-Glycolylneuraminic Acid Xenoantigen Contamination of Human Embryonic and Mesenc - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Annamari Heiskanena, Tero Satomaaa, Sari Tiitinenb, Anita Laitinenb, Sirkka Mannelinb, Ulla Impolab, Milla Mikkolac, Cia Olssonc,d, Halina Miller-Podrazae, Maria Blomqvista, Anne Olonena, Hanna Saloa, Petri Lehenkarif, Timo Tuuric,d, Timo Otonkoskic,g, Jari Natunena, Juhani Saarinena, Jarmo Laineb


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      【摘要】$ y5 p7 ?% m( R. Q5 G6 i
   Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies. 7 T9 c' \9 i4 N" {5 U( o
      【关键词】 Stem cell Xenoantigen N-Glycolylneuraminic acid Clinical trial5 c% j% ?% l3 w9 M9 X& h: m
   INTRODUCTION6 m4 M7 L! A  ]4 }* P& l

Stem cell therapies based on both human (h) ESCs and MSCs are expected to provide novel approaches and significant improvements to the treatment of a large number of patients suffering form several debilitating diseases. For these hopes to be realized, it is essential that stem cells are handled and cultured in a manner that guarantees the efficacy and safety of the cellular therapy product. One such aspect is the choice of cell culture media and supplements. In principle, most investigators agree that all animal material should be avoided to maximize product safety. Currently, however, hESCs and MSCs are still usually cultured in the presence of animal-derived material for research purposes. In addition, the clinical efficacy of MSCs in human disease has been investigated using MSCs cultured with fetal bovine serum (FBS) in a number of clinical trials .

It was recently reported that hESC lines grown in contact with mouse feeder cells and animal-derived cell culture medium components are contaminated with the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) . The authors also suggested that the immunogenic contamination would be difficult to completely eliminate from cell lines and thus negatively influence their use for human therapy. Since almost all currently existing hESC lines have been derived under conditions where they have become exposed to animal material, this would represent a major setback for the development of cellular therapies based on hESCs. The extent of the potential contamination has remained unclear, because it is not known whether similar contamination would afflict other stem cell types or hESCs cultured on human feeder cells.

We have undertaken a project of glycome analysis of various human stem cell types, during which we encountered several Neu5Gc-containing glycoconjugates in stem cells. In the present work, in vitro-cultured human hESC and MSC lines were investigated to study the Neu5Gc contamination at the molecular level, identify sources of contamination, and establish whether the contamination would be reversible in stem cell progeny.$ J5 z# W# g0 U0 E" I! g$ |
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MATERIALS AND METHODS
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hESCs
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The generation of the four human hESC lines FES 21, FES 22, FES 29, and FES 30 has been described elsewhere ; Gibco cell culture systems, Invitrogen, Paisley, U.K., http://www.invitrogen.com) supplemented with 2 mM L-glutamine/penicillin streptomycin (Sigma-Aldrich), 20% Knockout Serum Replacement (Invitrogen), 1x nonessential amino acids (Invitrogen), 0.1 mM ß-mercaptoethanol (Invitrogen), 1x insulin-transferrin-selenium supplement (ITS) (Sigma-Aldrich), and 4 ng/ml basic fibroblast growth factor (bFGF) (Sigma). To induce the formation of EBs, the hESC colonies were first allowed to grow for 10¨C14 days, after which the colonies were cut in small pieces and transferred on nonadherent Petri dishes to form suspension cultures. The formed EBs were cultured in suspension for the next 10 days in standard culture medium (see above) without bFGF. For further differentiation into stage III differentiated cells, EBs were transferred onto gelatin-coated (Sigma-Aldrich) adherent culture dishes in media consisting of D-MEM/F-12 mixture (Gibco) supplemented with ITS, fibronectin (Sigma-Aldrich), L-glutamine, and antibiotics. The attached cells were cultured for 10 days, after which they were harvested. The cells were collected mechanically, washed, and stored frozen prior to glycan analysis.
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Bone-Marrow Derived Mesenchymal Stem Cell Lines

Bone marrow (BM)-derived MSCs were obtained as described previously . In brief, bone marrow obtained during orthopedic surgery was cultured in minimum essential -medium (-MEM), supplemented with 20 mM HEPES, 10% FBS, 1x penicillin-streptomycin, and 2 mM L-glutamine (all from Gibco). After a cell attachment period of 2 days, the cells were washed with Ca2  and Mg2 -free phosphate-buffered saline (PBS) (Gibco), subcultured further by plating the cells at a density of 2,000 to 3,000 cells/cm2 in the same media and removing half of the media and replacing it with fresh media twice a week until near confluence. For analysis of the effects of human serum on Neu5Gc decontamination, the cells were cultured for 3 weeks in the media, where FBS was replaced by 10% heat-inactivated blood group AB Rh-negative human serum from a male donor.

