Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells

Hongmei Mou; Vladimir Vinarsky; Purushothama Rao Tata; Karissa Brazauskas; Soon H Choi; Adrianne K Crooke; Bing Zhang; George M Solomon; Brett Turner; Hermann Bihler; Jan Harrington; Allen Lapey; Colleen Channick; Colleen Keyes; Adam Freund; Steven Artandi; Martin Mense; Steven Rowe; John F Engelhardt; Ya-Chieh Hsu; Jayaraj Rajagopal

doi:10.1016/j.stem.2016.05.012

Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells

Cell Stem Cell. 2016 Aug 4;19(2):217-231. doi: 10.1016/j.stem.2016.05.012. Epub 2016 Jun 16.

Authors

Hongmei Mou¹, Vladimir Vinarsky², Purushothama Rao Tata³, Karissa Brazauskas⁴, Soon H Choi⁵, Adrianne K Crooke⁵, Bing Zhang⁶, George M Solomon⁷, Brett Turner⁸, Hermann Bihler⁹, Jan Harrington⁹, Allen Lapey¹⁰, Colleen Channick¹¹, Colleen Keyes¹¹, Adam Freund¹², Steven Artandi¹², Martin Mense⁹, Steven Rowe¹³, John F Engelhardt⁵, Ya-Chieh Hsu⁶, Jayaraj Rajagopal¹⁴

Affiliations

¹ Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Pediatric Pulmonary Medicine, Massachusetts General Hospital for Children, Boston, MA 02114, USA.
² Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
³ Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
⁴ Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Division of Pediatric Pulmonary Medicine, Massachusetts General Hospital for Children, Boston, MA 02114, USA.
⁵ Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
⁶ Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA.
⁷ Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35233, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
⁸ Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
⁹ CFFT Lab, Cystic Fibrosis Foundation Therapeutics, Lexington, MA 01730, USA.
¹⁰ Division of Pediatric Pulmonary Medicine, Massachusetts General Hospital for Children, Boston, MA 02114, USA.
¹¹ Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
¹² Departments of Medicine and Biochemistry, Stanford University, Stanford, CA 94305, USA.
¹³ Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35233, USA; Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35233, USA; Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, AL 35294, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
¹⁴ Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Pediatric Pulmonary Medicine, Massachusetts General Hospital for Children, Boston, MA 02114, USA; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Division of Otology and Laryngology, Massachusetts Eye and Ear, Boston, MA 02114, USA. Electronic address: jrajagopal@partners.org.

Abstract

Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.

Keywords: TGFβ/BMP4/SMAD signaling; dedifferentiation; differentiation; dual SMAD signaling inhibition; epithelial basal and stems cells; p63(+) basal cells; replicative exhaustion; senescence; stemness; telomeres.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Cell Proliferation
Cell Self Renewal
Cellular Senescence
Cilia / metabolism
Epithelial Cells / cytology*
Epithelial Cells / metabolism*
Epithelium / metabolism
Humans
Keratinocytes / cytology
Keratinocytes / metabolism
Lung / cytology
Mice, Inbred C57BL
Mucus / metabolism
Signal Transduction*
Smad Proteins / metabolism*
Telomere / metabolism

Substances

Smad Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding