scholarly journals Microporous scaffolds drive assembly and maturation of progenitors into β-cell clusters

2019 ◽  
Author(s):  
Richard L. Youngblood ◽  
Joshua P. Sampson ◽  
Kimberly R. Lebioda ◽  
Graham Spicer ◽  
Lonnie D. Shea
Keyword(s):  
Basement Membrane ◽  
Suspension Cultures ◽  
Cell Interactions ◽  
Cell Maturation ◽  
Β Cells ◽  
Β Cell ◽  
Cell Clusters ◽  
Matrix Deposition ◽  
Cell Cell

AbstractHuman pluripotent stem cells (hPSCs) represent a promising cell source for the development of β-cells for use in therapies for type 1 diabetes. Current culture approaches provide the signals to drive differentiation towards β-cells, with the cells spontaneously assembling into clusters. Herein, we adapted the current culture systems to cells seeded on microporous biomaterials, with the hypothesis that the pores can guide the assembly into β-cell clusters of defined size that can enhance maturation. The microporous scaffold culture allows hPSC-derived pancreatic progenitors to form clusters at a consistent size as cells undergo differentiation to immature β-cells. By modulating the scaffold pore sizes, we observed 250-425 µm pore size scaffolds significantly enhance insulin expression and key β-cell maturation markers compared to suspension cultures. Furthermore, when compared to suspension cultures, the scaffold culture showed increased insulin secretion in response to glucose stimulus indicating the development of functional β-cells. In addition, scaffolds facilitated cell-cell interactions enabled by the scaffold design and cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells. We further investigated the influence of ECM on cell development by incorporating an ECM matrix on the scaffold prior to cell seeding; however, their presence did not further enhance maturation. These results suggest the microporous scaffold culture facilitates 3D cluster formation, supports cell-cell interactions, and provides a matrix similar to a basement membrane to drive in vitro hPSC-derived β-cell maturation and demonstrates the feasibility of these scaffolds as a biomanufacturing platform.

scholarly journals In vivo Imaging β-cell Function Reveals Two Waves of β-cell Maturation

2017 ◽  
Author(s):  
Jia Zhao ◽  
Weijian Zong ◽  
Yi Wu ◽  
Jiayu Shen ◽  
Dongzhou Gou ◽  
...  
Keyword(s):  
Cell Function ◽  
Light Sheet ◽  
Cell Maturation ◽  
Β Cells ◽  
Β Cell ◽  
New Strategy ◽  
Β Cell Function ◽  
Insulin Secreting Cells

AbstractThe insulin-secreting cells generated from stem cells in vitro are less glucose responsive than primary β-cells. To search for the missing ingredients that are needed for β-cell maturation, we have longitudinally monitored function of every β-cell in Tg (ins:Rcamp1.07) zebrafish embryos with a newly-invented two-photon light-sheet microscope. We have shown that β-cell maturation begins from the islet mantle and propagates to the islet core during the hatching period, coordinated by the islet vascularization. Lower concentration of glucose is optimal to initiate β-cell maturation, while increased glucose delivery to every cell through microcirculation is required for functional boosting of the β-cells. Both the initiation and the boosting of β-cell maturation demands activation of calcineurin/NFAT by glucose. Calcineurin activator combined with glucose promotes mouse neonatal β-cells cultured in vitro to mature to a functional state similar to adult β-cells, suggesting a new strategy for improving stem cell-derived β-like cell function in vitro.

scholarly journals Suppression of Peroxisome Proliferator-Activated Receptor γ-Coactivator-1α Normalizes the Glucolipotoxicity-Induced Decreased BETA2/NeuroD Gene Transcription and Improved Glucose Tolerance in Diabetic Rats

Endocrinology ◽  
2009 ◽  
Vol 150 (9) ◽  
pp. 4074-4083 ◽  
Author(s):  
Ji-Won Kim ◽  
Young-Hye You ◽  
Dong-Sik Ham ◽  
Jae-Hyoung Cho ◽  
Seung-Hyun Ko ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Receptor Site ◽  
Diabetic Rats ◽  
Peroxisome Proliferator ◽  
Β Cells ◽  
Β Cell ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cell Dysfunction

Abstract Peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α) is significantly elevated in the islets of animal models of diabetes. However, the molecular mechanism has not been clarified. We investigated whether the suppression of PGC-1α expression protects against β-cell dysfunction in vivo and determined the mechanism of action of PGC-1α in β-cells. The studies were performed in glucolipotixicity-induced primary rat islets and INS-1 cells. In vitro and in vivo approaches using adenoviruses were used to evaluate the role of PGC-1α in glucolipotoxicity-associated β-cell dysfunction. The expression of PGC-1α in cultured β-cells increased gradually with glucolipotoxicity. The overexpression of PGC-1α also suppressed the expression of the insulin and β-cell E-box transcription factor (BETA2/NeuroD) genes, which was reversed by PGC-1α small interfering RNA (siRNA). BETA2/NeuroD, p300-enhanced BETA2/NeuroD, and insulin transcriptional activities were significantly suppressed by Ad-PGC-1α but were rescued by Ad-siPGC-1α. PGC-1α binding at the glucocorticoid receptor site on the BETA2/NeuroD promoter increased in the presence of PGC-1α. Ad-siPGC-1α injection through the celiac arteries of 90% pancreatectomized diabetic rats improved their glucose tolerance and maintained their fasting insulin levels. The suppression of PGC-1α expression protects the glucolipotoxicity-induced β-cell dysfunction in vivo and in vitro. A better understanding of the functions of molecules such as PGC-1α, which play key roles in intracellular fuel regulation, could herald a new era of the treatment of patients with type 2 diabetes mellitus by providing protection from glucolipotoxicity, which is an important cause of the development and progression of the disease.