Flow Cytometric Analysis of Mesenchymal Stem Cell Phenotype# Q. T0 e- K0 y" |

BM MSCs were phenotyped by flow cytometry (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against CD13, CD14, CD29, CD34, CD44, CD45, CD49e, CD73, and HLA-ABC (all from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), CD105 (Abcam Ltd., Cambridge, U.K., http://www.abcam.com), and CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) were used for direct labeling. Appropriate FITC- and PE-conjugated isotypic controls (BD Biosciences) were used. Unconjugated antibodies against CD90 and human leukocyte antigens (HLA)-DR (both from BD Biosciences) were used for indirect labeling. For indirect labeling, FITC-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich) was used as a secondary antibody. The cells were negative for CD14, CD34, CD45, and HLA-DR and positive for CD13, CD29, CD44, CD90, CD105, and HLA-ABC.1 Q5 F% W6 D( Q( V# D

Osteogenic Differentiation of Mesenchymal Stem Cells
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To induce the osteogenic differentiation of the BM-derived MSCs, the cells were seeded in their normal proliferation medium at a density of 3 x 103/cm2 on 24-well plates (Nalge Nunc, Roskilde, Denmark, http://www.nalgenunc.com/). The next day, the medium was changed to osteogenic induction medium, which consisted of -MEM (Gibco) supplemented with 10% FBS (Gibco), 0.1 µM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.nuncbrand.com), 10 mM ß-glycerophosphate, 0.05 mM L-ascorbic acid-2-phosphate (Sigma-Aldrich), and penicillin-streptomycin (Gibco). The cells were cultured for 3 weeks, changing the medium twice a week before preparing samples for glycome analysis.
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Cell Harvesting for N-Glycome Analysis" @* L: N9 }% c  f' W
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One milliliter of cell culture medium was saved for N-glycome analysis, and the rest of the medium was removed by aspiration. Cell culture plates were washed with PBS buffer, pH 7.2. PBS was aspirated and cells scraped and collected with 5 ml of PBS (repeated two times). At this point, a small cell fraction (10 µl) was taken for cell counting and the rest of the sample centrifuged for 5 minutes at 400g. The supernatant was aspirated and the pellet washed in PBS an additional two times. The cells were collected with 1.5 ml of PBS, transferred from a 50-ml tube into a 1.5-ml collection tube, and centrifuged for 7 minutes at 5,400 rpm. The supernatant was aspirated, and washing was repeated one more time. The cell pellet was stored at ¨C70¡ãC and used for N-glycome analysis.
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Glycan Isolation

Asparagine-linked glycans were detached from cellular glycoproteins by F. meningosepticum N-glycosidase F digestion (Calbiochem, San Diego, http://www.emdbiosciences.com), essentially as described . The released asparagine-linked glycans (N-glycans) were purified for analysis mainly by miniaturized solid-phase extraction steps as described (T. Satomaa, A. Helskanen, M. Mikkola, C. Olsson, M. Blomqvist, T. Jaatinen, O. Aitio, A. Olonen, J. Helin, J. Natunen, T. Tuuri, T. Otankaski, J. Saarinen, J. Laine, unpublished observations).0 a4 j9 {9 i* r5 B! }+ t+ ?4 {
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Mass Spectrometry
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Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry was performed with a Bruker Ultraflex TOF/TOF instrument (Bruker, Rheinstetten, Germany, http://www.bruker.de/) essentially as described . The mass spectrometric fragmentation analysis was done according to manufacturer's instructions.9 w+ }0 w/ V# ]$ d  V$ L( K0 u& m

Neu5Gc-Specific Antibody Procedures

The Neu5Gc-specific monoclonal antibody P3Q against the glycolipid antigen GM3(Gc) was a kind gift from Dr. Ana Maria V¨¢zquez and Dr. Ernesto Moreno from the Center of Molecular Immunology, Havana, Cuba. It is essentially similar in binding specificity as described previously . The plates were then incubated in 2% bovine serum albumin (BSA) in PBS for 1 hour and overlaid with the anti-N-glycolyl antibody diluted with 2% BSA in PBS. After 2 hours, the plates were washed with PBS and overlaid with a second antibody, horseradish peroxidase-labeled antibody (rabbit anti-human IgG  chains, P.0214; DAKO, Glostrup, Denmark, http://www.dako.com), diluted with 2% BSA in PBS. After an additional 2 hours, the plates were washed with PBS, and the binding fractions were visualized using 0.02% 3,3'-diaminobenzidine tetrahydrochloride in PBS containing 0.03% H2O2. All steps were performed at room temperature. The P3 antibody was confirmed to bind strongly to N-glycolyl-containing glycolipids and, in higher concentrations, also to sulfated glycolipids.3 Q" M  Y# _: x$ ?& Y. }

