Seeded Endodermal Cells Differentiate to NKX2-1⁺/SOX2⁺ Proximal Lung Progenitors with Culture on Decellularized Scaffolds
(A) Schematic representation of differentiation to lung progenitor cells.
(B and C) Real-time PCR analysis reveals an upregulation of Foxa2 and lung progenitor marker Nkx2-1 expression. Expression is presented relative to E13.5 mouse lungs, mean ± SEM, n = 4 experiments.
(D and E) Proximal airway progenitor marker Sox2 and distal alveolar progenitor marker Sox9 were both detected, n = 4 experiments; with extended culture (day 21), Sox2 expression is upregulated.
(F and G) NKX2-1^mcherry+ cells are quantified with flow cytometry. The proportion of NKX2-1⁺ cells increases with extended culture at day 21 to 46.42% ± 3.6%, n = 3 experiments.
(H and I) Immunostaining of day 7 cultures shows the presence of NKX2-1⁺/SOX2⁺/TRP63⁺ coexpressing airway basal stem cells. A small population of distal lineage NKX2-1⁺/SOX9⁺ progenitor cells is also present at this time point. (H) Scale bar represents 13 μm; (I) scale bar represents 25 μm.
(J and K) Immunostaining of day 21 cultures for NKX2-1, SOX2, and SOX9 reveals that the majority of NKX2-1⁺ cells coexpress SOX2, while SOX9 protein expression is rare. Scale bar represents 50 μm.
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Extended Culture of Endodermal Cells on Lung Scaffolds Promotes Differentiation to Ciliated, Club, and Basal Airway Epithelial Cells
(A) Schematic representation of ESC differentiation to airway epithelial populations.
(B and C) RT-PCR analysis reveals the expression of airway epithelial genes Foxj1, Scgb1a1, TRP63, and Cftr. Alveolar epithelial markers Aqp5, Sftpb, and Sftpc were detected at much lower levels, although Aqp5 and Sftpb increased with extended culture. Gene expression is presented relative to adult lungs, mean ± SEM, n = 4 experiments, ^∗p < 0.001.
(D) IF staining and confocal microscopic analysis of day 21 cell-matrix cultures showed mature airway epithelial populations: ciliated cells (TUBB4A⁺), club cells (SCGB1A1⁺), and basal cells (KRT5⁺). Scale bar represents 50 μm.
(E) Cell proportions were quantified as percent of total cell population at days 7, 14, and 21, mean ± SEM, n = 3 experiments.
(F and G) Scanning EM image of the surface epithelia of day 21 cultures (F, scale bar represents 2.5 μm) displays mature tight junction-coupled (white arrowhead) ciliated and nonciliated cells with similar morphology to native mouse airways (G, scale bar represents 5 μm).
(H and I) Transmission EM analysis of day 21 tissue sections confirms the presence of tight junction-coupled (white arrowhead) ciliated cells (ci), club cells (cc), and basal cells (bc). (H) Scale bar represents 4 μm. (I) Scale bar represents 2 μm.
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Proximal and Distal Lung Scaffolds Both Promote Airway Differentiation, while Isolate Matrix Proteins Alone Do Not Support Lung Lineage Commitment of Seeded Endodermal Cells
(A and B) Decellularized sections from both the proximal and distal regions of the lungs were generated and seeded with sorted endodermal cells. Tissue and IF staining show that both matrix sources promoted differentiation to airway ciliated (TUBB4A) and club (SCGB1A1) cells. Scale bars represent 25 μm.
(C) Immunostaining analysis demonstrates that endodermal cells seeded on lung scaffolds maintain definitive endoderm marker FOXA2 and do not express pluripotency marker POU5F1. Luminal structures are positive for epithelial cell markers CDH1 and panKRT. In contrast to cells seeded on scaffolds, endodermal cells cultured at ALI without the support of the lung matrix do not form organized structures and do not express epithelial cell markers. Scale bar represents 25 μm.
