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. 2017 Feb;6(2):622-633.
doi: 10.5966/sctm.2016-0192. Epub 2016 Sep 15.

Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling

Dan C Wilkinson  1 , Jackelyn A Alva-Ornelas  2 , Jennifer M S Sucre  2 , Preethi Vijayaraj  2 , Abdo Durra  2 , Wade Richardson  1 , Steven J Jonas  2 , Manash K Paul  2 , Saravanan Karumbayaram  3 , Bruce Dunn  1 , Brigitte N Gomperts  2   3
Affiliations

Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling

Dan C Wilkinson et al. Stem Cells Transl Med. 2017 Feb.

Abstract

Stem cell technologies, especially patient-specific, induced stem cell pluripotency and directed differentiation, hold great promise for changing the landscape of medical therapies. Proper exploitation of these methods may lead to personalized organ transplants, but to regenerate organs, it is necessary to develop methods for assembling differentiated cells into functional, organ-level tissues. The generation of three-dimensional human tissue models also holds potential for medical advances in disease modeling, as full organ functionality may not be necessary to recapitulate disease pathophysiology. This is specifically true of lung diseases where animal models often do not recapitulate human disease. Here, we present a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor-β1 to single cell-type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two-dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell-cell contact and recapitulating the lung microenvironment. Our bottom-up approach for synthesizing patient-specific lung tissue in a scalable system allows for the development of relevant human lung disease models with the potential for high throughput drug screening to identify targeted therapies. Stem Cells Translational Medicine 2017;6:622-633.

Keywords: Disease modeling; Lung; Three-dimensional cell culture; Tissue engineering.

