Osteoclasts secrete osteopontin into resorption lacunae during bone resorption.

Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone.

An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants.

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000 µg/L. The limit of quantitation (LOQ) was 0.5 µg/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.

Metabolic glycoengineering of mesenchymal stromal cells with N-propanoylmannosamine.

There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the N-glycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri- and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.

Novel data analysis tool for semiquantitative LC-MS-MS2 profiling of N-glycans.

Despite recent technical advances in glycan analysis, the rapidly growing field of glycomics still lacks methods that are high throughput and robust, and yet allow detailed and reliable identification of different glycans. LC-MS-MS(2) methods have a large potential for glycan analysis as they enable separation and identification of different glycans, including structural isomers. The major drawback is the complexity of the data with different charge states and adduct combinations. In practice, manual data analysis, still largely used for MALDI-TOF data, is no more achievable for LC-MS-MS(2) data. To solve the problem, we developed a glycan analysis software GlycanID for the analysis of LC-MS-MS(2) data to identify and profile glycan compositions in combination with existing proteomic software. IgG was used as an example of an individual glycoprotein and extracted cell surface proteins of human fibroblasts as a more complex sample to demonstrate the power of the novel data analysis approach. N-glycans were isolated from the samples and analyzed as permethylated sugar alditols by LC-MS-MS(2), permitting semiquantitative glycan profiling. The data analysis consisted of five steps: 1) extraction of LC-MS features and MS(2) spectra, 2) mapping potential glycans based on feature distribution, 3) matching the feature masses with a glycan composition database and de novo generated compositions, 4) scoring MS(2) spectra with theoretical glycan fragments, and 5) composing the glycan profile for the identified glycan compositions. The resulting N-glycan profile of IgG revealed 28 glycan compositions and was in good correlation with the published IgG profile. More than 50 glycan compositions were reliably identified from the cell surface N-glycan profile of human fibroblasts. Use of the GlycanID software made relatively rapid analysis of complex glycan LC-MS-MS(2) data feasible. The results demonstrate that the complexity of glycan LC-MS-MS(2) data can be used as an asset to increase the reliability of the identifications.

Nanoscale reversed-phase liquid chromatography-mass spectrometry of permethylated N-glycans.

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and ß-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope.

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galß1-3GlcNAcß1-3Galß1-4GlcNAc and Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-6(Galß1-3GlcNAcß1-3)Galß1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is ß1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-6(Galß1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galß1-3GlcNAcß1-3Galß1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.

Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms.

Human stem cells contain substantial amounts of the xenoantigen N-glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP-N-acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell-specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co-occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of beta-catenin in both CMAH overexpressing cells and bone marrow-derived mesenchymal stem cells, thereby inactivating Wnt/beta-catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc-induced alteration of crucial stem cell-specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells.

DNA-PK contributes to the phosphorylation of AIRE: importance in transcriptional activity.

The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage.

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and alpha2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen.

Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein-protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens.

Pivotal advance: analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin).

HMGB1 (amphoterin) is a 30-kDa heparin-binding protein that mediates transendothelial migration of monocytes and has proinflammatory cytokine-like activities. In this study, we have investigated proinflammatory activities of both highly purified eukaryotic HMGB1 and bacterially produced recombinant HMGB1 proteins. Mass analyses revealed that recombinant eukaryotic HMGB1 has an intrachain disulphide bond. In mass analysis of tissue-derived HMGB1, two forms were detected: the carboxyl terminal glutamic acid residue lacking form and a full-length form. Cell culture studies indicated that both eukaryotic and bacterial HMGB1 proteins induce TNF-alpha secretion and nitric oxide release from mononuclear cells. Affinity chromatography analysis revealed that HMGB1 binds tightly to proinflammatory bacterial substances. A soluble proinflammatory substance was separated from the bacterial recombinant HMGB1 by chloroform-methanol treatment. HMGB1 interacted with phosphatidylserine in both solid-phase binding and cell culture assays, suggesting that HMGB1 may regulate phosphatidylserine-dependent immune reactions. In conclusion, HMGB1 polypeptide has a weak proinflammatory activity by itself, and it binds to bacterial substances, including lipids, that may strengthen its effects.

