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1.
Front Endocrinol (Lausanne) ; 14: 1226615, 2023.
Article in English | MEDLINE | ID: mdl-37842306

ABSTRACT

Background: Diabetes mellitus is characterized by chronic hyperglycemia with loss of ß-cell function and mass. An attractive therapeutic approach to treat patients with diabetes in a non-invasive way is to harness the innate regenerative potential of the pancreas. The Islet Neogenesis-Associated Protein pentadecapeptide (INGAP-PP) has been shown to induce ß-cell regeneration and improve their function in rodents. To investigate its possible mechanism of action, we report here the global transcriptional effects induced by the short-term INGAP-PP in vitro treatment of adult rat pancreatic islets. Methods and findings: Rat pancreatic islets were cultured in vitro in the presence of INGAP-PP for 4 days, and RNA-seq was generated from triplicate treated and control islet samples. We performed a de novo rat gene annotation based on the alignment of RNA-seq reads. The list of INGAP-PP-regulated genes was integrated with epigenomic data. Using the new gene annotation generated in this work, we quantified RNA-seq data profiled in INS-1 cells treated with IL1ß, IL1ß+Calcipotriol (a vitamin D agonist) or vehicle, and single-cell RNA-seq data profiled in rat pancreatic islets. We found 1,669 differentially expressed genes by INGAP-PP treatment, including dozens of previously unannotated rat transcripts. Genes differentially expressed by the INGAP-PP treatment included a subset of upregulated transcripts that are associated with vitamin D receptor activation. Supported by epigenomic and single-cell RNA-seq data, we identified 9 previously unannotated long noncoding RNAs (lncRNAs) upregulated by INGAP-PP, some of which are also differentially regulated by IL1ß and vitamin D in ß-cells. These include Ri-lnc1, which is enriched in mature ß-cells. Conclusions: Our results reveal the transcriptional program that could explain the enhancement of INGAP-PP-mediated physiological effects on ß-cell mass and function. We identified novel lncRNAs that are induced by INGAP-PP in rat islets, some of which are selectively expressed in pancreatic ß-cells and downregulated by IL1ß treatment of INS-1 cells. Our results suggest a relevant function for Ri-lnc1 in ß-cells. These findings are expected to provide the basis for a deeper understanding of islet translational results from rodents to humans, with the ultimate goal of designing new therapies for people with diabetes.


Subject(s)
Diabetes Mellitus , Islets of Langerhans , RNA, Long Noncoding , Rats , Humans , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Pancreatitis-Associated Proteins/genetics , Pancreatitis-Associated Proteins/metabolism , Pancreatitis-Associated Proteins/pharmacology , Insulin/metabolism , Islets of Langerhans/metabolism , Peptides/metabolism , Diabetes Mellitus/metabolism , Vitamin D/metabolism
2.
BMC Genomics ; 22(1): 585, 2021 Aug 02.
Article in English | MEDLINE | ID: mdl-34340653

ABSTRACT

BACKGROUND: Loss of pancreatic insulin-secreting ß-cells due to metabolic or autoimmune damage leads to the development of diabetes. The discovery that α-cells can be efficiently reprogrammed into insulin-secreting cells in mice and humans has opened promising avenues for innovative diabetes therapies. ß-cell loss triggers spontaneous reprogramming of only 1-2% of α-cells, limiting the extent of regeneration. Most α-cells are refractory to conversion and their global transcriptomic response to severe ß-cell loss as well as the mechanisms opposing their reprogramming into insulin producers are largely unknown. Here, we performed RNA-seq on FAC-sorted α-cells to characterize their global transcriptional responses at different time points after massive ß-cell ablation. RESULTS: Our results show that α-cells undergo stage-specific transcriptional changes 5- and 15-days post-diphtheria toxin (DT)-mediated ß-cell ablation. At 5 days, α-cells transiently upregulate various genes associated with interferon signaling and proliferation, including Interferon Induced Protein with Tetratricopeptide Repeats 3 (Ifit3). Subsequently, at 15 days post ß-cell ablation, α-cells undergo a transient downregulation of genes from several pathways including Insulin receptor, mTOR and MET signaling. CONCLUSIONS: The results presented here pinpoint novel markers discriminating α-cells at different stages after acute ß-cell loss, and highlight additional signaling pathways that are modulated in α-cells in this context.


Subject(s)
Diabetes Mellitus , Glucagon-Secreting Cells , Insulin-Secreting Cells , Animals , Insulin , Mice , Transcriptome
3.
Stem Cell Reports ; 16(9): 2289-2304, 2021 09 14.
Article in English | MEDLINE | ID: mdl-34450036

ABSTRACT

Heterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting ß-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.


