文摘
Stem-cell-based tracheal replacement has been used to treat patients with end-stage airway disease in compassionate cases. Clinical experience suggests that restoration of an epithelial barrier is a priority to improve outcomes, but recent data suggest that a substantial number of autologous epithelial cells are required for effective engraftment. Existing cell culture methods fail to expand the airway epithelial progenitor pool, presenting a considerable hurdle for progression to clinical trials. We aimed to assess feeder layers and a Rho-associated kinase inhibitor as a method to expand human respiratory epithlieum for airway bioengineering.p>
Methods
<p id="spara20">Human epithelial cells from endobronchial biopsy samples were cultured on mouse 3T3-J2 fibroblast feeder layers in medium containing Y-27632, a Rho-associated kinase inhibitor (3T3+Y). Air-liquid interface and tracheosphere assays were done to determine differentiation capacity, and high-speed video microscopy to observe ciliary behaviour. Telomerase expression was measured with immunofluorescence and western blotting and telomere length with real-time PCR. Cells were karyotyped and engraftment potential tested in ex-vivo and in-vivo xenograft models.p>Findings
<p id="spara30">3T3+Y allowed for rapid and sustained expansion of airway epithelial basal cells. Population doublings demonstrated that cultures derived from donor biopsy samples could provide sufficient cell numbers to recellularise human tracheal scaffolds. At clinically applicable passages, cells were capable of airway differentiation in vitro, forming both ciliated and goblet lineages. Ciliary beat frequency and beat pattern were within normal range. Cells were karyotypically normal despite extensive expansion. Protein expression of telomerase was increased in 3T3+Y compared with that in conventional growth medium, and telomere length appeared to stabilise. Cultured cells repopulated a decellularised tracheal scaffold ex vivo and restored a differentiated epithelium in an in-vivo tracheal transplantation xenograft model.p>Interpretation
<p id="spara40">The findings show that 3T3+Y generates large numbers of airway basal epithelial stem cells that retain qualities desirable for clinical transplantation. These data have important implications for personalised autologous airway epithelial cell therapy because cells could be obtained rapidly and in an appropriate number. Preclinical and clinical validation is required.p>Funding
<p id="spara50">Wellcome Trust.
