Isolation of individual cellular components from lung tissues of patients with lymphangioleiomyomatosis

Katsutoshi Ando, Naoya Fujino, Keiko Mitani, Chiharu Ota, Yoshinori Okada, Takashi Kondo, Teruaki Mizobuchi, Masatoshi Kurihara, Kenji Suzuki, Yoshito Hoshika, Hiroki Ebana, Etsuko Kobayashi, Kazuhisa Takahashi, Hiroshi Kubo, Kuniaki Seyama

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4 Citations (Scopus)

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 fluorescenceactivated 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-/podoplaninhi subset), alveolar type II cells (EpCAMhi/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 LAMaffected lung tissues, providing new research opportunities in this field.

Original languageEnglish
Pages (from-to)L899-L908
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume310
Issue number10
DOIs
Publication statusPublished - 2016 May 15

Keywords

  • Fluorescence-activated cell sorting
  • Loss of heterozygosity
  • Lymphangioleiomyomatosis cells
  • Lymphatic endothelial cells
  • The tuberous sclerosis complex genes

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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  • Cite this

    Ando, K., Fujino, N., Mitani, K., Ota, C., Okada, Y., Kondo, T., Mizobuchi, T., Kurihara, M., Suzuki, K., Hoshika, Y., Ebana, H., Kobayashi, E., Takahashi, K., Kubo, H., & Seyama, K. (2016). Isolation of individual cellular components from lung tissues of patients with lymphangioleiomyomatosis. American Journal of Physiology - Lung Cellular and Molecular Physiology, 310(10), L899-L908. https://doi.org/10.1152/ajplung.00365.2015