American Association for Cancer Research
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Supplementary Materials and Methods and References from Human Pluripotent Stem Cell–Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis

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posted on 2023-03-31, 00:48 authored by Lisa M. Julian, Sean P. Delaney, Ying Wang, Alexander A. Goldberg, Carole Doré, Julien Yockell-Lelièvre, Roger Y. Tam, Krinio Giannikou, Fiona McMurray, Molly S. Shoichet, Mary-Ellen Harper, Elizabeth P. Henske, David J. Kwiatkowski, Thomas N. Darling, Joel Moss, Arnold S. Kristof, William L. Stanford

This file contains descriptions of materials and methods used in this study pertaining to cell and tissue staining, Western blot and mRNA analysis, and cell size and genotyping analyses. Corresponding references are also included.

Funding

LAM

United States Department of Defense

Salary

National Institute of Arthritis and Musculoskeletal and Skin Diseases

Canadian Institutes of Health Research

Intramural Research Program

NIH

National Heart, Lung, and Blood Institute

LAM Foundation

Lucy J. Engles TSC/LAM Research Program

Ontario Institute for Regenerative Medicine

Ministry of Research, Innovation and Science

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CIHR

Richard and Edith Strauss

History

ARTICLE ABSTRACT

Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation–bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/− mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491–502. ©2017 AACR.

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    Cancer Research

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    Keywords

    AngiogenesisAngiogenic factors and receptorsBiomarkersCell Death And SenescencePreclinical ModelsStem Cell Biology

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