Pneumologie 2015; 69 - A46
DOI: 10.1055/s-0035-1556638

Differentiation of human pluripotent stem cells into airway epithelial cells

S Ulrich 1, S Weinreich 1, R Haller 1, S Menke 1, R Olmer 1, U Martin 1
  • 1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Germany; REBIRTH Cluster of Excellence; Member of the German Center for Lung Research (DZL); Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH)

Introduction: Pluripotent stem cells (PSCs), like embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer promising new therapeutic options to treat lung diseases, such as cystic fibrosis or surfactant deficiencies, based on cellular/tissue replacement therapies, disease modelling and drug screening. These regenerative approaches crucially rely on the efficient differentiation of PSCs into lung epithelial cells which is the aim of our present study.

Methods: We made use of the human (h)ESC reporter cell line hES3 NKX2.1-eGFP (kindly provided by the lab of A. Elefanty) expressing eGFP under the endogenous NKX2.1 promoter to establish and optimize the respiratory differentiation. Immunofluorescence stainings, qRT PCR analysis and flow cytometric quantification were used to analyze the generated definitive endoderm (DE) and NKX2.1-eGFPpos cell populations. Additionally, we made use of TALEN (transcription activator-like effector nucleases) technology for the establishment of a traceable reporter cell line.

Results: With our current differentiation strategy we can reliably generate a highly enriched DE population of about 70.9%± 2.9% CXCR4pos/c-Kitpos cells (Mean ± SEM, n = 3) additionally verified by coexpression of the transcription factors SOX17pos/FOXA2pos. Subsequent differentiation resulted in the generation of about 35% NKX2.1-eGFPpos cells related to the entire differentiation culture. Characterization of the purified NKX2.1-eGFPpos population indicated their lung epithelial phenotype, so far. In addition, we successfully generated a traceable hES3 NKX2.1-eGFP AAVS1-CAGRedStarnuc reporter cell line as a useful tool for further maturation experiments e.g. in co-culture/transplantation experiments.

Discussion: We were able to generate a highly enriched DE population followed by the induction of a distinct population of NKX2.1-eGFPpos most likely associated with the lung lineage. Future work will focus on additional optimization of the differentiation strategy and further maturation of NKX2.1-eGFPpos cells into airway epithelial cell types, like e.g. Club cells or ciliated cells.

*Presenting author