Immunohistochemistry

BM MSCs on passages 9 to 14 were grown on 0.01% poly-L-lysine (Sigma-Aldrich)-coated glass eight-chamber slides (Lab-TekII; Nalge Nunc) at 37¡ãC with 5% CO2 for 2¨C4 days. After culturing, cells were rinsed five times with PBS (10 mM sodium phosphate, pH 7.2, 140 mM NaCl) and fixed with 4% PBS-buffered paraformaldehyde, pH 7.2, at room temperature (RT) for 10 minutes, followed by washings three times 5 minutes with PBS. Some MSC samples were extracted with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes at RT before blocking. In addition, specificity of the staining was confirmed by sialidase treatment after fixation. In brief, fixed MSC samples were incubated with 10 mU of sialidase (Arthrobacter ureafaciens; Glyko, San Leandro, CA, http://www.prozyme.com) in 50 mM sodium acetate buffer, pH 5.5, overnight at 37¡ãC. After detergent extraction or sialidase treatment, cells were washed three times with PBS. After fixation and different treatments, the nonspecific binding sites were blocked with 3% human serum albumin (HAS)-PBS (Finnish Red Cross Blood Service, Helsinki, Finland, http://www.redcross.fi/en_GB/etusivu/) for 30 minutes at RT. Primary antibodies were diluted in 1% HSA-PBS and incubated for 60 minutes at RT, followed by washing three times for 10 minutes with PBS. Secondary antibody, FITC-labeled goat-anti-mouse (Sigma-Aldrich), was diluted 1:300 in 1% HSA-PBS and incubated for 60 minutes at RT in the dark. Furthermore, cells were washed three times for 5¨C10 minutes with PBS and mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) stain (Vector Laboratories, Peterborough, U.K., http://www.vectorlabs.com/). Immunostainings were analyzed with Zeiss Axioskop 2-plus fluorescence microscope (Carl Zeiss Vision GmbH, Jena, Germany, http://www.zeiss.com) with FITC and DAPI filters. Images were taken with a Zeiss AxioCam MRc camera and with AxioVision Software 3.1/4.0 (Carl Zeiss) with the 400x magnification.- ?# c5 `) f* G9 W8 F
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Biological Reagents
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Bovine serum apotransferrin and fetuin were from Sigma-Aldrich.
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RESULTS

Mass Spectrometric Analysis of Stem Cell Glycan Structures" `( P8 V+ H) C2 D2 i2 ]- ?; j
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Four hESC lines, four bone marrow-derived MSC lines, and cells differentiated from these stem cells, as well as the culture media and supplements used, were analyzed. Protein-incorporated Neu5Gc was analyzed by enzymatically detaching N-glycans from total cellular or media proteins, fractionating the released glycans into neutral and acidic N-glycan fractions, and analyzing them by MALDI-TOF mass spectrometry as described in Figure 1. The identification of Neu5Gc was based on detection of sialic acid residues with 16-Da larger mass than the human-type sialic acid, N-acetylneuraminic acid (Neu5Ac).2 J% e( {% l3 `  E, R8 w
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Figure 1. Matrix-assisted laser desorption ionization/time of flight mass spectrometric detection of representative sialylated asparagine-linked glycans (N-glycans) containing N-glycolylneuraminic acid (Neu5Gc). (A): Human (h) ESCs grown on human feeder cells in serum replacement medium, (B) MSCs grown in fetal bovine serum (FBS) containing medium, (C) serum replacement cell culture medium, (D) bovine serum transferrin, (E) MSC culture medium with FBS, and (F) fetuin isolated from FBS. (G): MSC grown for 2 weeks in human serum. The m/z and relative molar abundancy (%) of the observed glycans is indicated on the x- and y-axes of the panels, respectively. The glycan signals at m/z 1,946.67 (top panel), corresponding to the ¨C ion of Neu5Ac2Hex5HexNAc4, do not contain Neu5Gc. Schematic N-glycan structures corresponding to these signals are depicted on the left: light yellow circles, hexose; dark gray squares, N-acetylhexosamine; dark red diamonds, Neu5Ac; light blue diamonds, Neu5Gc. Abbreviation: m/z, mass-to-charge ratio.

