Production of high purity alveolar-like cells from iPSCs through depletion of uncommitted cells after AFE induction

Abstract

Protocols to differentiate induced pluripotent stem cells (iPSCs) into specialized cells are continually evolving. iPSCs can be differentiated to alveolar cells with protocols that focus on development, specifically by inducing differentiation into definitive endoderm (DE), anterior foregut endoderm (AFE) and then lung bud progenitor intermediaries. However, current protocols result in a relatively low yield of the desired alveolar cells. The aim of this study was to evaluate whether depleting uncommitted cells after AFE induction would have a beneficial effect on alveolar cell yield. iPSCs were differentiated on Matrigel-coated plates for 25days. At each stage, phenotype was assessed using flow cytometry, immunofluorescence and qRT-PCR. Additionally, samples were dissociated in trypsin following AFE induction to improve the purity of the cells for the subsequent lung differentiation phase. Finally, the efficacy of dissociating the samples was confirmed comparing the expression of markers indicative of pluripotency and apoptosis. The ability to differentiate iPSCs to DE was 96% and to AFE was 97% utilizing our current protocol. After depletion of uncommitted cells and 12 days in culture, the purity of lung bud progenitors was 99%. Finally, the percentage of alveolar types I and II at the end of differentiation was 74% as compared to 31% in control cultures that had not been depleted of uncommitted cells after AFE induction. In conclusion, depletion of uncommitted cells after AFE induction, improves terminal differentiation of alveolar cells from 31% to 74%.

Keywords: Alveolar cell; Anterior foregut endoderm; Differentiation; Flow cytometry; Induced pluripotent stem cell.