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

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.

Reversal of Surfactant Protein B Deficiency in Patient Specific Human Induced Pluripotent Stem Cell Derived Lung Organoids by Gene Therapy.

Surfactant protein B (SFTPB) deficiency is a fatal disease affecting newborn infants. Surfactant is produced by alveolar type II cells which can be differentiated in vitro from patient specific induced pluripotent stem cell (iPSC)-derived lung organoids. Here we show the differentiation of patient specific iPSCs derived from a patient with SFTPB deficiency into lung organoids with mesenchymal and epithelial cell populations from both the proximal and distal portions of the human lung. We alter the deficiency by infecting the SFTPB deficient iPSCs with a lentivirus carrying the wild type SFTPB gene. After differentiating the mutant and corrected cells into lung organoids, we show expression of SFTPB mRNA during endodermal and organoid differentiation but the protein product only after organoid differentiation. We also show the presence of normal lamellar bodies and the secretion of surfactant into the cell culture medium in the organoids of lentiviral infected cells. These findings suggest that a lethal lung disease can be targeted and corrected in a human lung organoid model in vitro.

Conversion of human and mouse fibroblasts into lung-like epithelial cells.

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.

Efficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

Three-dimensional culture and FGF signaling drive differentiation of murine pluripotent cells to distal lung epithelial cells.

Reciprocal signaling between the lung mesenchyme and epithelium is crucial for differentiation and branching morphogenesis. We hypothesized that the combination of signaling pathways comprising early epithelial-mesenchymal interactions and a 3D spatial environment are necessary for an efficient induction of embryonic and induced pluripotent stem cells (ESCs and iPSCs) into a lung cell phenotype with hallmarks of the distal niche. Aggregating early, but not late, embryonic lung mesenchyme with endoderm-induced mouse ESCs and iPSCs for 6 days resulted in organization into tubular structures and differentiation of the tubular lining cells to an NKX2-1(+)/SOX2(-)/SOX9(+)/proSFTPC(+) lineage. Over 80% of the endoderm-induced cells committed to an NKX2-1(+) lineage. Electron microscopy analysis demonstrated numerous multivesicular bodies and glycogen deposits in the tubular lining cells, characteristic features of type II epithelial cell progenitors. Using soluble FGFR2 receptor antagonists, we demonstrate that reciprocal fibroblast growth factor (FGF) 2, 7, and 10 signaling is essential for differentiation of endoderm-induced cells to an NKX2-1(+)/proSFTPC(+) phenotype within 3D aggregates. Only FGF2 was able to commit endoderm-induced cells in monolayer cultures to an NKX2-1(+) lineage, however with a significant lower efficiency (∼16%) than seen with mesenchyme. Thus, while FGF2 signaling alone can induce a primed population of ESCs and iPSCs, the cells do not differentiate to distal lung epithelial progenitors with the same efficiency and level of maturity that is achieved when the complex tissue and 3D environment of the developing lung is more accurately recapitulated.

Identification of a proximal progenitor population from murine fetal lungs with clonogenic and multilineage differentiation potential.

Lung development-associated diseases are major causes of morbidity and lethality in preterm infants and children. Access to the lung progenitor/stem cell populations controlling pulmonary development during embryogenesis and early postnatal years is essential to understand the molecular basis of such diseases. Using a Nkx2-1(mCherry) reporter mouse, we have identified and captured Nkx2-1-expressing lung progenitor cells from the proximal lung epithelium during fetal development. These cells formed clonal spheres in semisolid culture that could be maintained in vitro and demonstrated self-renewal and expansion capabilities over multiple passages. In-vitro-derived Nkx2-1-expressing clonal spheres differentiated into a polarized epithelium comprised of multiple cell lineages, including basal and secretory cells, that could repopulate decellularized lung scaffolds. Nkx2-1 expression thus defines a fetal lung epithelial progenitor cell population that can be used as a model system to study pulmonary development and associated pediatric diseases.