Proteomic identification of biomarkers expressed by human pluripotent stem cells

The ability to effectively monitor the behaviour of pluripotent stem cells and their differentiation is key to their use in basic and clinical research. Molecules expressed in particular cell types can be used to report the status of cell differentiation and is a recognised means of assessing the behaviour of cell cultures. There are currently few useful markers of stem cells and there is no rapid way to accurately determine their level of expression. In this study, we describe for the first time the potential of surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) to identify novel biomarkers of human pluripotent embryonal carcinoma stem cells and their differentiated derivatives. This approach allows the rapid and sensitive screening of cell samples without the need to purify the specimen prior to analysis. The identification of proteins expressed in specific cell populations will provide valuable tools for monitoring cellular development.     

The type III-9 repeat of human fibronectin is encoded by a single exon which is not alternatively spliced.

Type III homologies of human fibronectin are generally encoded by two exons, with the exception of the ED-A and ED-B repeats which are encoded by a single exon undergoing alternative splicing. We report that also the type III-9 homology is encoded by a single exon. Further more, RT-PCR analysis, performed on mRNA purified from fetal and adult tissues and from normal and tumor-derived cell types, showed that the III-9 region is not undergoing alternative splicing in all samples tested.     

Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8- 10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin- like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.     

Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.     

Cell fate potential of human pluripotent stem cells is encoded by histone modifications

Human embryonic stem cells (hESCs) expressing pluripotency markers are assumed to possess equipotent developmental potential. However, disparate responses to differentiation stimuli functionally illustrate that hESCs generate a spectrum of differentiated cell types, suggestive of lineage bias. Here, we reveal specific cell surface markers that allow subfractionation of hESCs expressing hallmark markers of pluripotency. By direct de novo isolation of these?subsets, we demonstrate that propensities for lineage differentiation are balanced with reduced clonogenic self-renewal. Histone modification marks of gene loci associated with pluripotency versus lineage specificity predicted cell fate potential of these subfractions, thereby supporting the absence of uniform bivalency as a molecular paradigm to describe cell fate determination of pluripotent cells. Our study reveals that cell fate potential is encoded within cells comprising hESC cultures, highlighting them as a means to understand the mechanisms of lineage specification of pluripotent cells.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

An alternatively spliced variant of cathepsin E in human gastric adenocarcinoma cells

Splice variant and authentic mRNAs for procathepsin E were measured at a ratio of 5:1 and 1:2 in Kato 3 and AGS cells, two human gastric adenocarcinoma cell lines. As a result of the precise splicing of the 3'-end of exon 6 to the 5'-end of exon 8, the variant lacked the 142 bp of exon 7 which encodes the second of the Asp residues that operate the catalytic mechanism of aspartic proteinases.     

A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

High-throughput karyotyping of human pluripotent stem cells

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner.     

Engineering human cells and tissues through pluripotent stem cells

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