Subject(s)
Cell Differentiation/genetics , Hepatocyte Nuclear Factor 1-beta/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Organogenesis/genetics , Pancreas/embryology , Pancreas/metabolism , Biomarkers , CRISPR-Cas Systems , Cell Lineage/genetics , Diabetes Mellitus/etiology , Disease Susceptibility , Fluorescent Antibody Technique , Gene Editing , Gene Expression Profiling , Gene Expression Regulation, Developmental , Haploinsufficiency , Hepatocyte Nuclear Factor 1-beta/metabolism , Humans , Immunophenotyping , Insulin-Secreting Cells/metabolism , Signal Transduction
4.
Front Endocrinol (Lausanne) ; 12: 692596, 2021.
Article in English | MEDLINE | ID: mdl-34295307

ABSTRACT

The occurrence of diabetes mellitus is characterized by pancreatic ß cell loss and chronic hyperglycemia. While Type 1 and Type 2 diabetes are the most common types, rarer forms involve mutations affecting a single gene. This characteristic has made monogenic diabetes an interesting disease group to model in vitro using human pluripotent stem cells (hPSCs). By altering the genotype of the original hPSCs or by deriving human induced pluripotent stem cells (hiPSCs) from patients with monogenic diabetes, changes in the outcome of the in vitro differentiation protocol can be analyzed in detail to infer the regulatory mechanisms affected by the disease-associated genes. This approach has been so far applied to a diversity of genes/diseases and uncovered new mechanisms. The focus of the present review is to discuss the latest findings obtained by modeling monogenic diabetes using hPSC-derived pancreatic cells generated in vitro. We will specifically focus on the interpretation of these studies, the advantages and limitations of the models used, and the future perspectives for improvement.


Subject(s)
Diabetes Mellitus , Insulin-Secreting Cells/cytology , Models, Biological , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Humans , Pancreas/cytology , Pancreas/growth & development
5.
J Cell Biol ; 219(9)2020 09 07.
Article in English | MEDLINE | ID: mdl-32673398

ABSTRACT

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA-mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.


Subject(s)
Argonaute Proteins/genetics , Estrogens/genetics , Eukaryotic Initiation Factors/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Cell Line , Cell Line, Tumor , Enhancer Elements, Genetic/genetics , Estradiol/genetics , Gene Expression Regulation, Neoplastic/genetics , HEK293 Cells , Humans , MCF-7 Cells , Promoter Regions, Genetic/genetics , Protein Binding/genetics
6.
Nucleic Acids Res ; 47(20): 10645-10661, 2019 11 18.
Article in English | MEDLINE | ID: mdl-31598691

ABSTRACT

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genome, Human , Receptors, Glucocorticoid/metabolism , Receptors, Progesterone/metabolism , Base Sequence , Binding Sites , Breast Neoplasms/pathology , Cell Dedifferentiation/drug effects , Cell Dedifferentiation/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromatin/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Glucocorticoids/pharmacology , Humans , Progestins/pharmacology , Promegestone/pharmacology , Protein Binding/drug effects , Transcription, Genetic/drug effects
7.
Endocr Relat Cancer ; 26(1): 13-29, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30121620

ABSTRACT

Preclinical and clinical studies support that Notch signaling may play an important oncogenic role in cancer, but there is scarce information for pituitary tumors. We therefore undertook a functional study to evaluate Notch participation in pituitary adenoma growth. Tumors generated in Nude mice by subcutaneous GH3 somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower prolactin and GH tumor content and a decrease in angiogenesis. Furthermore, in silico transcriptomic and epigenomic analyses uncovered several tumor suppressor genes related to Notch signaling in pituitary tissue, namely Btg2, Nr4a1, Men1, Zfp36 and Cnot1. Gene evaluation suggested that Btg2, Nr4a1 and Cnot1 may be possible players in GH3 xenograft growth. Btg2 mRNA expression was lower in GH3 tumors compared to the parental line, and DAPT increased its expression levels in the tumor in parallel with the inhibition of its volume. Cnot1 mRNA levels were also increased in the pituitary xenografts by DAPT treatment. And the Nr4a1 gene was lower in tumors compared to the parental line, though not modified by DAPT. Finally, because DAPT in vivo may also be acting on tumor microenvironment, we determined the direct effect of DAPT on GH3 cells in vitro. We found that DAPT decreases the proliferative, secretory and migration potential of GH3 cells. These results position selective interruption of Notch signaling as a potential therapeutic tool in adjuvant treatments for aggressive or resistant pituitary tumors.


