Biology subjects

Lyon, J.

Publications and source records attributed to Lyon, J..

6 recordsLinked to original sources

bioRxiv2023

Complete Suspension Differentiation of Human Pluripotent Stem Cells into Pancreatic Islets Using Vertical-Wheel Bioreactors

Advanced protocols to produce human pluripotent stem cell (SC)-derived islets show promise in functional, metabolic, and transcriptional maturation of cell therapy product to treat diabetes. Available protocols are either developed as complete planar (2D) or, in later stages, combined with suspension cultures (3D). Despite marked progress, both approaches have clear limitations for scalability, cell loss and batch to batch heterogeneity during differentiation. Using a Vertical Wheel(R) bioreactor system, we present a highly efficient and scalable complete suspension protocol across all stages for directed differentiation of human pluripotent stem cells into functional pancreatic islets. Here, we generate homogeneous, metabolically functional, and transcriptionally enriched SC-islets and compared against adult donor islets. Generated SC-islets showed enriched endocrine cell composition ([~]63% CPEP+NKX6.1+ISL1+) and displayed functional maturity for glucose stimulated insulin secretion ([~]5-fold) during in vitro and post transplantation. Comprehensive stage-specific single-cell mass flow cytometry characterization with dimensional reduction analysis at stage-4 and -6 confirmed optimal maturation was achieved without heterogeneity. Notably, by 16-weeks transplantation follow-up, normal glycemic homeostasis was restored, and glucose responsive human c-peptide secretion response (2-fold) was achieved. Four months post engraftment, graft-harvested single cells displayed islet hormonal cell composition with flow cytometry, improved functional maturity by in vivo glucose-stimulated insulin secretion (GSIS) and enhanced transcriptional landscape with real-time expression that closely resembled patterns comparable to adult human islets. Our comprehensive evaluation of a complete suspension method applied across all stages using Vertical Wheel(R) bioreactors for SC-islets generation highlight progressive molecular and functional maturation of islets while reducing potential cell loss and cellular heterogeneity. Such a system could potentially be scaled to deliver clinical grade SC-islet products in a closed good manufacturing practice type environment. One Sentence SummaryThis study describes all-stages complete suspension protocol for SC-islets generation.

bioengineering↗

bioRxiv2022

The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study

BackgroundEvidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here we describe the generation of HepG2-derived transmitochondrial cybrids in order to investigate the impact of mtDNA variation upon mitochondrial (dys)function and susceptibility to iDILI against a constant nuclear background. In this study, cybrids were created to contain mitochondrial genotypes of haplogroup H and haplogroup J for comparison. MethodsBriefly, HepG2 cells were depleted of mtDNA to make rho zero cells before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 distinct transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide and tolcapone, by ATP assays and extracellular flux analysis. FindingsOverall, baseline mitochondrial function was similar between haplogroups H and J. However, haplogroup specific responses to mitotoxic drugs were observed; haplogroup J was more susceptible to the inhibition of respiratory complexes I and II, and also to the effects of tolcapone, an uncoupler of mitochondrial respiration. ConclusionsThis study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest, thus providing a practical and reproducible system to investigate the cellular consequences of variation in mitochondrial genome against a constant nuclear background. Additionally the results support that that inter-individual variation in mitochondrial genotype and haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. FundingThis work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

pharmacology and toxicology↗

bioRxiv2021

Impacts of the COVID-19 Pandemic on a Human Research Islet Program

Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specialising in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, recipient groups reported poorer feedback regarding the samples. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.

physiology↗

bioRxiv2021

Heterogenous impairment of α-cell function in type 2 diabetes is linked to cell maturation state

In diabetes, glucagon secretion from pancreatic -cells is dysregulated. We examined -cells from human donors and mice using combined electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses -cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat-feeding of mice, -cells shift towards a {beta}-cell-like electrophysiologic profile in concert with an up-regulation of the {beta}-cell Na+ channel isoform Scn9a and indications of impaired -cell identity. In human -cells we identify links between cell membrane properties and cell surface signalling receptors, mitochondrial respiratory complex assembly, and cell maturation. Cell type classification using machine learning of electrophysiology data demonstrates a heterogenous loss of electrophysiologic identity in -cells from donors with T2D. Indeed, a sub-set of -cells with impaired exocytosis is defined by an enrichment in progenitor markers suggesting important links between -cell maturation state and dysfunction in T2D. Key findingsO_LI-cell exocytosis is suppressed by glucose-dependent inhibition of P/Q-type Ca2+ currents C_LIO_LIDysfunction of -cells in type 2 diabetes is associated with a {beta}-cell-like electrophysiologic signature C_LIO_LIPatch-seq links maturation state, the mitochondrial respiratory chain, and cell surface receptor expression to -cell function C_LIO_LI-cell dysfunction occurs preferentially in cells enriched in endocrine lineage markers C_LI

cell biology↗

bioRxiv2019

Aging of human endocrine pancreatic cell types is heterogeneous and sex-specific

The human endocrine pancreas must regulate glucose homeostasis throughout the human lifespan, which is generally decades. We performed meta-analysis of single-cell, RNA-sequencing datasets derived from 36 individuals, as well as functional analyses, to characterize age-associated changes to the major endocrine pancreatic cell types. Increasing age was associated with shifts in pancreatic alpha and beta cell identity and loss of nuclear integrity in non-diabetic humans. In non-diabetic individuals [≥] 50 years old, 80% of their beta cells exhibited a transcriptional signature similar to cells from type-2 diabetic (T2D) donors. Surprisingly, [~]5% of beta cells from T2D donors retained a youthful, N.D. transcriptional profile. Furthermore, beta cell function was reduced by 50% during aging in men but not women, which may explain sex-associated differences in diabetes etiology. These analyses reveal that aging of the human endocrine pancreas is sex- and cell-type specific.

physiology↗

bioRxiv2019

Pancreas patch-seq links physiologic dysfunction in diabetes to single-cell transcriptomic phenotypes

Pancreatic islet cells regulate glucose homeostasis through insulin and glucagon secretion; dysfunction of these cells leads to severe diseases like diabetes. Prior single-cell transcriptome studies have shown heterogeneous gene expression in major islet cell-types; however it remains challenging to reconcile this transcriptomic heterogeneity with observed islet cell functional variation. Here we achieved electrophysiological profiling and single-cell RNA sequencing in the same islet cell (pancreas patch-seq) thereby linking transcriptomic phenotypes to physiologic properties. We collected 1,369 cells from the pancreas of donors with or without diabetes and assessed function-gene expression networks. We identified a set of genes and pathways that drive functional heterogeneity in {beta}-cells and used these to predict {beta}-cell electrophysiology. We also report specific transcriptional programs that correlate with dysfunction in type 2 diabetes (T2D) and extend this approach to cryopreserved cells from donors with type 1 diabetes (T1D), generating a valuable resource for understanding islet cell heterogeneity in health and disease. Key findingsO_LIPancreas patch-seq provides a single-cell survey of function-transcriptome pairing in 1,369 islet cells from donors with and without diabetes C_LIO_LIExpression of a specific subset of genes predicts {beta}-cell electrophysiology in transcriptome-function networks. C_LIO_LICompromised {beta}-cell function in T2D correlates with altered ETV1 expression and inflammatory pathways C_LIO_LIFunctional heterogeneity in -cells maps to ER stress and islet lineage markers C_LIO_LIApplication of patch-seq to cells from rare cryopreserved islets from donors with T1D C_LI

genomics↗