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.

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.

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis.

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.

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.

Expression of the affected A gamma globin gene associated with Greek nondeletion hereditary persistence of fetal hemoglobin.

The overexpressed A gamma globin gene in the Greek type of nondeletion hereditary persistence of fetal hemoglobin has a unique single-base substitution located at position -117 relative to the site of transcription initiation. This gene and its normal counterpart were transferred into cultured cell lines by using a retroviral vector. The only difference in expression between the transferred normal and mutant gamma genes was observed in the human erythroleukemia cell line KMOE after exposure of the cells to cytosine arabinoside, a condition that resulted in an adult pattern of endogenous globin gene expression by the cells and was associated with increased expression of the mutant gene.

Histone positions within the nucleosome using platinum labeling and the scanning transmission electron microscope.

An approach to studying the organization of macromolecular complexes using heavy-atom labeling has been developed and applied to the problem of determining the positions of the histone proteins within the nucleosome. The approach is based on the capability of the scanning transmission electron microscope to image heavy atoms. Nucleosomes containing histones labeled with heavy atoms were prepared by lysine modification of selected histones with methyl (methylthio)acetimidate, followed by reconstitution of the modified histones into nucleosomes, and reaction of the reconstituted nucleosomes with chloroglycyl-1-methioninatoplatinum (II). Micrographs of the platinum-labeled nucleosomes were obtained using the scanning transmission electron microscope, and analyzed using both computer and manual techniques. The results of the analysis were 24 A resolution maps of the distribution of high electron scattering density picture elements (representative of platinum atoms) indicating the position of each histone. The significance of those results and the general applicability of the platinum-labeling techniques are discussed. Finally, a description of the histone positions within the nucleosomes is presented and discussed in relation to the current literature on nucleosome structure.

Erythropoietin and hydroxyurea can act on early erythroid progenitors from adult human peripheral blood to modulate fetal globin mRNA levels.

Fetal hemoglobin production in adult human erythroid progenitors can be influenced by exogenous factors. In this report, we examine the effects of erythropoietin (Epo) and hydroxyurea (HU) in serum-free media on fetal (gamma-) globin mRNA levels when present during the first week of peripheral blood burst-forming unit-erythroid (BFU-E) culture. Fetal calf serum (FCS) is known to elevate gamma-globin mRNA levels in BFU-E culture and has been shown to do so even when added after the first week of culture. Removal of FCS from the first week of our BFU-E cultures did not significantly reduce gamma-globin mRNA levels, in agreement with these earlier findings. Addition of Epo to BFU-E cultures 4 days before addition of FCS led to a significant drop in gamma-globin mRNA levels; Epo addition also led to a significant increase in the number of erythroid bursts. Culture conditions, including Epo in serum-free liquid medium for 1 week followed by plating in semisolid medium containing FCS and Epo, resulted in low gamma-globin mRNA levels, similar to levels found in vivo, and high burst numbers. These conditions were used to demonstrate significant elevation of gamma-globin mRNA levels by HU present during the first week of BFU-E culture. Our findings suggest that gamma-globin mRNA levels can be influenced early in erythropoiesis by Epo and HU.

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.

Retroviral transfer of genes into erythroid progenitors derived from human peripheral blood.

Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.

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.

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.

Johns Hopkins University, Baltimore, Maryland.

The STEM can be used in one of three modes: 1) to image individual atoms; 2) to measure mass or molecular weight; 3) to collect electron energy loss spectra or x-ray fluorescence data. Heavy atom imaging is used to identify chemical groups in a molecule or macromolecules in an assembly. Specific labels have been developed for bases in nucleic acids. These permit localization of bound proteins on single strand nucleic acids. Pt(gly-L-met)Cl is a specific label for methionine residues of proteins as shown with the SLS aggregate of collagen. Lysine can be labeled as well if first methyl (methyl-thio-acetimidate) is coupled. This labeling procedure permits the localization of individual histones within a nucleosome. Mass determination can be used to answer crucial questions about biological assemblies. This is demonstrated by examples from muscle structure.

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.

Retroviral transfer of a human fetal globin gene carrying the -202 G gamma beta (+)-HPFH mutation into the human erythroleukemia line, KMOE.

The presence of point mutations at position -202 relative to the mRNA Cap site of both human fetal gamma-globin genes is linked with elevated fetal globin levels in adults. The question addressed in this study is whether the -202 mutation affects gamma-globin gene expression in the same manner as the -117 hereditary persistence of fetal hemoglobin (HPFH) A gamma-globin mutation. The -117 mutation was found to cause over-expression and confer inducibility of a retrovirally transferred gamma-globin gene in cytosine arabinoside (araC)-treated KMOE cells in an earlier study. In this study, fetal globin genes driven by either the normal G gamma or -202 HPFH G gamma-globin promoter were retrovirally transferred into human erythroid KMOE cells. The -202 HPFH mutation did not cause over-expression or confer inducibility of the transferred gamma-globin gene in araC-treated KMOE cells. Thus, the -202 HPFH mutation affects gamma-globin gene expression by a different mechanism than the -117 HPFH mutation. Furthermore, this study provides evidence against a general increasing of gamma-globin gene expression as might be expected from the -202 mutation altering binding of a ubiquitous factor such as Sp1.

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.