SummaryBackground-Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up.

Sequencing data have been deposited at the European Genome-Phenome Archive (http://www.ebi.ac.uk/ega/) under accession numbers EGAD00001005193. Somatic mutation calls, including single base substitutions, indels and structural variants, from all 632 samples have been deposited on Mendeley Data with the identifier: http://dx.doi.org/10.17632/b53h2kwpyy.2. Code Availability Detailed method and custom R scripts for the analysis of mutational burden in bronchial epithelium are available in Supplementary Code. Other packages used in the analysis are listed below:

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Rationale: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts.Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T31Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.