Abstract
Introduction Alpha-1 antitrypsin (AAT) deficiency (AATD) is the most frequent form of genetically determined emphysema. Type II alveolar cells (AEC2) are capable of producing AAT but little is known about their role in AATD pathogenesis. Aims and objectives We hypothesise that AEC2s play an intrinsic role in AATD pathogenesis and therefore we sought to engineer a novel in vitro model to enable the derivation of lung epithelial cells to define the developmental timing of AAT expression in emerging AEC2s. Methods We targeted 2 fluorescent reporters into the endogenous NKX2.1 and surfactant protein C loci of a human induced pluripotent stem cell (iPSC) line from an individual without AATD. Using a directed differentiation approach that recapitulates embryonic development we generated lung progenitors followed by putative AEC2s. A microarray RNA expression profile was produced and RT-qPCR was used to further quantify mRNA expression. Results Sorted lung progenitors demonstrate significant expression of AAT at day 15 of differentiation and subsequently increase up to day 34. Sorting more mature cells that co-express both reporters demonstrates expression of several AEC2-specific genes and increased expression of AAT mRNA. Flow cytometry for intracellular AAT reveals increased protein expression in AEC2s. Conclusions We demonstrate that differentiation of a novel dual reporter iPSC line can generate early lung progenitors and later AEC2s and that these cells express AAT in significant quantities at both the mRNA and protein levels. We intend to target fluorescent reporters into iPSC from individuals with AATD therefore generating a novel model of disease in AEC2s.
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