Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N -glycans were released by PNGase F digestion and O -glycans by β-elimination. Released oligosaccharides were analyzed by liquid chromatography – tandem mass spectrometry. In total, 102 N -glycans and 40 O -glycans were identified in animal heart tissue lysates. The N - and O -glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N - and O -linked glycans, N,N´ -diacetyllactosamine (LacdiNAc) on N -glycans only and sulfated O -glycans. The relative amounts of α-Gal-containing N -glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O -glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N -glycans. The identified N- and O- linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major alpha Gal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection.

N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major αGal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection.

BACKGROUND:Pericardial tissue from various animal species is utilized for the production of the bioprosthetic heart valves (BHV) used clinically. Experimental data show that the eventual breakdown of BHV is partly due to immunological interactions with carbohydrate tissue antigens. To understand these processes, we have examined the glycolipid-based carbohydrate antigens in naïve porcine, bovine, and equine pericardia. EXPERIMENTAL:Total non-acid and acid glycosphingolipid fractions were isolated from porcine, bovine, and equine pericardia, and individual glycolipid compounds were characterized by thin-layer chromatography, mass spectrometry, and binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays. RESULTS:The non-acid glycolipid fractions from all species contained glycosphingolipids based on the globo- and neolacto-series, including pentaglycosylceramides with terminal Galα3 determinants. Terminal blood group A and H (O) structures based on type 2 core chains were present in porcine pericardium, while the Forssman pentaosylceramide was found in equine pericardium. All acid glycolipid fractions contained sulfatide and several gangliosides with both N-acetyl- and N-glycolyl-neuraminic acid as terminal saccharide chain determinants. CONCLUSION:Several carbohydrate antigens which are potential targets for the human immune system have been identified in the animal pericardial tissues used for the production of BHV. Which of these antigens are left in the tissues after industrial BHV production processes, as well as their potential role in eventual BHV degradation, remains to be elucidated.

One prerequisite for a successful clinical outcome of human pluripotent stem cell (hPSC) based therapies is immune compatibility between grafted cells/tissue and recipient. This study explores immune determinants of human embryonic stem cell lines (hESC) and induced human pluripotent stem cell (hiPSC) lines and hepatocyte-and cardiomyocyte-like cells derived from these cells. HLA class I was expressed on all pluripotent hPSC lines which upon differentiation into hepatocyte-like cells was considerably reduced in contrast to cardiomyocyte-like cells which retained class I antigens. No HLA class II antigens were found in the pluripotent or differentiated cells. Histo-blood group carbohydrate antigens SSEA-3/SSEA-4/SSEA-5, Globo H, A, Le(x)/Le(y) and sialyl-lactotetra were expressed on all hPSC lines. Blood group AB(O) H antigen expression was in accordance with ABO genotype. Interestingly, only a subpopulation of A1O1 cells expressed A. During differentiation of hPSC, some histo-blood group antigens showed congruent alteration patterns while expression of other antigens differed between the cell lines. No systematic difference in the hPSC cell surface tissue antigen expression was detected. In conclusion, hPSC and their derivatives express cell surface antigens that may cause an immune rejection. Furthermore, tissue antigen expression must be established for each individual stem cell line prior to clinical application.

The application of human stem cell technology offers theoretically a great potential to treat various human diseases. However, to achieve this goal a large number of scientific issues remain to be solved. Cell surface carbohydrate antigens are involved in a number of biomedical phenomena that are important in clinical applications of stem cells, such as cell differentiation and immune reactivity. Due to their cell surface localization, carbohydrate epitopes are ideally suited for characterization of human pluripotent stem cells. Amongst the most commonly used markers to identify human pluripotent stem cells are the globo-series glycosphingolipids SSEA-3 and SSEA-4. However, our knowledge regarding human pluripotent stem cell glycosphingolipid expression was until recently mainly based on immunological assays of intact cells due to the very limited amounts of cell material available. In recent years the knowledge regarding glycosphingolipids in human embryonic stem cells has been extended by biochemical studies, which is the focus of this review. In addition, the distribution of the human pluripotent stem cell glycosphingolipids in human tissues, and glycosphingolipid changes during human stem cell differentiation, are discussed.