N-glycan signals indicating the presence of Neu5Gc were detected in all the four different hESC lines cultured in commonly used hESC serum replacement medium on human feeder cell layers (Fig. 1A). Approximately 6%¨C11% of total sialylated N-glycans in the undifferentiated hESCs contained Neu5Gc. The N-glycan signals depicted in Figure 1 expressed above average levels of Neu5Gc, possibly because of mechanisms of Neu5Gc incorporation to the cells. Therefore, these glycan signals were used as indicative signals in the present Neu5Gc analyses. When these N-glycans were subjected to mass spectrometric fragmentation analysis, they produced fragment ions corresponding to Neu5Gc (data not shown). Similar Neu5Gc structures as in hESCs were observed in all the MSC lines cultured in FBS containing culture medium (Fig. 1B), indicating that in addition to hESCs, also bone marrow-derived MSCs are susceptible to Neu5Gc contamination.

Immunohistochemical Detection of Neu5Gc in Stem Cells

To verify the results obtained by mass spectrometry, immunohistochemical analyses using a Neu5Gc-specific monoclonal antibody  against the glycolipid antigens Neu5Gc3LacCer (GM3Gc) and Neu5Gc3nLc4Cer were performed (Fig. 2). Glycans containing Neu5Gc were detected on both bone marrow-derived MSCs (Fig. 2A¨C2D) and hESCs (Fig. 2E). Cell permeabilization and extraction with detergent at room temperature abolished the staining, suggesting antigen localization in detergent-soluble areas on the cell surface (Fig. 2B). The results suggest that in addition to glycoprotein glycans, Neu5Gc contamination can also be present in lipid-linked glycans in stem cells.

Figure 2. Immunochemical detection of N-glycolylneuraminic acid (Neu5Gc) in a bone marrow-derived MSC line. (A): MSCs cultured in the presence of fetal bovine serum were stained by Neu5Gc-specific antibody. (B): Detergent extraction of the sample abolished staining, indicating that Neu5Gc epitopes were present in detergent-soluble areas of the cell surface. (C): The staining was decreased after sialidase treatment, indicating that antibody binding was dependent on sialic acids such as Neu5Gc. (D): Negative control without primary antibody. Scale bar in (A¨CD): 20 µm. (E): Human (h) ESC line FES 30 grown on mouse feeder cells and in the presence of serum replacement cell culture supplement stained by Neu5Gc-specific antibody. Neu5Gc epitopes were detected in both hESCs (densely growing cells in the lower part of the picture) and feeder cells.
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Analysis of Cell Culture Media for Sources of Neu5Gc. L5 ], P! E9 \* M) X4 N
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To identify the cell culture media components contributing to Neu5Gc contamination, the culture media and their main protein components were analyzed. The hESC samples were cultured on human fibroblast feeder cells in commercial serum-free medium .
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Taken together, all the media used in stem cell culture contained Neu5Gc, but there were significant differences in the relative concentrations. Significantly, the commercial serum replacement medium proved to be a more potent source of Neu5Gc than FBS. The relative Neu5Gc contents in the culture media correlated with the observed high Neu5Gc levels in hESCs (Fig. 1A) and significantly lower Neu5Gc levels in MSCs (Fig. 1B).; S5 F; N% A) a! _# `4 u* I

Neu5Gc in Stem Cell Progeny- ?6 f/ a8 V& d+ G, j8 T& e

To determine whether the progeny of Neu5Gc-containing hESC would be permanently contaminated by Neu5Gc, hESCs were differentiated into embryoid bodies (EBs) and further into stage III spontaneously differentiated cells. EBs were cultured in the same medium as hESCs (without bFGF) in the absence of feeder cells and further differentiated cells on gelatin-coated dishes in media supplemented with ITS and fibronectin. The Neu5Gc content was found to decrease during differentiation into EB, and no Neu5Gc was detected in stage III differentiated cells. In the present analyses, Neu5Gc-containing N-glycan signals corresponding to 0.5% of total sialylated N-glycans could be reliably detected. The findings suggested that at least the majority of Neu5Gc contamination in hESC was reversible and that Neu5Gc could be gradually removed from hESC progeny in cell culture. Furthermore, the results suggested that hESC differentiation diminished the susceptibility of the cells to Neu5Gc contamination from the culture medium.4 B: v/ o& C6 n5 C, M7 D( S6 ~- o
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To determine whether similar decontamination as observed in hESCs also occurs in MSCs, bone marrow-derived MSCs were induced to undergo osteogenic differentiation in the presence of FBS. It was found that the differentiated cells had on average approximately 50% lower Neu5Gc contents than the original MSCs, and in one out of four experiments, N-glycan structures containing Neu5Gc could not be detected after cell culture in differentiation medium (data not shown). The results suggested that also MSC progeny could be at least partially decontaminated with respect to Neu5Gc.

