Testing organ-specific responses to therapies in tissues differentiated from Cystic Fibrosis patient derived iPSCs.

Cystic Fibrosis (CF) is a life-shortening disease that is caused by mutations in the CFTR gene, a gene that is expressed in multiple organs. There are several primary tissue models of CF disease, including nasal epithelial cultures and rectal organoids, that are effective in reporting the potential efficacy of mutation-targeted therapies called CFTR modulators. However, there is the well-documented variation in tissue dependent, therapeutic response amongst CF patients, even those with the same CF-causing mutation.

Truth and Reconcilition Commissions and Health Care System Responses for Indigenous Peoples: A Scoping Review.

Grounded in human rights approaches, truth and reconciliation commissions (TRCs) explore an event or process that did widespread and systematic intentional harm to a group of people. Health as a fundamental right is an important component addressed by TRCs. Yet despite TRCs often having recommendations for health care systems, it is unknown how well these recommendations are being translated within health care settings.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells.

Background: Cystic Fibrosis causing mutations in the gene , reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations.

A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.

Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs.

Clinical and functional efficacy of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis carrying the N1303K mutation.

Background: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations.

Correlation of Electrophysiological and Fluorescence-Based Measurements of Modulator Efficacy in Nasal Epithelial Cultures Derived from People with Cystic Fibrosis.

It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber.

Cost-effectiveness analysis of genetic tools to predict treatment response in patients with cystic fibrosis.

Background: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapies show variable efficacy for patients with CF. Patient-derived predictive tools may identify individuals likely to respond to CFTRs, but are not in routine use. We aimed to determine the cost-utility of predictive tool-guided treatment with CFTRs as add-on to standard of care (SoC) for individuals with CF.

Local memory allocation recruits memory ensembles across brain regions.

Memories are thought to be stored in ensembles of neurons across multiple brain regions. However, whether and how these ensembles are coordinated at the time of learning remains largely unknown. Here, we combined CREB-mediated memory allocation with transsynaptic retrograde tracing to demonstrate that the allocation of aversive memories to a group of neurons in one brain region directly affects the allocation of interconnected neurons in upstream brain regions in a behavioral- and brain region-specific manner in mice.

Viscoelastic Notch Signaling Hydrogel Induces Liver Bile Duct Organoid Growth and Morphogenesis.

Cholangiocyte organoids can be used to model liver biliary disease; however, both a defined matrix to emulate cholangiocyte self-assembly and the mechano-transduction pathways involved therein remain elusive. A series of defined viscoelastic hyaluronan hydrogels to culture primary cholangiocytes are designed and it is found that by mimicking the stress relaxation rate of liver tissue, cholangiocyte organoid growth can be induced and expression of Yes-associated protein (YAP) target genes could be significantly increased. Strikingly, inhibition of matrix metalloproteinases (MMPs) does not significantly affect organoid growth in 3D culture, suggesting that mechanical remodeling of the viscoelastic microenvironment-and not MMP-mediated degradation-is the key to cholangiocyte organoid growth.

Winnipeg's North End Wellbeing Measure: Using Social Innovation to Drive Community Measurements.

Background: The Winnipeg Boldness Project, a social innovation initiative addressing early childhood outcomes in the underserved community of Point Douglas, worked alongside the community to develop a meaningful measurement tool, the North End Wellbeing Measure (NEWM). This article describes the context, the research and pilot, and the lessons learned.

Objectives: To develop a community-based tool called the NEWM, which evaluates what is important to Point Douglas families.

A Fluorescence-based Assay of Membrane Potential for High-throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions.

A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes-based on the structure of ivacaftor-to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin.

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness.

Stage-Specific Generation of Human Pluripotent Stem Cell Derived Lung Models to Measure CFTR Function.

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms.

Antisense oligonucleotide splicing modulation as a novel Cystic Fibrosis therapeutic approach for the W1282X nonsense mutation.

Background: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23.

Methods: ∼80 ASOs were screened in 16HBEge W1282X cells.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow.

Perspectives on the translation of in-vitro studies to precision medicine in Cystic Fibrosis.

Recent strides towards precision medicine in Cystic Fibrosis (CF) have been made possible by patient-derived in-vitro assays with the potential to predict clinical response to small molecule-based therapies. Here, we discuss the status of primary and stem-cell derived tissues used to evaluate the preclinical efficacy of CFTR modulators highlighting both their potential and limitations. Validation of these assays requires correlation of in-vitro responses to in-vivo measures of clinical biomarkers of disease outcomes.

A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients.

For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development.

Riociguat for the treatment of Phe508del homozygous adults with cystic fibrosis.

Background: Riociguat is a first-in-class soluble guanylate cyclase stimulator for which preclinical data suggested improvements in cystic fibrosis transmembrane conductance regulator (CFTR) function.

Methods: This international, multicenter, two-part, Phase II study of riociguat enrolled adults with cystic fibrosis (CF) homozygous for Phe508del CFTR. Part 1 was a 28-day, randomized, double-blind, placebo-controlled study in participants not receiving CFTR modulator therapy.