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

Abstract

SUMMARY Functional modeling of many adult epithelia is limited by the difficulty of 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 found 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 inhibition in a feeder-free culture system we were able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium that is physiologically responsive to clinically relevant drugs such as CFTR modulators. This approach is effective for clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers, and may therefore be broadly applicable for modeling of epithelia.

Lung organoids: current uses and future promise

“…3A). With the BMP inhibitors dorsomorphin and DMH1, no change in the proportion of ciliated and secretory cells was seen, although other studies have suggested that inhibition of BMP signaling inhibits basal cell differentiation (Mou et al, 2016;Tadokoro et al, 2016). Addition of BMP4 ligand results in small spheres with mostly basal cells and few Krt8 + cells (Fig.…”

“…Various methods have been developed for isolating and growing these basal cells from different regions of the normal human respiratory system, including nasal epithelium, ʻlarge airways', which include the trachea, primary bronchi and intralobar bronchi down to about the third or fourth generation, and from bronchial brushings (Hackett et al, 2011;Randell et al, 2011). The most efficient methods for expanding and cloning TRP63 + KRT5 + human basal cells involves culturing them either on irradiated mouse 3T3-J2 fibroblasts in the presence of the Rho kinase inhibitor Y-27632 (Butler et al, 2016;Kumar et al, 2011;Suprynowicz et al, 2012), or with a Rho kinase inhibitor together with inhibitors of Smaddependent signaling through the BMP and TGFβ pathways and an activator of Wnt signaling (Mou et al, 2016). The progenitor properties and differentiation capacity of these basal cells can then be followed in organoid cultures.…”

Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells

“…Nevertheless, expansion of donorderived cell supplies for preclinical studies of CFTR modulators will be enhanced by the CRC method. Recently, inhibition of SMAD signaling pathways in a feeder-free culture system was used to expand airway basal stem cells (17). However, decreases in CFTR rescue as a function of cell expansion were quite profound in ALI cultures generated with this method.…”

Take a deep breath and digest the material: organoids and biomaterials of the respiratory and digestive systems

“…[14] Human esophagus can be cultured on an air-liquid interface or on the matrix of decellularized esophageal tissue. [100,101] Yet, these cultures lack the supporting tissue around the esophagus along with a tubular structure that an esophageal organoid could incorporate. The proper matrix and Biomaterials for 3D Cell Biology Prospective Article media composition will need to be determined in order to generate esophageal organoids from primary tissue and hPSCs.…”