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Figures

Figure 1
Figure 1
Generation and characterization of 3D pulmonary organoids. Organoids are generated by the agglomeration of cell‐coated alginate beads either in a slowly rotating HARV bioreactor or in a 96‐well plate format. (Ai): Alginate bead graphic. (Aii): White light micrograph of alginate beads (scale bar = 400 μm). (Bi): Graphic showing alginate beads coated with collagen I. (Bii): Collagen I immunofluorescence showing a conformal coating of collagen I on the bead surface. Inset, confocal z‐stack of a single collagen I‐coated bead (scale bar = 400 μm). (Ci): Loading and function of HARV bioreactor. One milliliter of sedimented, functionalized alginate beads were loaded into a 4‐ml vessel. Two million fetal lung fibroblasts were seeded into the vessel. The vessel was attached to the rotary base and rotation initiated. (Cii): Time‐lapse image of beads moving together in the 4‐ml HARV bioreactor as a single unit at 4 rpm. (Ciii): Image of beads moving independently in the 4‐ml HARV bioreactor at 16 rpm. (Civ): Graphical summary of bead flow patterns over several rpm values. (Di): Graphic of fetal lung fibroblast‐coated beads after incubation in the HARV bioreactor. (Dii): Fluorescence micrograph of calcein AM (viability dye) showing labeled fetal lung fibroblasts evenly coating functionalized beads (scale bar = 100 μm). (Ei): Graphic of aggregated, fetal lung fibroblast‐coated beads. (Eii): Typical mesenchymal 3D lung organoid generated in the 96‐well bioreactor after 3 days in culture (scale bar = 3 mm). Abbreviations: 3D, three‐dimensional; HARV, high‐aspect‐ratio vessel.
Figure 2
Figure 2
Successful integration of iPSC‐derived fibroblasts into organoid model. (Ai): Representative organoid generated using fetal lung fibroblasts. (Aii, Aiii): Confocal immunofluorescence micrographs of fetal lung fibroblast organoid sections for vimentin, collagen I, α‐SMA, and DAPI. (Bi): Representative organoid generated using iPSC‐derived lung fibroblasts. (Bii, Biii): Confocal immunofluorescence micrographs of iPSC‐derived lung fibroblast organoid sections for vimentin, collagen I, α‐SMA, and DAPI. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; iPSC, induced pluripotent stem cell; α‐SMA, α‐smooth muscle actin.
Figure 3
Figure 3
Characterization of the mechanism of organoid formation. (Ai–Aiv): Representative images taken of organoid formation after 0.7 hours. (Av–Aviii): Organoid position over time is highlighted indicating cyclic deformation with a period of transit of 0.05 Hz. (Aix): Red and black arrows indicate user‐tracked dimensions of organoid. (Ax): Measured strain versus time plot of indicated organoid dimensions at the 0.7‐hour time point (colors coordinate to dimensions specified in Aix). This process increases bead‐bead interactions aiding organoid formation. (Bi–Bv): Images of organoids at various time points during organoid formation. Superimposed is a blue track comprised of velocity vectors arrived at during the tracking process. At the 1.5‐hour time point, the organoid developed a defect, artificially increasing the measured strain for that time sequence. This rip was repaired shortly after, indicating the active role fibroblasts play in organoid formation. (Bvi): Plot of organoid speed over 50 seconds at 2 different time series. (Bvii): Plot of observed force applied to organoid during the 13‐hour period. This increase in force is caused by increased organoid elasticity; as the organoid stiffens, less energy is dissipated by bead‐bead friction and the organoid speeds up. (Bviii): Organoid stiffness versus time plot. (Ci–Civ): Effect of blebbistatin, a myosin II heavy chain phosphorylation inhibitor, on organoid contraction. Organoid contraction either slowed or was completely inhibited by adding increasing amounts of blebbistatin to culture media (scale bars = 3mm). (Cv): Plot of organoid area versus time at different concentrations of blebbistatin.
Figure 4
Figure 4
Effect of TGF‐β1 on organoid contraction and development of a fibrotic phenotype. (Ai, Aii): Representative control organoid imaged on days 6 and 8 after seeding (scale bar = 3 mm). (Aiii, Aiv): Representative organoid treated with TGF‐β1 during the same 2‐day period. The organoid contracted forming a saddle‐like geometry with the focal point near the bottom of the image indicated by an arrow (scale bars = 3 mm). (Av): Aggregate analysis of 20 organoids (10 experimental, 10 control) analyzed during the 8‐day experiment. TGF‐β1 was administered on day 6; thereafter, a clear separation between experimental and control organoid contraction was observed; ∗, p < .05. (Avi): Expression levels of two key genes involved in fibrosis, collagen I and α‐SMA, on treatment with 10 ng/ml TGF‐β1 by quantitative polymerase chain reaction; ∗, p < .05. (Bi–Biv): Confocal immunofluorescence micrographs of representative control organoid sections for vimentin, collagen I, α‐SMA, and DAPI. (Bv–Bviii): Confocal immunofluorescence micrograph of representative TGF‐β1 treated organoid sections for vimentin, collagen I, α‐SMA, and DAPI. Fibrotic areas show increased accumulation of cells that stain positive for collagen I and α‐SMA resembling fibrotic foci, the hallmark of idiopathic pulmonary fibrosis. (Scale bars = 400 μm [Bi, Biii, Bv, Bvii], 200 μm [Bii, Biv, Bvi, Bviii].) (Ci): Merged, rotated confocal z‐stack of patient, iPSC‐derived, α‐SMA reporter line control organoid. Inset, white light image of organoid. (Cii): Merged confocal z‐stack of patient, iPSC‐derived, α‐SMA reporter line organoid treated with TGF‐β1. Inset, white light image of organoid showing high degree of contraction. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; α‐SMA, α‐smooth muscle actin; TGF, transforming growth factor.
Figure 5
Figure 5
Immunostaining of 3D, multicellular organoids compared with adult human distal lung. (A): Confocal micrograph of cross sections of 3D multicellular lung organoids with immunofluorescence for CD31 (HUVECs), vimentin (FLFs), and pro‐SPB and pro‐SPC (type II alveolar epithelial cells) and T1a (type I alveolar epithelial cells; scale bar = 100 μm). (B): Confocal micrograph of multicellular 3D lung organoids with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs). FLFs were also seeded. (C): Confocal micrograph of a cross‐section of normal adult human lung with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs; scale bar = 100 μm). Abbreviations: 3D, three‐dimensional; DAPI, 4′,6‐diamidino‐2‐phenylindole; FLFs, fetal lung fibroblasts; HUVECs, human umbilical vein endothelial cells; SAECs, small airway epithelial cells; SPB, Surfactant Protein B; SPC, Surfactant Protein C.
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References

    1. NIH. NHLBI. Chapter 4 Disease Statistics. NHLBI FACT BOOK, FISC. YEAR 2012 2012:33–52. Available at: http://www.nhlbi.nih.gov/about/factbook/chapter4.htm#gr36\nhttp://www.nhlbi.nih.gov/about/documents/factbook/2012. Accessed November 10, 2015
    1. Hubbard R. The burden of lung disease. Thorax 2006;61:557–558. - PMC - PubMed
    1. Rackley CR, Stripp BR. Building and maintaining the epithelium of the lung. J Clin Invest 2012;122:2724–2730. - PMC - PubMed
    1. Moeller A, Ask K, Warburton D et al. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?. Int J Biochem Cell Biol 2008;40:362–382. - PMC - PubMed
    1. Selman M, King TE, Pardo A; American Thoracic Society; European Respiratory Society; American College of Chest Physicians. Idiopathic pulmonary fibrosis: Prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151. - PubMed

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