Subject(s)
Adenoma/pathology , Pituitary Neoplasms/pathology , Receptors, Notch/antagonists & inhibitors , Adenoma/metabolism , Animals , Cell Line, Tumor , Diamines/pharmacology , Female , Mice, Inbred BALB C , Mice, Nude , Pituitary Neoplasms/metabolism , Prolactin/metabolism , Rats , Receptors, Notch/metabolism , Signal Transduction , Thiazoles/pharmacology , Tumor Burden
9.
Nat Cell Biol ; 17(5): 615-626, 2015 May.
Article in English | MEDLINE | ID: mdl-25915126

ABSTRACT

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/metabolism , Multipotent Stem Cells/metabolism , Nuclear Proteins/metabolism , Pancreas/metabolism , Phosphoproteins/metabolism , Signal Transduction , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Animals, Genetically Modified , Cell Differentiation , Cell Lineage , Cell Proliferation , Cells, Cultured , Computational Biology , DNA-Binding Proteins/genetics , Databases, Genetic , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Humans , Mice, Inbred C57BL , Nuclear Proteins/genetics , Organogenesis , Pancreas/embryology , Phenotype , Phosphoproteins/genetics , RNA, Messenger/metabolism , TEA Domain Transcription Factors , Time Factors , Transcription Factors/genetics , YAP-Signaling Proteins , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism
10.
Clin Cancer Res ; 20(15): 4086-95, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24842953

ABSTRACT

PURPOSE: Clinical and epidemiologic data suggest that obesity is associated with more aggressive forms of prostate cancer, poor prognosis, and increased mortality. C-terminal-binding protein 1 (CtBP1) is a transcription repressor of tumor suppressor genes and is activated by NADH binding. High calorie intake decreases intracellular NAD(+)/NADH ratio. The aim of this work was to assess the effect of high-fat diet (HFD) and CtBP1 expression modulation over prostate xenograft growth. EXPERIMENTAL DESIGN: We developed a metabolic syndrome-like disease in vivo model by feeding male nude mice with HFD during 16 weeks. Control diet (CD)-fed animals were maintained at the same conditions. Mice were inoculated with PC3 cells stable transfected with shCtBP1 or control plasmids. Genome-wide expression profiles and Gene Set Enrichment Analysis (GSEA) were performed from PC3.shCtBP1 versus PC3.pGIPZ HFD-fed mice tumors. RESULTS: No significant differences were observed in tumor growth on CD-fed mice; however, we found that only 60% of HFD-fed mice inoculated with CtBP1-depleted cells developed a tumor. Moreover these tumors were significantly smaller than those generated by PC3.pGIPZ control xenografts. We found 823 genes differentially expressed in shCtBP1 tumors from HFD-fed mice. GSEA from expression dataset showed that most of these genes correspond to cell adhesion, metabolic process, and cell cycle. CONCLUSIONS: Metabolic syndrome-like diseases and CtBP1 expression cooperate to induce prostate tumor growth. Hence, targeting of CtBP1 expression might be considered for prostate cancer management and therapy in the subset of patients with metabolic syndromes.


Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Diet, High-Fat/adverse effects , Metabolic Syndrome/prevention & control , Obesity/prevention & control , Prostatic Neoplasms/prevention & control , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Adhesion , Cell Proliferation , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , Chromatin Immunoprecipitation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Gonadal Steroid Hormones/pharmacology , Humans , Immunoenzyme Techniques , Male , Metabolic Syndrome/etiology , Metabolic Syndrome/pathology , Mice , Mice, Nude , Obesity/etiology , Obesity/pathology , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
11.
Nat Genet ; 46(2): 136-143, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24413736

ABSTRACT

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Gene Regulatory Networks/genetics , Islets of Langerhans/metabolism , Transcription Factors/metabolism , Base Sequence , Chromatin/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation , Diabetes Mellitus, Type 2/metabolism , Electrophoretic Mobility Shift Assay , Formaldehyde , Genome-Wide Association Study , Humans , Molecular Sequence Data , Sequence Analysis, RNA , Transcription Factors/genetics , Web Browser
12.
Nat Genet ; 46(1): 61-64, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24212882

ABSTRACT

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ~400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease.


Subject(s)
Enhancer Elements, Genetic/genetics , Mutation , Pancreas/abnormalities , Pancreatic Diseases/congenital , Transcription Factors/genetics , Chromosomes, Human, Pair 10 , Embryonic Stem Cells/physiology , Epigenomics/methods , Female , Genes, Recessive , Humans , Male , Pancreatic Diseases/genetics , Pedigree
13.
J Mater Sci Mater Med ; 24(1): 189-98, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23080375

ABSTRACT

Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.