(D) TUNEL staining for identification of apoptotic cells on tissue sections was completed for different time points. Cell counts of immunostained sections shows the percentage of apoptotic (TUNEL⁺) cells at days 7, 14, and 21 cultures. Scale bars represent 25 μm. Mean ± SEM, n = 3 experiments.
(E) Immunostaining for Ki67 shows a decline in proliferation with progressive differentiation of seeded cells to airway epithelia from day 7 to day 21 (scale bar represents 25 μm); mean ± SEM, n = 3 experiments.
(F) Schematic representation of isolated matrix-protein recellularization. Sorted endodermal cells are seeded on specific matrix proteins or Matrigel and cultured at ALI in base supportive media.
(G) Real-time PCR analysis reveals upregulation of other endodermal lineage (Pax8, Foxa3, Pdx1) and neuroectoderm (Olig2) markers with culture of definitive endoderm on isolated matrix proteins compared with lung scaffolds. Gene expression is presented relative to definitive endoderm; mean ± SEM, n = 3 experiments.
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Differentiated Airway Epithelial Cultures Consist of Mature Airway Epithelia with Functional CFTR Protein
(A) H&E staining of day 21 scaffold cultures shows a sheet of ciliated cells lining a luminal structure. Scale bar represents 25 μm.
(B) Transmission EM image of cilia shows formation of the appropriate 9+2 dynein arms of motile respiratory cilia. Scale bar represents 100 nm.
(C) Transmission EM of day 21 cultures shows characteristic secretory vesicles inside differentiated club cells with microvilli lining the cell surface (black arrowheads). Scale bar represents 1 μm.
(D) Western blot of d21 culture media shows secreted SCGB1A1, while it is not detected in endodermal cultures without the lung scaffold. Coomassie stain is used as loading control; presented band is at 55 kDa.
(E) IF staining of cell cultures shows mature epithelial sheets with established tight junctions, represented by positive TJP1 staining of CDH1⁺ epithelial cells. Scale bar represents 50 μm.
(F) Stacked confocal images (X-Z plane) show polarized CFTR expression on the apical cell membranes. Scale bar represents 25 μm.
(G) Iodide flux was measured after cAMP agonist-induced CFTR activity in day 21 scaffold cultures. cAMP agonists, forskolin and 3-isobutyl-1-methylxanthine, and a CFTR potentiator, genistein (together as forskolin, 3-isobutyl-1-methylxanthine [IBMX], and genistein) (FIG), were added (0 min, red arrow) and replaced periodically for 8 minutes while iodide flux was measured every minute. Peak efflux, 70.6 ± 7.7 μM, was reached immediately with addition of FIG at 1 min. No efflux was detected with added vehicle control (DMSO). n = 3 experiments; mean ± SEM.
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Organization and Differentiation of Endodermal Cells is Dependent on HS Proteoglycans on Decellularized Lung Scaffolds
(A) Acellular scaffolds were recellularized after heparitinase I or chondroitinase ABC treatment. H&E staining of day 21 seeded scaffold cultures show limited organization and differentiation in the heparitinase I-treated group, while chondroitinase ABC-treated cultures resemble control groups. Scale bar represents 50 μm.
(B) Scanning EM analysis of cultures show a lack of epithelial morphology and tight junction coupling of seeded cells in heparitinase I-treated scaffolds, where cells appear rounded with no resemblance to a lung phenotype. Scale bar represents 10 μm.
(C) Proteome profiler antibody array detects 31 proteins from the array profile that are remaining on lung scaffolds (black). Comparison of the protein profile from decellularized scaffolds treated with or without heparitinase I revealed several HS-bound proteins that are removed from scaffolds and found in the wash supernatant after enzyme treatment (red rectangles): CXCL12, serpinE1, PDGF-AB, HGF, MMP8, FGF2, proliferin, IL10, and CCL3. Data presented are average of two arrays from separate experiments.
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