Stellate cells are in utero markers of pancreatic disease in cystic fibrosis.

BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.

Early developmental phenotypes in the cystic fibrosis sheep model.

Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/-  animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.

Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR -/- sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder.