Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice missing critical morphogenetic genes. While epithelial cell lineages were efficiently generated from ESC, other cell types were mosaic. A complete contribution of donor ESC to lung tissue has never been achieved. The mouse lung has never been generated in a rat.
Objective: To generate the mouse lung in a rat.
Methods: CRISPR/Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat 1-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESC into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESC to the lung tissue was examined by immunostaining, flow cytometry and single-cell RNA sequencing.
Measurements and main results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR/Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESC restored pulmonary and thyroid structures in mouse-rat chimeras leading to a near 99% contribution of ESC to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat.
Conclusions: A combination of CRISPR/Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors".
Keywords: CRISPR/Cas9; NKX2-1; Neonatal lung tissue; blastocyst complementation; embryonic stem cells.