TY - JOUR
T1 - Establishment of a conditionally reprogrammed primary eccrine sweat gland culture for evaluation of tissue-specific CFTR function
AU - Eastman, Alice C.
AU - Rosson, Gedge
AU - Kim, Noori
AU - Kang, Sewon
AU - Raraigh, Karen
AU - Goff, Loyal A.
AU - Merlo, Christian
AU - Lechtzin, Noah
AU - Cutting, Garry R.
AU - Sharma, Neeraj
N1 - Publisher Copyright:
© 2024 European Cystic Fibrosis Society
PY - 2024
Y1 - 2024
N2 - Background: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). Methods: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. Results: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. Conclusions: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.
AB - Background: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). Methods: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. Results: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. Conclusions: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.
KW - CFTR function
KW - Conditional reprogramming
KW - Eccrine sweat glands
KW - Genotype-phenotype
KW - Modulator heterogeneity
KW - scRNA-seq
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U2 - 10.1016/j.jcf.2024.06.013
DO - 10.1016/j.jcf.2024.06.013
M3 - Article
C2 - 38969603
AN - SCOPUS:85197602192
SN - 1569-1993
JO - Journal of Cystic Fibrosis
JF - Journal of Cystic Fibrosis
ER -