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.

The monosialoganglioside GM1a protects against complement attack.

The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface, ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, we here generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell death in vitro. Glycolipid-analysis by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF) allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenously administered GM1a integrated into the plasma membrane of trophoblasts, substantially increased binding of complement factor H (FH) and was sufficient to protect the cells from complement attack and cell death. GM1a treatment also rescued human endothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantly reduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). This study demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo.

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb-/- mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

Differential expression patterns of glycosphingolipids and C-type lectin receptors on immune cells in absence of functional regulatory T cells.

BACKGROUND: Glycosylation is a common and complex type of protein posttranslational modification. Altered glycosylation of immunoglobulins in autoimmune diseases has led to the "altered glycan hypothesis" postulating existence of a unique glycan signature on immune cells and extracellular proteins characterized by site-specific relative abundances of individual glycan structures and glycosylation patterns. However, it is not clear how glycosylation on leukocyte subpopulations differ between states of health or inflammation. HYPOTHESIS: Glycosphingolipid patterns on immune cells of forkhead-box-P3-deficient scurfy mice differs from those on wild-type immune cells. METHODS: T cells and dendritic cells were isolated from spleens of either wild-type or age-matched scurfy mice. Glycosphingolipids of CD4+ T cells and splenic dendritic cells from wild-type and scurfy mice were then analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). In addition, flow cytometry and ChipCytometry were used to access expression patterns of various C-type lectin receptors on antigen-presenting cells from various organs of both wild-type and scurfy mice. RESULTS: We, hereby report differential expression of glycosphingolipids in health and under inflammatory conditions as reflected in wild-type and scurfy mice. Furthermore, we observed that the absence of functional regulatory T cells correlated with elevated expression of CLEC-7A and CD205 but a reduction in levels of CLEC12A and CD206 on antigen-presenting cells. CONCLUSION: We hereby show that the absence of functional regulatory T cells affects expression pattern and quantities of glycosphingolipids on immune cells. Thus, glycosphingolipids could serve as biomarkers for mapping genetical and homeostatic perturbances such as those resulting from a diseased condition.

Systematic analysis of the human tumor cell binding to human vs. murine E- and P-selectin under static vs. dynamic conditions.

Endothelial E- and P-selectins promote metastasis formation by interacting with sialyl-Lewis X and A (sLeX/sLeA) on circulating tumor cells. This interaction precedes extravasation and can take place under dynamic and static conditions. Metastasis formation is often studied in xenograft models. However, it is unclear whether species differences exist in the ligand specificity of human (h) vs. murine (m) selectins and whether different ligands are functional under dynamic vs. static conditions. We systematically compared the h vs. m E- and P-selectin (ESel/PSel) binding of a range of human tumor cells under dynamic vs. static conditions. The tumor cells were categorized by their sLeA/X status (sLeA+/sLeX+, sLeA-/sLeX+ and sLeA-/sLeX-). The general biological nature of the tumor-selectin interaction was analyzed by applying several tumor cell treatments (anti-sLeA/X blockade, neuraminidase, pronase and inhibition of O/N-glycosylation). We observed remarkable differences in the static vs. dynamic interaction of tumor cells with h vs. m ESel/PSel depending on their sLeA/X status. The tumor cell treatments mostly affected either static or dynamic as well as either h- or m-selectin interaction. mESel showed a higher diversity of potential ligands than hESel. Inhibition of O-GalNAc-glycosylation also affected glycosphingolipid synthesis. Summarized, different ligands on human tumor cells are functional under static vs. dynamic conditions and for the interaction with human vs. murine ESel/PSel. Non-canonical selectin ligands lacking the sLeA/X glycan epitopes exist on human tumor cells. These findings have important implications for the current development of glycomimetic, antimetastatic drugs and encourage the development of immunodeficient mice with humanized selectins.

Reduced sialylation triggers homeostatic synapse and neuronal loss in middle-aged mice.

Sialic acid-binding Ig-like lectin (Siglec) receptors are linked to neurodegenerative processes, but the role of sialic acids in physiological aging is still not fully understood. We investigated the impact of reduced sialylation in the brain of mice heterozygous for the enzyme glucosamine-2-epimerase/N-acetylmannosamine kinase (GNE+/-) that is essential for sialic acid biosynthesis. We demonstrate that GNE+/- mice have hyposialylation in different brain regions, less synapses in the hippocampus and reduced microglial arborization already at 6 months followed by increased loss of neurons at 12 months. A transcriptomic analysis revealed no pro-inflammatory changes indicating an innate homeostatic immune process leading to the removal of synapses and neurons in GNE+/- mice during aging. Crossbreeding with complement C3-deficient mice rescued the earlier onset of neuronal and synaptic loss as well as the changes in microglial arborization. Thus, sialic acids of the glycocalyx contribute to brain homeostasis and act as a recognition system for the innate immune system in the brain.

Cell-autonomous impact of polysialic acid-producing enzyme ST8SIA2 on developmental migration and distribution of cortical interneurons.

In humans, variations in the polysialic acid-producing enzyme ST8SIA2 and disturbances in the cortical inhibitory system are associated with neurodevelopmental psychiatric disorders such as schizophrenia and autism. In mice, the ST8SIA2-dependent formation of polysialic acid during embryonic development is crucial for the establishment of interneuron populations of the medial prefrontal cortex. However, the spatial pattern and the neurodevelopmental mechanisms of interneuron changes caused by loss of ST8SIA2 function have not been fully characterized. Here, we use immunohistochemical analysis to demonstrate that densities of parvalbumin-positive interneurons are not only reduced in the medial prefrontal cortex, but also in the adjacent motor and somatosensory cortices of St8sia2-deficient male mice. These reductions, however, were confined to the rostral parts of the analyzed region. Mice with conditional knockout of St8sia2 under the interneuron-specific Lhx6 promoter, but not mice with a deletion under the Emx1 promoter that targets cortical excitatory neurons and glia, largely recapitulated the area-specific changes of parvalbumin-positive interneurons in the anterior cortex of St8sia2-/- mice. Live imaging of interneuron migration in slice cultures of the developing cortex revealed a comparable reduction of directional persistence accompanied by increased branching of leading processes in slice cultures obtained from St8sia2-/- embryos or from embryos with interneuron-specific ablation of St8sia2. Together, the data demonstrate a cell-autonomous impact of ST8SIA2 on cortical interneuron migration and the distribution of parvalbumin-positive interneurons in the anterior cortex. This provides a neurodevelopmental mechanism for how dysregulation of ST8SIA2 may lead to disturbed inhibitory balance as observed in schizophrenia and autism.

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.

The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells.

The WNT/ß-catenin signaling pathway is a prominent player in many developmental processes, including gastrulation, anterior-posterior axis specification, organ and tissue development, and homeostasis. Here, we use human pluripotent stem cells (hPSCs) to study the dynamics of the transcriptional response to exogenous activation of the WNT pathway. We describe a mechanism involving the WNT target gene SP5 that leads to termination of the transcriptional program initiated by WNT signaling. Integration of gene expression profiles of wild-type and SP5 mutant cells with genome-wide SP5 binding events reveals that SP5 acts to diminish expression of genes previously activated by the WNT pathway. Furthermore, we show that activation of SP5 by WNT signaling is most robust in cells with developmental potential, such as stem cells. These findings indicate a mechanism by which the developmental WNT signaling pathway reins in expression of transcriptional programs.