days in these conditions, M2 cells were seen to have begun downregulating GalT expression. Continuing expression may reflect the transient persistence of epitope after downregulation of transcription, the persistence of undifferentiated hESCs, or the possible retention of hTERT expression in some progenitor populations. There was no evidence of persistent expression on phenotypically differentiated neuronal progenitor cells after a directed differentiation protocol (Fig. 5Bb).
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-gal at similar time points, as determined by flow cytometry (Fig. 5C). Hence, at day 21 of differentiation, we observed 8% calcein release (Fig. 5D), and at day 22 of differentiation, 8.8% of cells were expressing -gal (Fig. 5Cf). The M2 population in Figure 5Ca (green) can be seen as distinct from the parental H9 population (pale green), but with time the two populations gradually merge (Fig. 5Cf), indicating that by 22 days of differentiation, GalT expression is minimal in M2 cells. Figure 5C also indicates (red overlays) a similar pattern for the downregulation of an hESC-specific marker, Tra-1-81, with differentiation.
GalT-Engineered hESCs (M2) Are Karyotypically Normal and Have Normal Potentiality
Fifty chromosome spreads of M2 cells were examined at 10 passages after transfection and were shown to have a normal 46XX karyotype (data not shown). Cells at the same passage were injected intramuscularly into SCID mice and gave rise to broadly differentiated tumors (Fig. 6A). M2 cells differentiated in vitro in a similar manner to the parental H9 line and gave rise to cells staining for representative markers of all three germ layers (Fig. 6B). We conclude that there is no overt evidence of abnormality or restriction of potentiality.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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M2 cells exposed to human serum for 1 hour still contained small numbers of undifferentiated cells as indicated by lectin binding of the colonies arising after 7 days (Fig. 3). This result would be anticipated in a variegated population if a subset of undifferentiated cells, not expressing the transgene at the time of exposure, subsequently reinitiated transcription and formally shows that survival did not arise as a consequence of loss of the transgene. Alternatively, colonies arising after lysis could simply reflect incomplete lysis. In either event, multiple exposures might be expected to eliminate these cells. Minority populations of M2 cells, when mixed with HEK 293 cells, were efficiently killed. When the same mixed populations were exposed to heat-inactivated serum and the cells grown in conditions that allow outgrowth of only hESC colonies, we could detect the number of potentially tumorigenic cells in the mixed population. Hence, at 0.8% hESCs, an average of 308 colonies arose from the mixed population exposed to inactive serum, whereas no colonies survived in a sister culture exposed to active serum (Fig. 4i). Sporadic colonies (a single colony in three replicates) appear when the hESC population increases to 12.5% of the population, although it is not possible at this density to count colonies in the heat-inactivated control (Fig. 4e).
Although the majority of GalT-expressing cells in a population of M2 cells are lysed in human serum within 1 hour, the proportion of M2 hESCs that express GalT remains less than 100%. If nonexpressing cells have lost GalT expression as a consequence of variegated transgene expression, such cells could present a continuing risk of tumorigenesis. However, we show that increasing the exposure to human serum further reduces the number of surviving cells, suggesting that these cells are sensitive to active serum and continue to express the transgene. In addition, the mixing experiments provide evidence that, in practice, M2s that fail to express GalT and hence are insensitive to human serum and appear at very low frequency after incubation. Nevertheless, this theoretical risk could, in principle, be reduced by targeting the transgene (using homologous recombination to acquire the native hTERT or another ESC-specific promoter), by targeting an hTERT/GalT cassette to a "neutral" genomic site unassociated with position effect variegation, or by incorporating insulator elements to confer site-independent expression.
