Pancreatic islet and progenitor cell surface markers with cell sorting potential.

AIMS/HYPOTHESIS: The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation. METHODS: Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates. RESULTS: In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)⁺/NK6 homeobox 1 (NKX6-1)⁺ multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)⁺ cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting. CONCLUSIONS/INTERPRETATION: Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.

Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Characterization of unknown adult stem cell samples by large scale data integration and artificial neural networks.

Stem cells represent not only a potential source of treatment for degenerative diseases but can also shed light on developmental biology and cancer. It is believed that stem cells differentiation and fate is triggered by a common genetic program that endows those cells with the ability to differentiate into specialized progenitors and fully differentiated cells. To extract the stemness signature of several cells types at the transcription level, we integrated heterogeneous datasets (microarray experiments) performed in different adult and embryonic tissues (liver, blood, bone, prostate and stomach in Homo sapiens and Mus musculus). Data were integrated by generalization of the hematopoietic stem cell hierarchy and by homology between mouse and human. The variation-filtered and integrated gene expression dataset was fed to a single-layered neural network to create a classifier to (i) extract the stemness signature and (ii) characterize unknown stem cell tissue samples by attribution of a stem cell differentiation stage. We were able to characterize mouse stomach progenitor and human prostate progenitor samples and isolate gene signatures playing a fundamental role for every level of the generalized stem cell hierarchy.

RAD and the RAD Study-Annotator: an approach to collection, organization and exchange of all relevant information for high-throughput gene expression studies.

MOTIVATION: Gene expression array technology has become increasingly widespread among researchers who recognize its numerous promises. At the same time, bench biologists and bioinformaticians have come to appreciate increasingly the importance of establishing a collaborative dialog from the onset of a study and of collecting and exchanging detailed information on the many experimental and computational procedures using a structured mechanism. This is crucial for adequate analyses of this kind of data. RESULTS: The RNA Abundance Database (RAD; http://www.cbil.upenn.edu/RAD) provides a comprehensive MIAME-supportive infrastructure for gene expression data management and makes extensive use of ontologies. Specific details on protocols, biomaterials, study designs, etc. are collected through a user-friendly suite of web annotation forms. Software has been developed to generate MAGE-ML documents to enable easy export of studies stored in RAD to any other database accepting data in this format (e.g. ArrayExpress). RAD is part of a more general Genomics Unified Schema (http://www.gusdb.org), which includes a richly annotated gene index (http://www.allgenes.org), thus providing a platform that integrates genomic and transcriptomic data from multiple organisms. This infrastructure enables a large variety of queries that incorporate visualization and analysis tools and have been tailored to serve the specific needs of projects focusing on particular organisms or biological systems.

Maintaining data integrity in microarray data management.

Gene expression microarrays are a relatively new technology, dating back just a few years, yet they have already become a very widely used tool in biology, and have evolved to a wide range of applications well beyond their original design intent. However, while the use of microarrays has expanded, and the issues of performance optimization have been intensively studied, the fundamental issue of data integrity management has largely been ignored. Now that performance has improved so greatly, the shortcomings of data integrity control methods constitute a greater percent of the stumbling blocks for investigators. Microarray data are cumbersome, and the rule up to this point has mostly been one of hands-on transformations, leading to human errors which often have dramatic consequences. We show in this review that the time lost on such mistakes is enormous and dramatically affects results; therefore, mistakes should be mitigated in any way possible. We outline the scope of the data integrity issue, to survey some of the most common and dangerous data transformations, and their shortcomings. To illustrate, we review some case studies. We then look at the work done by the research community on this issue (which admittedly is meager up to this point). Some data integrity issues are always going to be difficult, while others will become easier-one of our goals is to expedite the use of integrity control methods. Finally, we present some preliminary guidelines and some specific approaches that we believe should be the focus of future research.

Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.

Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.

High-resolution BAC-based map of the central portion of mouse chromosome 5.

The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable.

