Generation of alveolar epithelial type 1 (AT1) cells
Inventors
BURGESS, Claire • Kotton, Darrell
Assignees
Publication Number
US-12324818-B2
Publication Date
2025-06-10
Expiration Date
2043-12-15
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Abstract
The technology described herein is directed to methods of producing or differentiating AT1 cells, and AT1 cells made by the methods described herein.
Core Innovation
The technology described provides methods for producing or differentiating alveolar epithelial type I (AT1) cells in vitro. Central to the innovation is the use of either inducing ectopic Yes Associated Protein (YAP) expression in alveolar epithelial type II (AT2) cells and/or lung epithelial progenitor cells, or maintaining these cells in a medium containing a Large Tumor Suppressor Kinase (LATS) inhibitor. These procedures result in the differentiation and expansion of AT1 cells that express key molecular, morphologic, and functional characteristics reminiscent of natural human AT1 cells.
The patent addresses the challenge that AT1 cells, due to their thin, flat shape and quiescent, terminally differentiated phenotype, are difficult to isolate and maintain in culture. This limitation has hindered the understanding of AT1 cell biology, their origins, and their roles in lung health and disease. By providing reliable in vitro methods to generate and study AT1 cells, the patent solves a long-standing problem in lung biology research.
These methods leverage manipulation of the Hippo-LATS-YAP signaling pathway. This is accomplished either through overexpression of nuclear-activated YAP or by using LATS inhibitors in serum-free defined media. The resulting cells can be genetically modified to include fluorescent reporters, allowing for tracking and purification. The system can efficiently generate stable AT1 cells in both two-dimensional and three-dimensional culture systems, including air-liquid interface cultures, providing a useful in vitro model and source of AT1 cells for further study.
Claims Coverage
The patent claims cover several inventive features centered around methods for differentiating AT1 cells from AT2 or lung progenitor cells, including composition aspects and various process details. The primary independent claim (Claim 1) focuses on culturing AT2 and/or progenitor cells in a medium comprising a LATS inhibitor to induce differentiation into AT1 cells.
Culturing AT2 or progenitor cells in a medium comprising a LATS inhibitor results in differentiation into AT1 cells
Alveolar epithelial cell type II (AT2) cells and/or lung epithelial progenitor cells are cultured in a medium containing a Large Tumor Suppressor Kinase (LATS) inhibitor, which leads to differentiation of these cells into alveolar epithelial cell type I (AT1) cells. - The method applies to at least one AT2 cell and/or lung epithelial progenitor cell. - The culturing step uses a LATS inhibitor to trigger differentiation. - The resulting cells are AT1 cells.
Complete serum free defined medium as the culturing medium
The culturing medium used to differentiate AT1 cells is a complete serum free defined medium, supporting the generation and maintenance of the differentiated cells.
LATS inhibitor selection
The LATS inhibitor included in the culturing medium is selected from the group consisting of LATS-IN-1 (TRULI; N-(3-benzylthiazol-2(3H)-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide), GA-017, and TDI-011536.
Exclusion of Wnt and KGF signaling components
The culturing medium does not comprise, or the AT2 and/or lung epithelial progenitor cells are not in contact with, CHIR99021 and/or keratinocyte growth factor (KGF) protein.
Exclusion or inclusion of specific growth factors
The culturing medium may either not comprise, or the cells are not in contact with, ectopic epidermal growth factor (EGF) protein and/or ectopic fibroblast growth factor 10 (FGF10) protein; alternatively, the method can include further contacting with EGF and/or FGF10.
Induction of YAP signaling via LATS inhibition
Culturing AT2 and/or lung epithelial progenitor cells in a medium containing a LATS inhibitor induces YAP signaling, which is necessary for AT1 cell differentiation.
Broad applicability to various AT2 and/or progenitor cell sources
The method is applicable to human or primary AT2 cells, induced AT2 cells, human lung epithelial progenitor cells, primary lung epithelial progenitor cells, or induced lung epithelial progenitor cells.
Additional contacting steps for downstream analysis and modeling
The method can further comprise contacting the AT2 or lung epithelial progenitor cell, or a cell produced by the method, with: - a nucleic acid encoding a reporter protein; - a virus; - smoke; - Transforming Growth Factor Beta (TGFB) and/or bleomycin.
Use with cells harboring pulmonary fibrosis inducing mutations
The method covers use with AT2 or lung epithelial progenitor cells that comprise a pulmonary fibrosis (PF) inducing mutation.
Inducing ectopic Yes Associated Protein (YAP) expression as an additional or alternative differentiation strategy
The method may further comprise inducing ectopic YAP expression in AT2 and/or lung epithelial progenitor cells, optionally with: - YAP comprising mutations of the serine in at least one HXRXXS consensus sequence, including to alanine; - mutations corresponding to S61, S109, S127, S164, and S397 of SEQ ID NO: 1. - Inducing can include contacting with an expression vector encoding a YAP protein.
These inventive features define methods to reliably generate AT1 cells from AT2 or lung epithelial progenitor cells, employing LATS inhibitor-based differentiation, optional YAP overexpression strategies, selection of media components, and flexibility for various genetic or disease-related contexts.
Stated Advantages
Provides a reliable and efficient method for generating AT1 cells in vitro, overcoming challenges in isolating and maintaining primary AT1 cells.
Enables the creation of large, stable, and pure populations of AT1 cells suitable for both basic research and potential clinical applications.
Offers a serum-free, feeder-free, and defined system for AT1 cell differentiation, allowing for reproducibility and scalability.
Allows modeling and study of lung biology and diseases, including those linked to AT1 cell dysfunction, using human-derived cells.
Documented Applications
In vitro modeling of human AT1 cell biology and alveolar epithelial differentiation.
Study of alveolar epithelial injury including exposure to viral infection, smoke, Transforming Growth Factor Beta (TGFB), and bleomycin.
Disease modeling of conditions such as idiopathic pulmonary fibrosis associated with aberrant differentiation of AT2 to AT1 cells.
Potential use of produced AT1 cells in the treatment of lung diseases, including acute respiratory distress syndrome (ARDS), pulmonary fibrosis, interstitial lung diseases, acute inhalational lung injuries, childhood interstitial lung diseases, and bronchopulmonary dysplasia.
Identification of candidate therapeutic agents for lung diseases through screening with AT1 cells derived by the described methods.
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