Biology subjects

Dost, A. F. M.

Publications and source records attributed to Dost, A. F. M..

4 recordsLinked to original sources

bioRxiv2025

WS6 enables scalable ex vivo expansion and gene editing of epithelial basal stem cells

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro. Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as conditional reprogramming. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium ( EpMED; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation.

cell biology↗

bioRxiv2025

A human organoid model of alveolar regeneration reveals distinct epithelial responses to interferon-gamma

Chronic obstructive pulmonary disease is characterized by inflammation and emphysema, leading to progressive alveolar destruction. Currently, no therapies effectively regenerate the alveolar epithelium. Here, we describe a feeder- and serum-free primary adult human organoid model to investigate how inflammation influences alveolar regeneration. We achieve long-term expansion of multipotent progenitor-like cells, while an alveolar type 2 (AT2) maturation protocol enhances surfactant production and supports tubular myelin formation. Introducing a LATS inhibitor to the expansion condition induces alveolar type 1 (AT1) differentiation while maintaining AT2 cells. Using this platform, we find that interferon-gamma exerts cytotoxic effects on AT1 cells while promoting the growth of regenerating AT2 cells, illustrating how a single inflammatory stimulus can have divergent effects on alveolar epithelial cell types. These findings underscore the nuanced influence of pro-inflammatory cytokines on alveolar regeneration. Our organoid model provides a reductionist platform for mechanistic studies, aimed to identify strategies to enhance alveolar regeneration.

cell biology↗

bioRxiv2024

Early-stage lung adenocarcinoma is driven by an injury-associated, plastic cell state dependent on a KRAS-ITGA3-SRC axis

Glycine 12 mutations in the GTPase KRAS (KRASG12) are a known initiating event for lung adenocarcinoma (LUAD) with broad clinical relevance. KRASG12 mutations promote cell-intrinsic rewiring of the lung alveolar type II progenitor (AT2) cells, but to what extent such changes interplay with pathways essential for lung homeostasis and cell fate is unclear. We used single-cell RNA-seq (scRNA-seq) from AT2-mesenchyme organoid co-cultures, mouse models, and stage IA LUAD patient samples to identify conserved regulators of AT2 cell transcriptional dynamics and the impact of KRASG12D with temporal resolution. In AT2WT organoids, a transient injury/plasticity state preceded AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics most noted by retention of the injury/plasticity state. At later time points in tumorigenesis, AT2KRAS cells consisted of heterogeneous populations that could be defined by either the injury state or high expression of an AT2 cell signature. The injury state in AT2KRAS cells of LUAD in patients, mice, and organoids was distinguishable from AT2WT states by altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors to target the oncogenic injury cell state impaired AT2KRAS organoid growth. Thus, an injury/plasticity signature characterized as an essential step in lung repair is used during alveolar cell self-renewal and during initiation and progression of LUAD. Early-stage lung cancer may be susceptible to intervention by targeting the oncogenic-specific nature of this cell state.

cancer biology↗

bioRxiv2022

Druggable Growth Dependencies and Tumor Evolution Analysis in Patient-Derived Organoids of Neuroendocrine Cancer

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors and poorly-differentiated carcinomas. Treatment options for patients with NENs are limited, in part due to lack of accurate models. To address this need we established the first patient-derived tumor organoids (PDTOs) from pulmonary neuroendocrine tumors and derived PDTOs from an understudied NEN subtype, large cell neuroendocrine carcinoma (LCNEC). PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through drug sensitivity analyses, we uncover therapeutic sensitivities to an inhibitor of NAD salvage biosynthesis and to an inhibitor of BCL-2. Finally, we identify a dependency on EGF in pulmonary neuroendocrine tumor PDTOs. Consistent with these findings, analysis of an independent cohort showed that approximately 50% of pulmonary neuroendocrine tumors expressed EGFR. This study identifies a potentially actionable vulnerability for a subset of NENs, and further highlights the utility of these novel PDTO models for the study of NENs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/514549v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@14bed0borg.highwire.dtl.DTLVardef@1b7791forg.highwire.dtl.DTLVardef@7120a6org.highwire.dtl.DTLVardef@116b21d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPDTOs of pulmonary NETs and LCNEC were established C_LIO_LIPDTOs recapitulate intra-tumoral heterogeneity and evolution of parental tumors C_LIO_LIDrug assays reveal therapeutic vulnerabilities and biomarkers C_LIO_LIPulmonary NET PDTOs are dependent on EGF C_LI

cancer biology↗