A relational schema for both array-based and SAGE gene expression experiments.

MOTIVATION AND RESULTS: A relational schema is described for capturing highly parallel gene expression experiments using different technologies. This schema grew out of efforts to build a database for collaborators working on different biological systems and using different types of platforms in their gene expression experiments as well as different types of image quantification software. The tables are conceptually organized into three categories of information: Platform, Experiment (which includes image scanning and quantification), and Data. The strengths of the schema are: (i) integrating information on array elements using a gene index; (ii) describing samples using ontologies; (iii) reducing an experiment to a single RNA source for precise descriptions yet not losing the relationships between experiments done at the same time or for the same project; and (iv) maintaining both raw and processed (e.g. cleansed and normalized) data and recording how the data is processed. The result is a novel schema, which can hold both array and non-array data, is extensible for detailed experimental descriptions that are precise and consistent, and allows for meaningful comparisons of genes between experiments.

Generation of patterns from gene expression data by assigning confidence to differentially expressed genes.

MOTIVATION: A protocol is described to attach expression patterns to genes represented in a collection of hybridization array experiments. Discrete values are used to provide an easily interpretable description of differential expression. Binning cutoffs for each sample type are chosen automatically, depending on the desired false-positive rate for the predictions of differential expression. Confidence levels are derived for the statement that changes in observed levels represent true changes in expression. We have a novel method for calculating this confidence, which gives better results than the standard methods. Our method reflects the broader change of focus in the field from studying a few genes with many replicates to studying many (possibly thousands) of genes simultaneously, but with relatively few replicates. Our approach differs from standard methods in that it exploits the fact that there are many genes on the arrays. These are used to estimate for each sample type an appropriate distribution that is employed to control the false-positive rate of the predictions made. Satisfactory results can be obtained using this method with as few as two replicates. RESULTS: The method is illustrated through applications to macroarray and microarray datasets. The first is an erythroid development dataset that we have generated using nylon filter arrays. Clones for genes whose expression is known in these cells were assigned expression patterns which are in accordance with what was expected and which are not picked up by the standards methods. Moreover, genes differentially expressed between normal and leukemic cells were identified. These included genes whose expression was altered upon induction of the leukemic cells to differentiate. The second application is to the microarray data by Alizadeh et al. (2000). Our results are in accordance with their major findings and offer confidence measures for the predictions made. They also provide new insights for further analysis.

Maturation and developmental stage-related changes in fetal globin gene expression are reproduced in transiently transfected primary adult human erythroblasts.

A novel system is described, which uses transfection of primary human erythroblasts for the study of gene regulation in differentiating human red cells. This system includes a protocol for liquid culture of erythroid progenitors, which reproduces developmental differences in globin gene expression found between adult and cord blood as well as the maturation-related changes in fetal globin levels observed in adult cells. Reporter constructs driven by globin gene promoters were electroporated into adult and cord blood-derived erythroblasts at different time points during culture. Both the developmental stage and maturation-related differences in endogenous fetal and adult globin gene expression could be reproduced by the transiently transfected reporter constructs. Transfection of primary human erythroblasts during differentiation provides a previously unavailable opportunity to study dynamic aspects of erythropoiesis.

EpoDB: a prototype database for the analysis of genes expressed during vertebrate erythropoiesis.

EpoDB is a database of genes expressed in vertebrate red blood cells. It is also a prototype for the creation of cell and tissue-specific databases from multiple external sources. The information in EpoDB obtained from GenBank, SWISS-PROT, Transfac, TRRD and GERD is curated to provide high quality data for sequence analysis aimed at understanding gene regulation during erythropoiesis. New protocols have been developed for data integration and updating entries. Using a BLAST-based algorithm, we have grouped GenBank entries representing the same gene together. This sequence similarity protocol was also used to identify new entries to be included in EpoDB. We have recently implemented our database in Sybase (relational tables) in addition to SICStus Prolog to provide us with greater flexibility in asking complex queries that utilize information from multiple sources. New additions to the public web site (http://www.cbil.upenn.edu/epodb) for accessing EpoDB are the ability to retrieve groups of entries representing different variants of the same gene and to retrieve gene expression data. The BLAST query has been enhanced by incorporating BLASTView, an interactive and graphical display of BLAST results. We have also enhanced the queries for retrieving sequence from specified genes by the addition of MEME, a motif discovery tool, to the integrated analysis tools which include CLUSTALW and TESS.

