Fig 1: Differential infection kinetics and cellular tropism of CCCoVs and SARS‐CoV‐2 in human lung implants. A) Schematic diagram illustrating of human lung implant infection by multiple human coronaviruses. B–F) Viral titers in infected human lung implants: B) 229E at 2 d (n = 6) and 7 d (n = 3). C) NL63 at 2 d (n = 3) and 7 d (n = 3). D) OC43 at 2 d (n = 4) and 7 d (n = 4). E) HKU1 at 2 d (n = 4) and 7 d (n = 3). F) SARS‐CoV‐2 at 2 d (n = 3) and 7 d (n = 3). G–K) Representative immunofluorescence co‐staining showing virus‐infected cells (red) and markers of specific human cell types (green): CK19 (epithelial cells), vimentin (mesenchymal cells), CD34 (endothelial cells), CC10 (club cells), CK5 (basal cells), MUC5B (goblet cells) and FOXJ1 (ciliated cells), SFTPC (AT2). Scale bars: 50 µm. L) Summary of the susceptible cell subtypes for 229E, NL63, OC43, HKU1, and SARS‐CoV‐2 shown in panels (G–K). M) Schematic of the experimental design. Lung‐humanized mice were orally administered Paxlovid or vehicle starting at the time of HKU1 exposure (0 h) followed by dosing every 12 hours. Human lung implants were harvested 48 h after infection. N) HKU1 titers in the human lung implants administered Paxlovid (n = 8) or vehicle (n = 8) 48 h after exposure to virus. O) Representative immunofluorescence staining of viral nucleoprotein in human lung implants from Paxlovid‐ and vehicle‐treated mice. Scale bars: 50 µm. P) Quantification of HKU1 infected area as a percentage of the total field in Paxlovid‐treated (n = 4) and vehicle‐treated (n = 5) groups. Q) Representative histopathological features of HKU1 infected human lung implants following Paxlovid or vehicle treatment. Scale bars: 50 µm. Data presented as means ± SD. Statistical analyses were performed using a nonpaired two‐tailed Student's t‐test.
Fig 2: Progressive structural maturation and multilineage differentiation in cryopreserved human lung implants. A) H&E staining showing changes in the alveolar regions of human fetal lung tissue before transplantation and at 1, 4, 8, and 12 weeks after transplantation. Scale bars: 100 µm (top) and 50 µm (down). B–D) Quantification of (B) alveolar area, (C) MLI, and (D) alveolar wall thickness. n = 4 biological replicates, 20 technical replicates. E) Representative H&E staining of human lung implants displaying bronchioles (Br), alveolar structures (Av), cartilage (Ca), and blood vessels (V). Scale bars: 200 µm (left) and 50 µm (right). F) Immunofluorescence analysis of cellular composition in subcutaneous implants, identifying human‐derived epithelial cells (CK19), mesenchymal cells (Vimentin), and endothelial cells (CD34). Scale bars: 50 µm. G) Immunofluorescence staining for human (H CD31) and mouse (M CD31) endothelial cells in human lung implants. Scale bars: 200 µm (left) and 50 µm (right). H) Immunofluorescence staining of airway and alveolar cell subsets, including ciliated cells (FOXJ1), club cells (CC10), basal cell (CK5), goblet cells (MUC5B), AT1 (PDPN), and AT2 (SFTPC). Scale bars: 50 µm. Data presented as means ± SD. Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons tests.
Fig 3: FGF/VEGFA treatment and fragment optimization improve cryopreserved human lung implants engraftment and alveolar remodeling. A) Schematic diagram illustrating the cryopreservation process of human fetal lung tissue, preparation of tissue fragments, treatment strategies, and subcutaneous implantation into immunodeficient mice. B) Immunohistochemical staining for Ki‐67 to evaluate proliferative activity one week after transplantation. Scale bars: 50 µm. n = 8 biological replicates. C) Co‐immunofluorescence staining of Ki‐67 (red) with CK19 (epithelial cells, green), Vimentin (mesenchymal cells, green), CD34 (endothelial cells, green), SFTPC (alveolar type 2 cells, AT2, green). Scale bars: 50 µm. D) Schematic illustrating transplantation of fragments with varying diameters to evaluate the effects of growth factor supplementation and fragment size. E) Growth kinetics, endpoint volumes, and fold expansion (relative to the initial volume) of human lung implants with different fragment diameters and treatments over 12 weeks. Group sizes: 3 mm × 1 + MG, n = 6; all other groups, n = 8. F) Schematic diagram showing transplantation of four 2 mm fragments to evaluate the effect of growth factor treatment. G) Representative images of human lung implants 12 weeks post‐transplantation with or without growth factor treatment. H) Growth curves and endpoint volumes of implants. ×4 + MG, n = 6; ×4 + MG + GF, n = 10. I) H&E staining comparing lung implants with or without growth factor treatment at 12 weeks post‐transplantation. Scale bars: 200 µm (left) and 50 µm (right). J–L) Quantification of (J) alveolar area, (K) mean linear intercept (MLI), and (L) alveolar wall thickness. n = 4 biological replicates, 20 technical replicates. M) Immunofluorescence staining of alveolar type 1 cells (AT1, PDPN, green) and AT2 (SFTPC, red) after 12 weeks of transplantation. Scale bars: 50 µm. N) Quantification of the percentage of SFTPC⁺ AT2. n = 6 biological replicates. Data presented as means ± SD. Statistical analyses were performed using a nonpaired two‐tailed Student's t‐test (B,H,J–L,N) and one‐way ANOVA followed by Tukey's multiple comparisons tests for (E). ×1: One cryopreserved human fetal lung tissue fragment. MG: Matrigel. GF: Growth factors. ×4: Four cryopreserved human fetal lung tissue fragments.
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