Background: The two common sialic acids (Sias) in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients.Methods: We investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG.Results: Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia linkages revealed Siaa2-3> Siaa2-6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaa2-3> Siaa2-6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express 4-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3 +/- 16.9 pmol Sia/mu g protein), with Neu5Ac at 8-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity toward Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts.Conclusions: We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation.

Introduction. The aetiology and pathogenesis of ulcerative colitis (UC) are essentially unknown. Galectins are carbohydrate-binding lectins involved in a large number of physiological and pathophysiological processes. Little is known about the role of galectins in human UC. In this immunohistochemical exploratory study, both epithelial and inflammatory cell galectin expression were studied in patients with a thoroughly documented clinical history and were correlated with inflammatory activity. Material and Methods. Surgical whole intestinal wall colon specimens from UC patients (n = 22) and controls (n = 10) were studied. Clinical history, pharmacological treatment, and modified Mayo-score were recorded. Tissue inflammation was graded, and sections were stained with antibodies recognizing galectin-1, galectin-2, galectin-3, and galectin-4. Results. Galectin-1 was undetectable in normal and UC colonic epithelium, while galectin-2, galectin-3, and galectin-4 were strongly expressed. A tendency towards diminished epithelial expression with increased inflammatory grade for galectin-2, galectin-3, and galectin-4 was also found. In the inflammatory cells, a strong expression of galectin-2 and a weak expression of galectin-3 were seen. No clear-cut correlation between epithelial galectin expression and severity of the disease was found. Conclusion. Galectin expression in patients with UC seems to be more dependent on disease focality and individual variation than on degree of tissue inflammation.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

recruited at our center (n = 53) and reveals that long term graft survival and renal function are not significantly different between the anti-EPC positive and negative patient groups beyond six months and during the four-year follow-up.

Genetic engineering of donor pigs to eliminate expression of the dominant xenogeneic antigen galactose α1,3 galactose (Gal) has created a sea change in the immunobiology of xenograft rejection. Antibody mediated xenograft rejection of GGTA-1 α-galactosyltransferase (GTKO) deficient organs is now directed to a combination of non-Gal pig protein and carbohydrate antigens. Glycan analysis of GTKO tissues identified no new neo-antigens but detected high levels of N-acetylneuraminic acid (Neu5Gc) modified glycoproteins and glycolipids. Humans produce anti-Neu5Gc antibody and in very limited clinical studies sometimes show an induced anti-Neu5Gc antibody response after challenge with pig tissue. The pathogenicity of anti-Neu5Gc antibody in xenotransplantation is not clear however as non-human transplant models, critical for modelling anti-Gal immunity, do not produce anti-Neu5Gc antibody. Antibody induced after xenotransplantation in non-human primates is directed to an array of pig endothelial cells proteins and to a glycan produced by the pig B4GALNT2 gene. We anticipate that immune suppression will significantly affect the T-cell dependent and independent specificity of an induced antibody response and that donor pigs deficient in synthesis of multiple xenogeneic glycans will be important to future studies.