EpoDB: a database of genes expressed during vertebrate erythropoiesis.

EpoDB is a database designed for the study of gene regulation during differentiation and development of vertebrate red blood cells. In building EpoDB, we have taken the in advance approach to the data integration problem: we have extracted data relevant to red blood cells from GenBank, SWISS-PROT, TRRD (transcriptional regulation data) and GERD (expression levels data) to create a single integrated, highly curated view. Tools have been developed to automate data extraction from online resources, cleanse data of errors, enter information manually from the primary literature, generate a uniform, canonical representation of information and maintain data currency. The database is organized around biological features, e.g., genes, rather than sequences, which are supported by a controlled and consistent vocabulary for gene names and gene family names. Beyond the standard database queries, the functionality of EpoDB includes the ability to extract features and subsequences, display sequences and features graphically using bioWidget viewers and integrated analysis tools. EpoDB may be accessed at: http://cbil.humgen.upenn.edu/epodb/

The myosin filament XV assembly: contributions of 195 residue segments of the myosin rod and the eight C-terminal residues.

A mixture of two peptides of approximately M(r) 13000 has been isolated from a papain digest of LC2 deficient myosin. The peptides assemble into highly ordered aggregates which in one view are made up of strands of pairs of dots with an average side to side spacing of 13.0 nm and an average axial repeat of 9.0 nm. In another view there are strands of single dots with a side-to-side spacing of 7.8 nm and an axial repeat of 9.1 nm. From N-terminal peptide sequencing, the two peptides have been shown to come from regions of the myosin rod displaced by 195 residues. We have shown that either peptide alone can assemble to form the same aggregates. The 195 residue displacement of the M(r) 13000 peptides corresponds closely to the 196 residue repeat of charges along the myosin rod. This finding permits us to designate 195 residue segments of the myosin rod and to relate assembly characteristics directly to the similar 195 residue segments and 196 residue charge repeat. The most C-terminal 195 residue segment carries information for assembly into helical strands. The contiguous 195 residue segment, in major part, carries information for the unipolar assembly, characteristic of the assembly in each half of the myosin filament. The next contiguous 195 residue segment, in major part, carries information for bipolar assembly which is characteristic of the bare zone region of the filament; and for the transition from the bipolar bare zone to unipolar assembly. The effect of the eight C-terminal residues of the myosin rod on the assembly of the contiguous 195 residues has also been studied. The entire fragment of 195 + eight C-terminal residues assembled to form helical strands with an axial repeat of 30 nm. Successive deletion of charged residues changed the axial repeat of the helical strands suggesting that the charged residues at the C-terminus are involved in determining the pitch in the helical assembly of the contiguous 195 residues.

Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.

Allogeneic transplantation of islets of Langerhans was facilitated by the cotransplantation of syngeneic myoblasts genetically engineered to express the Fas ligand (FasL). Composite grafting of allogeneic islets with syngeneic myoblasts expressing FasL protected the islet graft from immune rejection and maintained normoglycemia for more than 80 days in mice with streptozotocin-induced diabetes. Graft survival was not prolonged with composite grafts of unmodified myoblasts or Fas-expressing myoblasts. Islet allografts transplanted separately from FasL-expressing myoblasts into the contralateral kidney were rejected, as were similarly transplanted third-party thyroid allografts. Thus, the FasL signal provided site- and immune-specific protection of islet allografts.

Partial repression of human gamma-globin genes by LCR element HS3 when linked to beta-globin genes and LCR element HS2 in MEL cells.

