Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Quantitative Assessment of Sialo-Glycoproteins and N-Glycans during Cardiomyogenic Differentiation of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell-surface-exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N-glycome by capillary gel electrophoresis revealed three novel structures comprising ß1,3-linked galactose, α2,6-linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.

Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms.

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development.

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG).

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors andin vitrodifferentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCsin vitro Knock-down ofPMM2by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

Structure and biochemical characterization of bacteriophage phi92 endosialidase.

Surface-associated capsular polysaccharides (CPSs) protect bacteria against phage infection and enhance pathogenicity by interfering with the function of the host innate immune system. The CPS of enteropathogenic Escherichia coli K92 is a unique sialic acid polymer (polySia) with alternating α2,8- and α2,9-linkages. This CPS can be digested by the gene 143 encoded endosialidase of bacteriophage phi92. Here we report the crystal structure of the phi92 endosialidase in complex with a dimer of α2,9-linked sialic acid and analyze its catalytic functions. Unlike the well characterized and homologous endosialidase of phage K1F, the phi92 endosialidase is a bifunctional enzyme with high activity against α2,8- and low activity against α2,9-linkages in a polySia chain. Moreover, in contrast to the processive K1F endosialidase, the phi92 endosialidase degrades the polymer in a non-processive mode. Beyond describing the first endosialidase with α2,9-specificity, our data introduce a novel platform for studies of endosialidase regioselectivity and for engineering highly active α2,9-specific enzymes.

A multivalent adsorption apparatus explains the broad host range of phage phi92: a comprehensive genomic and structural analysis.

Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.