title: Isolation of individual cellular components from lung tissues of patients with lymphangioleiomyomatosis.
abstract: Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease entailing cystic destruction of the lungs and progressive respiratory failure. LAM lungs are histologically characterized by the proliferation of smooth muscle-like cells (LAM cells) and an abundance of lymphatic vessels. To elucidate the pathophysiological processes of LAM, cell-type-specific analyses are required. However, no method exists for isolating the individual types of cells in LAM lesions. Therefore, we established a fluorescence-activated cell sorting (FACS)-based method for the direct isolation of LAM cells and other various cellular components from LAM-affected lung tissue. We obtained LAM-affected lung tissue from resections or transplant recipients and prepared single-cell suspensions. FACS, immunohistochemical, and molecular analysis were used cooperatively to isolate HMB45-positive LAM cells with tuberous sclerosis complex (TSC) 2 loss of heterozygosity (LOH). Using a combination of antibodies against an epithelial cell adhesion molecule (EpCAM) and podoplanin, we fractionated CD45-negative lung cells into three groups: lymphatic endothelial cells (LEC) (EpCAM(-)/podoplanin(hi) subset), alveolar type II cells (EpCAM(hi)/podoplanin(-) subset), and mesenchymal cells (EpCAM(-)/podoplanin(-/low) subset). During subsequent analysis of HMB45 expression, as a LAM-specific marker, we clearly identified LAM cells in the mesenchymal cell population. We then discovered that CD90(+)/CD34(-) cells in the mesenchymal cell population are not only positive for HBM45 but also had TSC2 LOH. These isolated cells were viable and subsequently amenable to cell culture. This method enables us to isolate LAM cells and other cellular components, including LAM-associated LEC, from LAM-affected lung tissues, providing new research opportunities in this field.
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan; kando@juntendo.ac.jp.
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Ando
authors.affiliation: Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Sendai, Japan;
authors.forename: Naoya
authors.initials: N
authors.lastname: Fujino
authors.affiliation: Division of Human Pathology, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Keiko
authors.initials: K
authors.lastname: Mitani
authors.affiliation: Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Sendai, Japan;
authors.forename: Chiharu
authors.initials: C
authors.lastname: Ota
authors.affiliation: Department of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University Graduate School of Medicine, Miyagi, Japan;
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Okada
authors.affiliation: Department of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University Graduate School of Medicine, Miyagi, Japan;
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Pneumothorax Research Center and Division of Thoracic Surgery, Nissan Tamagawa Hospital, Tokyo, Japan; and.
authors.forename: Teruaki
authors.initials: T
authors.lastname: Mizobuchi
authors.affiliation: Pneumothorax Research Center and Division of Thoracic Surgery, Nissan Tamagawa Hospital, Tokyo, Japan; and.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kurihara
authors.affiliation: Division of Thoracic Surgery, Juntendo University Faculty of Medicine and Graduate School of Medicine, Bunkyo-Ku, Tokyo, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Hoshika
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ebana
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Etsuko
authors.initials: E
authors.lastname: Kobayashi
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Sendai, Japan;
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kubo
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan;
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Seyama
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation; D048430:Cell Shape; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000473:pathology*; D018192:Lymphangioleiomyomatosis / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors
keywords: fluorescence-activated cell sorting; loss of heterozygosity; lymphangioleiomyomatosis cells; lymphatic endothelial cells; the tuberous sclerosis complex genes