scholarly journals SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo

2021 ◽  
Author(s):  
Mattias Malaguti ◽  
Rosa Portero Migueles ◽  
Jennifer Annoh ◽  
Daina Sadurska ◽  
Guillaume Blin ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Lines ◽  
Modular Design ◽  
Cell Interactions ◽  
Cell Populations ◽  
Cell Contact ◽  
Pluripotent Cells ◽  
Cell Fate Decisions ◽  
Cell Cell

ABSTRACTCell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered “sender” and “receiver” cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions.

scholarly journals Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Isaac M. Richardson ◽  
Christopher J. Calo ◽  
Laurel E. Hind
Keyword(s):  
Cell Interactions ◽  
Tissue Microenvironment ◽  
Experimental Systems ◽  
Microphysiological Systems ◽  
Human Immune Response ◽  
Human Immune ◽  
Future Direction ◽  
Neutrophil Response ◽  
Cell Cell

Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.

scholarly journals Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability

2018 ◽  
Vol 314 (4) ◽  
pp. E308-E321 ◽  
Author(s):  
Peter A. Kropp ◽  
Jennifer C. Dunn ◽  
Bethany A. Carboneau ◽  
Doris A. Stoffers ◽  
Maureen Gannon
Keyword(s):  
Expression Patterns ◽  
Cell Maturation ◽  
Placental Lactogen ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Progenitors ◽  
Glucose Stimulated Insulin Secretion ◽  
Double Heterozygosity ◽  
And Function ◽  
The Impact

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in β-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the β-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal β-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal β-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous β-cells would have an impaired ability to respond to stress. Indeed, we observed that β-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

Novel insights into cell-cell interactions at the blood-brain barrier revealed by a fully-human flow-based in vitro model

2014 ◽  
Vol 275 (1-2) ◽  
pp. 32
Author(s):  
Birgit Obermeier ◽  
Bryan L. Benson ◽  
Haiyan Lu ◽  
Grahame Kidd ◽  
Simona Spampinato ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
In Vitro Model ◽  
Cell Interactions ◽  
Brain Barrier ◽  
Blood Brain ◽  
Vitro Model ◽  
Human Flow ◽  
Cell Cell

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: A study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles

1990 ◽  
Vol 145 (3) ◽  
pp. 414-427 ◽  
Author(s):  
Yvonne Munari-Silem ◽  
Marc Mesnil ◽  
Samia Seimi ◽  
Fran�oise Bernier-Valentin ◽  
Rachida Rabilloud ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescence Microscopy ◽  
Cell Interactions ◽  
Cell Cell

scholarly journals Physical confinement signals regulate the organization of stem cells in three dimensions

2016 ◽  
Vol 13 (123) ◽  
pp. 20160613 ◽  
Author(s):  
Sebastian V. Hadjiantoniou ◽  
David Sean ◽  
Maxime Ignacio ◽  
Michel Godin ◽  
Gary W. Slater ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Interactions ◽  
Three Dimensions ◽  
Cell Substrate ◽  
Cell Cell

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro , mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells.

scholarly journals Insulin secretion stimulated by l-arginine and its metabolite l-ornithine depends on Gαi2

2014 ◽  
Vol 307 (9) ◽  
pp. E800-E812 ◽  
Author(s):  
Veronika Leiss ◽  
Katarina Flockerzie ◽  
Ana Novakovic ◽  
Michaela Rath ◽  
Annika Schönsiegel ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Amino Acid ◽  
Metabolic Phenotype ◽  
Β Cells ◽  
Β Cell ◽  
Glucose Challenge ◽  
Intracellular Ca ◽  
Plasma Insulin Levels

Bordetella pertussis toxin (PTx), also known as islet-activating protein, induces insulin secretion by ADP-ribosylation of inhibitory G proteins. PTx-induced insulin secretion may result either from inactivation of Gαo proteins or from combined inactivation of Gαo, Gαi1, Gαi2, and Gαi3 isoforms. However, the specific role of Gαi2 in pancreatic β-cells still remains unknown. In global (Gαi2−/−) and β-cell-specific (Gαi2βcko) gene-targeted Gαi2 mouse models, we studied glucose homeostasis and islet functions. Insulin secretion experiments and intracellular Ca2+ measurements were used to characterize Gαi2 function in vitro. Gαi2−/− and Gαi2βcko mice showed an unexpected metabolic phenotype, i.e., significantly lower plasma insulin levels upon intraperitoneal glucose challenge in Gαi2−/− and Gαi2βcko mice, whereas plasma glucose concentrations were unchanged in Gαi2−/− but significantly increased in Gαi2βcko mice. These findings indicate a novel albeit unexpected role for Gαi2 in the expression, turnover, and/or release of insulin from islets. Detection of insulin secretion in isolated islets did not show differences in response to high (16 mM) glucose concentrations between control and β-cell-specific Gαi2-deficient mice. In contrast, the two- to threefold increase in insulin secretion evoked by l-arginine or l-ornithine (in the presence of 16 mM glucose) was significantly reduced in islets lacking Gαi2. In accord with a reduced level of insulin secretion, intracellular calcium concentrations induced by the agonistic amino acid l-arginine did not reach control levels in β-cells. The presented analysis of gene-targeted mice provides novel insights in the role of β-cell Gαi2 showing that amino acid-induced insulin-release depends on Gαi2.

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