Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells.
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.
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.
Hongmei
H
Mou
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.
Vladimir
V
Vinarsky
Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
Purushothama Rao
PR
Tata
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.
Karissa
K
Brazauskas
Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Soon H
SH
Choi
Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Adrianne K
AK
Crooke
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Bing
B
Zhang
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.
George M
GM
Solomon
Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
Brett
B
Turner
CFFT Lab, Cystic Fibrosis Foundation Therapeutics, Lexington, MA 01730, USA.
Hermann
H
Bihler
CFFT Lab, Cystic Fibrosis Foundation Therapeutics, Lexington, MA 01730, USA.
Jan
J
Harrington
Division of Pediatric Pulmonary Medicine, Massachusetts General Hospital for Children, Boston, MA 02114, USA.
Allen
A
Lapey
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
Colleen
C
Channick
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
Colleen
C
Keyes
Departments of Medicine and Biochemistry, Stanford University, Stanford, CA 94305, USA.
Adam
A
Freund
Departments of Medicine and Biochemistry, Stanford University, Stanford, CA 94305, USA.
Steven
S
Artandi
CFFT Lab, Cystic Fibrosis Foundation Therapeutics, Lexington, MA 01730, USA.
Martin
M
Mense
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.
Steven
S
Rowe
Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
John F
JF
Engelhardt
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Ya-Chieh
YC
Hsu
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.
Jayaraj
J
Rajagopal
D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D016922:Cellular Senescence; D002923:Cilia / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008168:Lung / Q000166:cytology; D008810:Mice, Inbred C57BL; D009093:Mucus / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D051785:Smad Proteins
TGFβ/BMP4/SMAD signaling; dedifferentiation; differentiation; dual SMAD signaling inhibition; epithelial basal and stems cells; p63(+) basal cells; replicative exhaustion; senescence; stemness; telomeres