Dataset Information

Human islet of Langerhans grown on nanosubstrates LC-MSMS

ABSTRACT: Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. In the present study we evaluate the contribution of the physical properties of the extracellular matrix in supporting long term culture of human islets in vitro. We used metal oxide layers with tailored nanoscale roughness to fabricate scaffolds. Their suitability to support long term culture of human islets was investigated through assessment of β-cell survival, differentiation and function by using a proteomic approach to evaluate the molecular mechanisms involved. Proteomic analysis confirmed that ns-ZrOx promoted the activation of a transcriptional and translational program leading to activation of pro-survival and pro-differentiation signalling pathways. The process was driven by a mechanotransductive pathway driven by nanostructured topology, via remodelling of the actin cytoskeleton nuclear architecture and increase of oxygen sensor PDH2 Our data suggest that β-cells relay on nanotopography features to regulate their differentiation and survival. Tailored nanostructured substrates may provide a unique strategy to identify novel hints for tissue engineering and molecular /pharmacological targets of intervention to treat diabetes mellitus.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Islet Of Langerhans

SUBMITTER: gabriella tedeschi

LAB HEAD: Gabriella Tedeschi

PROVIDER: PXD007569 | Pride | 2018-07-11

REPOSITORIES: Pride

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Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Galli Alessandra A Maffioli Elisa E Sogne Elisa E Moretti Stefania S Di Cairano Eliana Sara ES Negri Armando A Nonnis Simona S Norata Giuseppe Danilo GD Bonacina Fabrizia F Borghi Francesca F Podestà Alessandro A Bertuzzi Federico F Milani Paolo P Lenardi Cristina C Tedeschi Gabriella G Perego Carla C

Scientific reports 20180702 1

Ex vivo expansion and differentiation of human pancreatic β-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which β-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates ...[more]

PMID: 29967323

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Project description:The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet Î²-cell development, but its effects on adult Î²-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-Î²GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are Î² cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating Î²-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of Î²-cell mass in treatment of both major forms of diabetes. Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme. 5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

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