Disappearance of Neu5Gc from Mesenchymal Stem Cells Cultured in Human Serum

To investigate whether MSC lines contaminated with Neu5Gc can be decontaminated by culturing them without contact to animal materials, MSCs previously cultured with FBS were moved to the same media supplemented with heat-inactivated blood group AB Rh-negative human serum instead of FBS. In immunohistochemistry, the number of Neu5Gc-positive cells significantly decreased, but after 2 weeks of culture, individual Neu5Gc-positive cells were still occasionally detected (Fig. 3). This suggested that although most cells could be easily decontaminated with regard to Neu5Gc, absolute decontamination may be difficult to reach. Mass spectrometric analysis of the Neu5Gc content in MSCs transferred from FBS to human serum containing cell culture medium indicated that Neu5Gc contamination was significantly decreased in MSCs grown in human serum (Fig. 1G).
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Figure 3. Effect of culture with human serum on N-glycolylneuraminic acid (Neu5Gc) expression in mesenchymal stem cells. (A): Cells cultured in the presence of fetal bovine serum show staining with Neu5Gc-specific antibody. (B): Number of Neu5Gc positive cells decreased when cells were cultured for 1 week in human serum. (C): Individual Neu5Gc positive cells were still occasionally detected after 2-week cultivation in human serum. (D): Negative control without the primary Neu5Gc specific antibody used to detect Neu5Gc expression. Original magnification, x200.

DISCUSSION
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It has previously been reported that hESCs are capable of efficient Neu5Gc uptake from culture media components .
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In general, based on the current data, it is clear that all stem cell culture conditions should be controlled with respect to Neu5Gc contamination. There are reliable biochemical and immunochemical methods for analyzing Neu5Gc content in biological materials .! a$ q; `7 {; X% W: T' V2 u

Importantly, the present data suggest that culturing both hESCs and MSCs in appropriate conditions results in removal of Neu5Gc from stem cells or the stem cell progeny. Accordingly, recent studies have demonstrated by direct chemical analyses that hESCs lose the Neu5Gc contamination  during cell culture with human-derived supplements. This is in accordance with our finding that most MSCs became decontaminated after prolonged culture in media in which FBS was replaced by human serum. However, our results suggested that complete decontamination may be difficult to achieve by changing culture conditions since individual cells may express Neu5Gc even after long periods. For the generation of pure Neu5Gc-negative cell populations, a negative selection procedure using for example Neu5Gc-recognizing antibodies might prove feasible.

The molecular mechanism of Neu5Gc loss from stem cells remains to be clarified . It is known that cells are able to secrete sialic acid-containing glycoprotein materials into culture medium, suggesting that Neu5Gc-containing material can also be secreted. Another direct mechanism of Neu5Gc loss is dilution of the molecule in stem cell progeny during normal cell growth and division. Intriguingly, the present analyses showed significant reduction in Neu5Gc levels in both hESCs and MSCs during cell culture, especially after stem cell differentiation in differentiation-inducing culture conditions.
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Finally, with respect to the existing stem cells potentially contaminated with Neu5Gc, it should be noted that even normal human tissues may acquire minor amounts of Neu5Gc from dietary and other sources . Although it remains possible that such tissues may face low-intensity immune attack, we feel that it is unlikely that the vast majority of currently existing hESC and MSC lines that have been inadvertently exposed to Neu5Gc will be permanently contaminated and unfit for future applications if they are cultured in conditions avoiding renewed exposure to animal materials., j; X9 h5 h  f' U5 w2 R

DISCLOSURES, ]( J9 u* k: J) H8 L
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T.S., J.N., and J.S. own stock in Glykos Finland Ltd. A.H., T.S., M.B., A.O., H.S., J.N., and J.S. have indicated a financial interest in Glykos Finland Ltd. S.T., A.L., S.M., U.I., and J.L. have indicated a financial interest in the Finnish Red Cross Blood Service.! X6 G1 H5 A: L( [5 i$ y

ACKNOWLEDGMENTS: E" m' z  K; @4 S0 ^( a

We thank A.M. V¨¢zquez and E. Moreno from the Center of Molecular Immunology, Havana, Cuba, for antibody donation, and J. Helin for expertise in mass spectrometry. This study was supported by the Finnish Funding Agency for Technology and Innovation (TEKES), The Sigrid Juselius Foundation, The Juvenile Diabetes Foundation, and the Academy of Finland. J.S. and J.L. contributed equally to this work.
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