TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds

Claudia Bilodeau,Brian Cox,Daochun Luo,Ian M Rogers,Martin Post,Jinxia Wang,Sharareh Shojaie,Cameron Ackerley,Olivia Goltsis

doi:10.1038/s41536-021-00124-4

Abstract

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

Highlights

Extracellular matrix (ECM) is a complex three-dimensional network of proteins that is crucial for cell attachment, proliferation, and differentiation[1,2,3]
Most epithelial cells lining the tubule-like structures on the scaffolds at day 21 expressed the proximal lung lineage marker SOX2 with the Previously, we demonstrated that ES-derived definitive endoderm (DE) cells differentiated into proximal airway epithelial cells when seeded on acellular lung scaffolds under serum- and growth factor-free conditions[8]
We have shown that acellular lung ECM without supplementation promotes endoderm differentiation into proximal airway cells[8]

Summary

INTRODUCTION

Extracellular matrix (ECM) is a complex three-dimensional network of proteins that is crucial for cell attachment, proliferation, and differentiation[1,2,3]. The hierarchical differentiation process of endoderm cells to airway cells on the decellularized lung scaffold, remains to be elucidated. We demonstrate that TP63+ epithelial cells arise during early lung specification of definitive endoderm cells on acellular lung scaffolds. These multipotent TP63+ cells give rise to ciliated, secretory and mature basal cells making up a pseudostratified columnar airway epithelium that is abrogated by removal of TP63. And swift differentiation of DE cells into basal cells between days 4 and 7 of culture was substantiated by increased gene expression of Tp63, Krt[5], and Krt[14] (Supplementary Fig. 3b). Tp63 gene expression remained high compared to freshly sorted endoderm cells but dropped gradually with

RESULTS

Bilodeau et al 3

DISCUSSION

10 Rat Lung decellularization