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%.

Viral pandemic preparedness: A pluripotent stem cell-based machine-learning platform for simulating SARS-CoV-2 infection to enable drug discovery and repurposing

“…The aiLUNG simulations produced a similar expression profile when compared with actual human wild‐type lung cell specific factors outlined above (Figure 1A), consistent with previous studies 4,5,7,12,13,15,20,22,23 . The probability that all (N = 15) of these aiLUNG outcomes were correctly predicted by chance alone using the binomial test is .0021.…”

“…Once validated the aiPSC simulations were used to create the wild‐type lung cell simulations (aiLUNG) by activating NKX2.1 and Wnt5a in the presence of a simulated lung cell medium as described previously 12,20 . The unsupervised aiLUNG simulations converged quickly (24 iterations) to a new system wide steady state without evidence of overtraining after 1000 iterations.…”

“…These values were not locked on so that after the first iteration all values were determined by evolving system behavior. The lung cell models (aiLUNG) were created through direct conversion of the aiPSCs to aiLUNG cells using overexpression of NK2 Homeobox 1 (NKX‐2.1), and Wnt Family Member 5A (Wnt5a) in the presence of a simulated lung cell medium 12,20 . Once validated with published peer reviewed data, the aiLUNG simulations were exposed to simulated SARS‐CoV‐2 infection by turning on extracellular Spike‐RBP (RNA Binding Domain) in the presence of active Transmembrane Serine Protease 2 (TMPRSS2).…”

“…The initial goal of this project was to create computer simulations (aiPSCs) of human induced pluripotent stem cells (iPSC) and artificially induced lung (aiLUNG) cell models then validate these models using the results published by References 4,5,7,12,13,15,20,22,and 23 and others as described above. Briefly, for the aiPSC models, the input…”

“…Finally, the aiLUNG‐COVID19 simulations were applied to drug repurposing of a small group of approved drugs with well‐known mechanisms of action. The genomic and phenotypic profiles of wild‐type aiLUNG cells and aiLUNG‐COVID‐19 simulations were validated based on the currently available experimental wet lab and other data 6‐21 . Ideally, in the future, this new technology would be implemented as soon as the new viral genome has been identified and validated.…”