Carl-Gustav Groth (1933–2014) It was a sad day when we learned of the death (on February 16th, 2014) of the International Xenotransplantation Association's (IXA) first president. Carl-Gustav Groth, MD, PhD, was born in Helsinki, Finland, on October 15th, 1933, but was brought up in Sweden. He attended the Karolinska Institute as a medical student, graduating in 1961, and decided on a career in transplantation. After participating in a minor role in the first kidney transplant in Sweden in 1964, and successfully defending his PhD thesis in rheology in 1965, he gained further vital experience in Dr. Thomas Starzl's pioneering unit at the University of Colorado in Denver. In 1967, he came to the attention of the relatively small transplant community at that time when he played a significant role in the first successful series of liver transplants carried out by Dr. Starzl. Although Dr. Starzl had performed a handful of liver transplants in 1963, the 1967 series was the first in which some patients survived long term. Carl, a young clinical fellow, put an immense effort into the postoperative care of these patients. Carl returned to Stockholm to complete his surgical training, but spent another period in Denver between 1971 and 1972. After establishing himself back in Stockholm, he became head of the transplantation programme in 1976, being promoted to professor at the Karolinska Institute in 1983 (retiring in 2000). He pioneered important aspects of pancreas transplantation, and established an active and successful transplant programme at Huddinge Hospital, which included kidney, liver, pancreas, islet, and bone marrow transplantation. Carl retired from his clinical activities in 2000, but remained as Professor Emeritus until his death. For several years (1986–1999), he was a member of the Nobel Assembly (chairman in 1998), which elects Nobel prizewinners in Physiology or Medicine. His leading role in the world of transplantation resulted in his election to the presidency of the Transplantation Society (2000–2002), of which the IXA was the first official “section”, and he was a co-recipient of its most prestigious scientific award, the Medawar Prize, in 2006. He received many other honors, including Honorary Fellowship of the Royal College of Surgeons of England (1989), the Swedish King's Medal (1998), and the ASTS-Roche Pioneer Award (2008). His first major contribution in the field of xenotransplantation was in 1993 when he led the team that carried out a series of wild-type fetal pig islet transplants in patients with type I diabetes. Although none of these patients benefited clinically by improved control of their diabetes, prolonged porcine C-peptide was documented in several patients, and the study provided the first evidence of morphologically intact porcine islets in humans. Importantly, none of the recipients experienced any adverse events. The initiation of this trial demonstrated Carl's vision for the future, but he and his colleagues did not believe the results were good enough to justify further trials at that time. Subsequently, however, he wondered whether progress in xenotransplantation would have been quicker if he and his team had persisted. In view of the problems that have subsequently become identified, for example, with regard to the expression of the Galα1,3Gal antigen in neonatal pig islets, it is unlikely that any trial in the 1990s would have been fully successful. Perhaps Carl foresaw this when he closed the xeno-islet program. He had the ability to make a correct decision, based on feeling what is right, even when not all of the data were yet available. Nevertheless, he remained a strong advocate for pushing ahead cautiously with clinical trials, and criticized the ‘moratoria’ that various governments and organizations imposed on clinical xenotransplantation. When it was suggested by a number of people that there was sufficient activity in xenotransplantation research to establish a society that would concentrate attention in this field, the IXA was established and Carl was elected its first president in 1998. From that time, he played a gentle but persistent role in advancing this field of research and, although not very actively involved himself, was always lending support to other groups. For example, he gave considerable informal, but valuable, support to Dr. Wei Wang in Changsha, China, who had already initiated a clinical pig islet transplant trial. In 2007, Carl was a co-organizer of the “pig-to-man islet summit” in Stockholm, a valuable conference for invited researchers to discuss how islet xenotransplantation could be advanced into the clinic. In 2008, with Wei Wang and Luc Noel, he played a role in setting up the World Health Organization (WHO) meeting in Changsha to discuss regulatory requirements for clinical trials relating to xenotransplantation. His contributions to xenotransplantation were recognized by the award of Honorary Membership of the IXA in 2005. In 2008, he took on the editor-in-chief position of the IXA's official journal, Xenotransplantation, which he maintained during a difficult period until he relinquished it at the end of 2011. In 2013, the journal honored him by establishing the Carl Groth Xeno Prize for the best paper published each year. Carl was a very pleasant and diplomatic companion who quietly persisted in pushing the field forward. In more recent years, when in failing health, he was able to play a less significant role, but retained his great interest in the development of xenotransplantation. His many friends in xenotransplantation research will miss his wise advice and loyal support.