Identification of antibodies against HAI-1 and integrin alpha6beta4 as immunohistochemical markers of human villous cytotrophoblast.

Syncytiotrophoblast and invasive extravillous trophoblast arise from a common stem cell, namely villous cytotrophoblast, but have very different characteristics. The study of the differentiation process relies on the availability of suitable markers for these different cell types of developing placenta. In this work, we have produced monoclonal antibodies that are specific to human villous cytotrophoblast. Monoclonal antibody (MAb) MG2 was specific to villous cytotrophoblast across gestation, and recognizes hepatocyte growth factor activator inhibitor type 1. MAb MD10 stained villous cytotrophoblast across gestation and also some endothelial cells, particularly in the second or third trimester. MAb MD10 recognizes human integrin alpha6beta4. As a test for specificity, the novel MAbs were also used for staining of frozen tissue from human colon carcinoma. The results show that the two antibodies can be used as tools to study human villous cytotrophoblasts and also human tumors. The MG2 antibody seems most specific and promising for the study of various aspects of human villous cytotrophoblast.

Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by [35S]methionine labelling and two-dimensional gel electrophoresis.

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.

Comparison of the glycosylation of in vitro generated polyclonal human IgG and therapeutic immunoglobulins.

We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography-mass spectrometry, we analyzed IgGs' glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro.

Extracellular o-linked N-acetylglucosamine is enriched in stem cells derived from human umbilical cord blood.

Stem cells have a unique ability to self-renew and differentiate into diverse cell types. Currently, stem cells from various sources are being explored as a promising new treatment for a variety of human diseases. A diverse set of functional and phenotypical markers are used in the characterization of specific therapeutic stem cell populations. The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. We have now discovered by mRNA analysis that a novel glycosyltranferase, epidermal growth factor (EGF) domain-specific O-linked GlcNAc transferase (EOGT), is highly expressed in stem cells. EOGT is responsible for adding O-linked N-acetylglucosamine (O-GlcNAc) to folded EGF domains on extracellular proteins, such as those on the Notch receptors. We were able to show by immunological assays that human umbilical cord blood-derived mesenchymal stromal cells display O-GlcNAc, the product of EOGT, and that O-GlcNAc is further elongated with galactose to form O-linked N-acetyllactosamine. We suggest that these novel glycans are involved in the fine tuning of Notch receptor signaling pathways in stem cells.

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

BACKGROUND: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. METHODS: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. RESULTS: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. CONCLUSIONS: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

Production of a recombinant antibody specific for i blood group antigen, a mesenchymal stem cell marker.

Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology.

Lectin from Erythrina cristagalli supports undifferentiated growth and differentiation of human pluripotent stem cells.

Lectins are carbohydrate-binding proteins, which occur ubiquitously in nature and are abundant in all living organisms from bacteria to mammals. They have several biological functions among which cell adhesion is well known and characterized. Based on the characterization of the glycome of human embryonic stem cells (hESCs), we have investigated the properties of glycan-binding lectins as a novel class of culture support matrices supporting hESC culture. We report that an Erythrina cristagalli lectin (agglutinin) (ECA) matrix supported the undifferentiated growth and significantly increased the plating efficiency of both hESC and human induced pluripotent stem cells when used in conjunction with pinacidil, an antihypertensive drug with ROCK inhibition activity. As a matrix, ECA maintained pluripotency, robust proliferation with a normal karyotype, and the ability to differentiate both in vitro and in vivo. Therefore, our findings indicate that lectins are potential candidates for design of culture and differentiation methods, and that ECA is a potent simple defined matrix for human pluripotent stem cells.

Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.