Subject(s)
Cell Differentiation , Cellular Microenvironment , Animals , Bone Morphogenetic Protein 2/metabolism , CHO Cells , Cricetinae , Cricetulus , Humans , Mice , Protein Array Analysis
14.
Cell Metab ; 16(4): 435-48, 2012 Oct 03.
Article in English | MEDLINE | ID: mdl-23040067

ABSTRACT

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and ß cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the ß cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a ß cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to ß cell programming and diabetes pathophysiology.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/metabolism , RNA, Long Noncoding/metabolism , Animals , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins , Diabetes Mellitus, Type 2/pathology , Down-Regulation , Gene Expression Profiling , Genetic Loci , Humans , Mice , RNA, Messenger/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
15.
J Clin Invest ; 122(10): 3469-71, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23006323

ABSTRACT

Understanding the transcriptional mechanisms that underlie pancreas formation is central to the efforts to develop novel regenerative therapies for type 1 diabetes. Recently, mutations in the transcription factor GATA6 were unexpectedly shown to be the most common cause of human pancreas agenesis. In this issue of the JCI, Carrasco et al. and Xuan et al. investigate the role of Gata6 and its paralogue Gata4 in mouse embryonic pancreas and show that GATA factors are essential regulators of the proliferation, morphogenesis, and differentiation of multipotent pancreatic progenitors.

16.
Methods Mol Biol ; 791: 287-96, 2011.
Article in English | MEDLINE | ID: mdl-21913087

ABSTRACT

Noncoding regulatory genomic elements are central for cellular function, differentiation, and disease, but remain poorly characterized. FAIRE (formaldehyde-assisted isolation of regulatory elements) has emerged as a simple method to identify and analyze active regulatory sequences based on their decreased nucleosomal content. More recently FAIRE was combined with high-throughput sequencing (FAIRE-seq) to locate tissue-specific regulatory elements at a genome scale in purified human pancreatic islets. Here we describe the implementation of the FAIRE method in human pancreatic islet cells.


Subject(s)
Chromatin/genetics , Formaldehyde/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Cell Line , Chemical Fractionation , Chloroform/chemistry , DNA/genetics , DNA/isolation & purification , Humans , Islets of Langerhans/cytology , Phenol/chemistry , Sonication , Tissue Culture Techniques , Tissue Fixation
17.
J Biomed Mater Res A ; 98(2): 245-56, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21626656

ABSTRACT

Cellular microarray developments and its applications are the next step after DNA and protein microarrays. The choice of the surface chemistry of the substrates used for the implementation of this technique, that must favor proper protein immobilization while avoiding cell adhesion on the nonspotted areas, presents a complex challenge. This is a key issue since usually the best nonfouling surfaces are also the ones that retain immobilized the smallest amounts of printed protein. To quantitatively assess the amount of protein immobilization, in this study several combinations of fluorescently labeled fibronectin (Fn*) and streptavidin (SA*) were microspotted, with and without glycerol addition in the printing buffer, on several substrates suitable for cellular microarrays. The substrates assayed included chemically activated surfaces as well as Poly ethylene oxide (PEO) films that are nonfouling in solution but accept adhesion of proteins in dry conditions. The results showed that the spotted Fn* was retained by all the surfaces, although the PEO surface did show smaller amounts of immobilization. The SA*, on the other hand, was only retained by the chemically activated surfaces. The inclusion of glycerol in the printing buffer significantly reduced the immobilization of both proteins. The results presented in this article provide quantitative evidence of the convenience of using a chemically activated surface to immobilize proteins relevant for cellular microarray applications, particularly when ECM proteins are cospotted with smaller factors which are more difficult to be retained by the surfaces.


Subject(s)
Fibronectins/analysis , Immobilized Proteins/analysis , Myoblasts/metabolism , Streptavidin/analysis , Tissue Array Analysis/methods , Animals , Calibration , Cell Proliferation , Cell Survival , Fluorescence , Humans , Mice , Molecular Weight , Myoblasts/cytology
18.
J Mater Sci Mater Med ; 20(7): 1525-33, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19301106

ABSTRACT

Lately there has been an increasing interest in the development of tools that enable the high throughput analysis of combinations of surface-immobilized signaling factors and which examine their effect on stem cell biology and differentiation. These surface-immobilized factors function as artificial microenvironments that can be ordered in a microarray format. These microarrays could be useful for applications such as the study of stem cell biology to get a deeper understanding of their differentiation process. Here, the evaluation of several key process parameters affecting the cellular microarray fabrication is reported in terms of its effects on the mesenchymal stem cell culture time on these microarrays. Substrate and protein solution requirements, passivation strategies and cell culture conditions are investigated. The results described in this article serve as a basis for the future development of cellular microarrays aiming to provide a deeper understanding of the stem cell differentiation process.


Subject(s)
Cell Culture Techniques/instrumentation , Flow Cytometry/instrumentation , Microarray Analysis/instrumentation , Microfluidic Analytical Techniques/instrumentation , Proteome/analysis , Stem Cells/metabolism , Animals , Cell Culture Techniques/methods , Equipment Design , Flow Cytometry/methods , Humans , Microarray Analysis/methods , Microfluidic Analytical Techniques/methods , Stem Cells/cytology , Technology Assessment, Biomedical
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