BACKGROUND:Although xenotransplantation of vascularized organs/cells has not yet reached the clinic, glutaraldehyde-treated bioprosthetic heart valves (BHV), derived from porcine or bovine tissues, are today used for clinical replacement of diseased heart valves. However, the durability of these valve cusps is limited partly due to the onset of immune responses to the grafts. The xenoantigen-determinant Galα3Gal- and corresponding anti-Gal antibodies have been postulated to in part contribute to BHV damage. However, the presence of other non-Gal carbohydrate antigen determinants as well as the immune response to these non-Gal antigens and the inflammatory response generated by their interaction with the immune system has not been studied. In this study, we have isolated and structurally characterized both non-acid and acid glycosphingolipids from naïve porcine aortic and pulmonary valve cusps. METHODS:Total non-acid and acid glycosphingolipids were isolated from porcine aortic and pulmonalis valve cusps of 20 animals. Glycosphingolipid components were structurally characterized by thin-layer chromatography, liquid chromatography-mass spectrometry and binding of monoclonal antibodies and lectins. RESULTS:The non-acid glycosphingolipids were characterized as globotetraosylceramide, H-type 2 pentaosylceramide, fucosyl-gangliotetraosylceramide, and Galα3neolactotetraosylceramide. The acid glycosphingolipid fractions had both sulfatide and gangliosides (GM3, GM2, GM1, fucosyl-GM1, GD3 and GD1a), and all gangliosides contained N-acetyl-neuraminic acid. Significantly, the N-glycolyl-neuraminic acid (NeuGc) variant, a major component in many pig organs and to which humans can develop antibodies, was not detected among the gangliosides. CONCLUSIONS:Pig valve cusps contain several complex lipid-bound carbohydrate structures that may be targets for the human immune system. Notable, the NeuGc determinant was absent in the cusp gangliosides. This work forms a platform for further characterizing the antibody reactivity of patients with porcine-derived BHV.

Background: Carbohydrate epitopes are often used as markers for characterization of human pluripotent stem cells (hPSC). Results: Sialyl-lactotetra is highly expressed on the cell surface of hPSC lines, and the expression decreased upon early differentiation. Conclusion: Sialyl-lactotetra is a novel marker of undifferentiated human stem cells. Significance: These findings might impact future applications in regenerative medicine. Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 109 cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.

In recent years ABO incompatible kidney transplantation (KTx) has become a more or less clinical routine procedure with graft and patient survival similar to those of ABO compatible transplants. Antigen-specific immunoadsorption (IA) for anti-A and anti-B antibody removal constitutes in many centers an important part of the treatment protocol. ABO antibody titration by hemagglutination is guiding the treatment; both if the recipient can be transplanted as well as in cases of suspected rejections if antibody removal should be performed. Despite the overall success of ABO incompatible KTx, there is still room for improvements and an extension of the technology to include other solid organs. Based on an increased understanding of the structural complexity and tissue distribution of ABH antigens and the fine epitope specificity of the ABO antibody repertoire, improved IA matrices and ABO antibody diagnostics should be developed. Furthermore, under-standing the molecular mechanisms behind accommodation of ABO incompatible renal allografts could make it possible to induce long-term allograft acceptance also in human leukocyte antigen (HLA) sensitized recipients and, perhaps, also make clinical xenotransplantation possible.

Background: The human H-transferase (alpha 2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective.Methods: Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy. Results: Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis x and Lewis y determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis x and blood group H type 1 determinants on a lactotetraosyl core, respectively.Conclusions: Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective.

Background: Carbohydrate epitopes are often used as markers for characterization of human embryonic stem cells (hESC). Results: Several glycosphingolipids not previously found in hESC were characterized. Conclusion: The glycosylation of hESC is more complex than previously thought. Significance: These findings will help to understand the immunogenicity of hESC and might impact future applications in regenerative medicine. Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 109 cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Lex pentaosylceramide, and the Ley hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.