Clues for overcoming fetal (gamma-) globin gene repression in adult human erythroid cells may come from understanding why repression of isolated gamma-globin genes has not previously been achieved in the adult erythroid environment of mouse erythroleukemia cells (MEL). Repression of human gamma-globin genes has been demonstrated in MEL cells when transferred as part of the entire beta-globin gene cluster packaged in chromatin. Major differences in these approaches are prior packaging into chromatin and the presence of additional sequences, notably from the locus control region (LCR). In this report we focus on the contribution to gamma-globin gene repression that multiple elements of the LCR may have. We first show preferential activation of beta-globin genes over gamma-globin genes in MEL cells when linked to each other and to LCR sequences containing the core elements of DNase I hypersensitive sites 4, 3, and 2. Removal of the HS4 element had no effect, however, removal of the 225 bp HS3 core element resulted in a five-fold increase in gamma-globin gene expression. The enhancer 3' to the A gamma-globin gene also had no apparent effect on gamma-globin gene expression. These results provide first evidence of gamma-globin gene repression involving the core region of HS3 in the presence of the core region of HS2 and a beta-globin gene. A mechanism for repression involving sequestration of the gamma-promoter away from the strong enhancer activity of HS2 is proposed.

Immunomodulation of pancreatic islet allografts in mice with CTLA4Ig secreting muscle cells.

In an effort to create a model of in vivo production of immunosuppressants, we have transfected C2C12 muscle cells (H-2k) with the cDNA for CTLA4Ig, a fusion protein that prevents the activation of T cells by blocking the costimulatory signal transduced by the T cell receptors CD28 and CTLA4. CTLA4Ig-secreting clones were cotransplanted with islets as composite grafts in the renal subcapsular space of diabetic mice. When the myoblasts were syngeneic to C3H/HeJ hosts (H-2k), there was a significant prolongation of survival of allogeneic C57Bl/6J (H-2b) islets from a mean 11.0 days to 31.7 days. When the graft was completely allogeneic (H-2k myoblasts and islets into H-2b recipients), there was no benefit in survival. A transient blockade of LFA-1 with the mAb M17 was synergistic in this combination: 8 out of 12 C57Bl/6J recipients achieved long-term acceptance. Systemic CTLA4Ig levels were detected up to 60 days after transplantation. In conclusion, we have shown that C2C12 muscle cells can be genetically engineered to secrete functional CTLA4Ig and that they can be used as a gene reservoir for the continuous in vivo production of CTLA4Ig to modulate the survival of islet cell allografts.

Erythroid progenitors in the peripheral blood of children with sickle cell disease.

PURPOSE: The goals of this study were (a) to determine the number of peripheral blood burst forming units-erythroid (BFU-E); (b) to define the relationship between circulating BFU-E number and fetal hemoglobin (HbF) level; and (c) to define the relationship between BFU-E number and age in pediatric sickle cell disease (SCD) patients. PATIENTS AND METHODS: Fetal hemoglobin (HbF) level and peripheral blood BFU-E number were determined in children < 18 years of age with SCD in a steady state of their disease. These data were compared with those of normal children. RESULTS: An increased number of BFU-E was observed in the peripheral blood of children with SCD compared with normals (30.7 vs. 15.7 per 10(5) mononuclear cells, respectively; p = 0.009). Overall there was the suggestion of a direct relationship between HbF level and peripheral blood BFU-E number (regression coefficient = 0.445; p = 0.06). Additionally, a strong inverse relationship between BFU-E number and age (regression coefficient = -0.671; p < 0.0001) was observed. CONCLUSIONS: In children with SCD (a) there are an increased number of peripheral blood BFU-E compared with normal children; (b) the inverse relationship between HbF level and BFU-E number observed in adult SCD patients is not seen in children; and (c) there is a strong inverse relationship between age and BFU-E number. This information may help to further clarify the relationship between peripheral blood BFU-E and erythropoietic stress.