The core hTERT promoter sequence used in this study has higher transcriptional activity than the full hTERT promoter. When used to drive expression of neo in transfected hESCs, it provided higher colony number than neo cassettes transcribed from the PGK, SV40, or CMV promoters (data not shown). The hTERT gene is expressed in some adult progenitor cells; however, the core promoter is known to be downregulated in at least some of these populations, such as mesenchymal stem cells [16]. Hence, we anticipate that the hTERT/GalT transgene will be widely useful for application with multiple lineages. However, there may prove to be some lineages for which alternate promoters will be preferable, and it will be useful to generate additional modifications with GalT transcribed from promoters such as Oct4, Nanog, and Sox2.
A recent report has demonstrated that hESCs grown in medium containing serum replacement and other components of animal origin express an immunogenic nonhuman sialic acid, Neu5Gc, leading to complement-mediated cell death when exposed to human serum [17]. These authors attribute a major source of Neu5Gc to serum replacement. In contrast to that report, we did not detect cell death when H9 hESCs grown under our culture conditions (also including serum replacement) were incubated with human serum. In the absence of commercial sources of anti-Neu5Gc antiserum, we could not directly determine whether cells in our culture regime also express that epitope. Instead, we cultured H9 cells for 3 days with the addition of up to 3.0 mM Neu5Gc to the normal culture medium. After 3 days of exposure, cells were again incubated with human serum, and we again observed no difference in cell death in active versus heat-inactivated sera (data not shown). This result suggests that the sera used in this study did not contain significant levels of anti-Neu5Gc activity.
A novel feature of the pmGT approach is that M2 or similar cells encoding an hTERT/GalT transgene and engrafted into a patient may experience continuing in vivo immune surveillance [18–20] against residual hESCs or cells that re-express the GalT transgene due to dedifferentiation or as a consequence of secondary tumorigenesis. This possibility suggests an important advantage of the use of the GalT gene over TK-based approaches because in the latter case the tumor must be diagnosed (and hence well developed) before gancyclovir treatment can begin. With ongoing immune activity against GalT, it is anticipated that the tumors will simply never become established. In vivo surveillance for undifferentiated hTERT/GalT expression could be demonstrated in principle, using similarly modified murine hTERT/GalT ESCs in a syngeneic GalT knockout mouse, although this would not directly demonstrate the utility of M2 cells. More powerful still would be to induce tolerance in the GalT knockout mouse to M2 cells themselves, possibly by treatment with anti-CD4 antibodies or by inducing hematopoietic chimerism (reviewed in [21]). Such models could then be used to compare the tumorigenic potential of M2 versus parental cells.
These data demonstrate that hESCs can be routinely modified to provide clones with appropriate regulation of a GalT transgene and that such clones are sensitive to normal human serum when undifferentiated and insensitive after differentiation.
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SUMMARY |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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In addition, the presence of circulating antibody against the -gal epitope may provide continuing in vivo immune surveillance against cells expressing the GalT transgene. By harnessing the restricted expression of hTERT, nontumorigenic components of a graft would be preserved while undifferentiated or dedifferentiated cells would be targeted. This approach could be further improved by directing the transgene to a site free of position effect variegation using homologous recombination and by generating homozygous cells to guard against mutation or chromosome loss.
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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(1,2)fucosyltransferase and (1,3)galactosyltransferase alters the expression of H substance and Gal (1,3) Gal. J Biol Chem 1996;271:33105–33109.(1,4) Gal ligand. Biochem Biophys Res Commun 1995;216:814–820.[CrossRef][Medline]1,3Galactose-reactive human immunoglobulin G. Transplantation 1997;64:546–549.[CrossRef][Medline]1,3)Gal xenoantigen expression as a gene therapy approach to cancer. Gene Ther 1999;6:1073–1083.[CrossRef][Medline]gal epitope expression and decrease of tumorigenicity in pancreatic adenocarcinoma model. Mol Carcinog 2005;42:202–212.[CrossRef][Medline]This article has been cited by other articles:
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  S. J. Roberts, D. Howard, L. D. Buttery, and K. M. Shakesheff Clinical applications of musculoskeletal tissue engineering Br. Med. Bull., June 1, 2008; 86(1): 7 - 22. [Abstract] [Full Text] [PDF]
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