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Differentiation of CFTR knockout hPSCs into salivary gland epithelial progenitors models the development of cystic fibrosis patients

ABSTRACT: The differentiation of pluripotent stem cells has been broadly used in studying the disease mechanism and development process. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, to investigate whether the hPSCs-derived SGEPs are capable of modeling the characteristics of cystic fibrosis (CF), a disease impact SG function caused by mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene, the CFTR knockout hPSCs were differentiated into CF-SGEPs using the same protocol. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16-day of differentiation, the protein expression of CFTR was also decreased in SGEPs generated from CFTR knockout hPSCs. RNA-sequencing shows that multiple genes modulating SG development, function and were down-regulated and positive regulators of inflammation were up-regulated in CF-SGEPs, which was correlated with salivary phenotype in CF patients. These results demonstrated that suppression of CFTR disrupted the differentiation of hPSCs-derived SGEPs, which modeled the SG development of patients with CF. In summary, this study not only provided the proof that the hPSCs-derived SGEPs could serve as manipulatable and easily accessible models to study the SG developmental diseases, but also provided new avenues for the study of the CF mechanism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE224547 | GEO | 2023/12/06

REPOSITORIES: GEO

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Project description:Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) in cystic fibrosis (CF) results in exaggerated and chronic inflammation as well as increased susceptibility to chronic pulmonary infections, in particular with Pseudomonas aeruginosa. Based on the concept that host immune responses do not seem to be adequate to eradicate P.aeruginosa from the lungs of CF patients and that dendritic cells (DC) play an important role in initiating and shaping adaptive immune responses, this study analyzed the role of CFTR in bone marrow-derived murine DC from CFTR knockout (CF) mice with and without exposure to P.aeruginosa. DC expressed CFTR mRNA and protein, although at much lower levels compared to whole lung. Microarray analysis of gene expression levels in DC generated from CF and wild type (WT) mice revealed significantly different expression of 16 genes in CF DC compared to WT DC. Among the genes with lower expression in CF DC was Caveolin-1, a membrane lipid raft protein. Messenger RNA and protein levels of Caveolin-1 were decreased in the CF DC compared to WT DC. Consistently, the active form of sterol-responsive element binding protein (SREBP), a negative regulator of Caveolin-1 expression, was increased in CF DC. Following exposure to P.aeruginosa, gene expression levels in CF and WT DC changed for 912 genes involved in inflammation, chemotaxis, signaling, cell cycling and apoptosis more than 1.5-fold. Among the genes that showed a different response between WT and CF DC infected with P.aeruginosa, were 3Î²-hydroxysterol-Î7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP. These results suggest that CFTR dysfunction in non-epithelial cells results in changes in the expression of genes encoding factors involved in membrane structure and lipid-metabolism. These membrane alterations in immune cells may contribute to the abnormal inflammatory and immune response characteristic of CF. Experiment Overall Design: comparison of gene expression in dendritic cells of cystic fibrosis mice without or with Pseudosmonas aeruginosa infection vs wild type dendritic cell controls

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Differentiation of CFTR knockout hPSCs into salivary gland epithelial progenitors models the development of cystic fibrosis patients

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