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Pre-clinical Application Of Tissue-engineered Human Induced Pluripotent Stem Cell-derived Epithelial Grafts In A Porcine Airway Defect Model
Ratna Varma1,2, Alba E. Marin-Araujo3,4, Sara Rostami2, Thomas K. Waddell1,2, Golnaz Karoubi1,2, Siba Haykal5,2.
1Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 2Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada, 3Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON, Canada, 4Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, Toronto, ON, Canada, 5Division of Plastic & Reconstructive Surgery, Department of Surgery, University of Toronto, Toronto, ON, Canada.

PURPOSE
Tracheal injury, stenosis, and malignancy demand surgical management or transplantation, however, the latter fails due to the lack of a functioning epithelium. This issue also encompasses tissue engineering approaches such as decellularized matrices and synthetic biomaterials, wherein the absence of pseudostratified, mucociliary epithelia results in airway obstruction and life-long tracheostomies for patients. Our group has developed a composite biomaterial of Silk Fibroin and Collagen Vitrigel Membrane (SF-CVM), which provides high tensile strength for surgical manipulation and allows differentiation of primary human tracheal epithelial cells (HTECs) into functional ciliated and goblet cells. While HTECs are an endogenous source for recipient-derived grafts, they are limited in their ability to expand and differentiate reliably. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs), a promising alternative, to generate functional SF-CVM-based airway epithelial grafts. We further developed a porcine airway defect model to determine graft integration and survival across 3 days.
METHODS
We sequentially differentiated hiPSCs based on an established protocol towards definitive endoderm, lung progenitors, and airway progenitors, and analyzed them via flow cytometry. These airway progenitors were seeded on SF-CVM grafts under air-liquid interface culture for formation of tight junctions (ZO-1) and differentiation into ciliated (acetylated α-tubulin) and goblet cells (mucin 5AC), as assessed by immunocytochemistry.
We created a 2x4 cm tracheal defect and manipulated it according to the following four groups prior to defect closure: 1) no manipulation; 2) mucosa stripped; 3) mucosa stripped and replaced with bare SF-CVM graft; and 4) mucosa stripped and replaced with hiPSC-derived SF-CVM graft labeled with CMTMR dye. Post defect closure, all groups were wrapped in sternocleidomastoid muscle for vascularization. On post-operative day (POD) 3, all defects were assessed macroscopically, while Groups 2 to 4 were assessed for cell viability and death via calcein-AM and ethidium homodimer 1 staining, respectively.
RESULTS
We produced 92.62.1% CKIT+CXCR4+ definitive endoderm, 38.13.9% GFP-NKX2.1+ lung progenitors, and 65.62.4% P63+ airway progenitors, which differentiated into 64.67.8% ciliated and 2.11.4% goblet cells on SF-CVM (Figure 1A). This differentiation into ciliated cells on SF-CVM was significantly higher than that of HTECs (17.25.0%; P<0.05; Figure 1A) and physiologically relevant, being well within the 48-70% range present in human tracheae.
There were no respiratory complications or animal mortality. Group 1 resembled native epithelium, Group 2 had granulation tissue overgrowth, while Group 3 and 4 demonstrated SF-CVM integration with the surrounding tracheal tissue (Figure 1B). Groups 2 and 3 demonstrated epithelial infiltration with high cell mortality (Figure 1C). Group 4 retained the CMTMR label (magenta) which co-localized with calcein-AM (green), indicating that the hiPSC-derived epithelium was intact and alive on POD3 (Figure 1C).
CONCLUSIONS
We developed functional hiPSC-derived SF-CVM epithelial grafts which survived and integrated within porcine airway defects across 3 days. This is the first pre-clinical application of biomaterials-based airway grafts generated from hiPSCs that has significant clinical implications for treatment of small airway defects and full-length tracheal transplants.


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