Supplemental Table 1: Lung Donor InformationData represents the age (years), gender (male (M) or female (F)), Body Mass Index (BMI), and donor status (Donation after Cardiac Death (DCD) or Donation after Brain Death (DBD)). Age and BMI are presented with the summarized mean ± standard deviation, with HL38 excluded from these means as an outlier.

Supplemental Figure 1: Primary Endothelial Cell isolation and Culture

(A) Primary lung endothelial cells were isolated from large vessels and cultured for 5 days in EGM2 media prior to sorting for CD31+ population. Gating strategy demonstrates the exclusion of doublets and dead deals (Pacific Blue+), and the identification of the CD31+ population. Representative example presented. (B) Immunofluorescent staining of the sorted population in culture on gelatin-coated flasks, demonstrating endothelial (CD31+) purity. Scale bar = 100µm.

Supplemental Figure 2: Effect of ROCK Inhibitor on Cell Phenotype and Senescence.

(A) Gene expression analysis of n=3 unique donor-derived cell lines at passage 1 and passage 4, with or without the addition of ROCK inhibitor Y27632 (10uM). n=3 biological replicates analyzed per group. Error bars represent standard deviation. No significant differences between untreated and treated (+ROCK) were found by Mann-Whitney test, at each passage. (B) Representative image of cells at passage 1 and passage 4, with or without ROCK inhibitor, with Trypan Blue added to culture media. (C) Representative image of cells at passage 1 and passage 4, with or without ROCK inhibitor, stained for senescence-associated β-galactosidase activity at pH 6. Scale bars = 50µm.

Supplemental Figure 3: In Vitro Analysis of Basal Epithelial Stem Cell Capacity.

(A) Primary human lung epithelial stems cells at Air-Liquid Interface (ALI) in vitro, Day 21. Induction of ciliogenesis demonstrated by Haematoxyalin and Eosin staining. Scale bar = 25µm. (B) Immunofluorescent images demonstrate preservation of the basal stem cell population (KRT5+TP63+), functional ciliogenesis (FOXJ1 and Acetylated α-Tubulin (TUBA1B)) and adherens junction formation (E-Cadherin, CDH1) at ALI. Scale bar = 25µm. (C) 3-dimentional culture assay demonstrating sphere formation from single cells. Haematoxylin and Eosin stained spheres demonstrate luminal development by day 7. (D) Immunofluorescent images of spheres demonstrating a predominance of a KRT5+TP63+ phenotype in the spheroid. Scale bar = 50µm.

Supplemental Figure 4: Effect of Endothelium Cell Co-Culture on Basal Cell Population.

(A) Gene expression analysis of primary epithelial basal cells (passage 4) in co-culture with primary lung endothelial cells. Data represents the basal cell population (Epi+) plus the culture of endothelial cells on transwell inserts (Endo+), with or without the addition of VEGF in the culture media (VEGF+, 40ng/ml). (B) Expression of VEGF receptor by treated and untreated epithelium (undetected) and endothelium. Expression level is normalized to β-Actin (ACTA1) and expressed relative to normal lung tissue. n=3 biologic replicates analyzed in technical duplicate. Error bar = standard deviation. Analyzed by Kruskall-Wallis with Dunn’s post-test to untreated group (Epi+).

Supplemental Figure 5: Endothelial and Epithelial Cell Distribution.

Representative images of (A) Epithelial (KRT5) and (B) Endothelial (CD31) cell coverage on rat lung scaffolds (Collagen 1) following 7 days of ex vivo biomimetic culture.