Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes generation of the whole lung

Abstract

Millions worldwide suffer from incurable lung diseases, and organ transplantation remains their only hope. However, there has been an absolute shortage of donor lungs and unmet needs for transplantable lung generation composed of tissue-specific mesenchyme, epithelium, and endothelium. Elucidation of lung precursor traits during development can lead to solving this issue. Using lineage-tracing mice, we discovered that gastrulating Foxa2 lineage+ Pdgfrα+ mesendoderm forms the competitive mesenchymal lung niche. We further evidenced that Foxa2+Pdgfrα+ mesendoderm is an evolutionarily-conserved niche during human iPSC-derived lung differentiation. The Fgfr2 gene depletion, specifically in the Foxa2 lineage, showed lung agenesis phenotype. Strikingly, donor iPSCs injection into those blastocysts complemented endodermal and mesodermal defective lung organ niches that efficiently led to the entire lung generation. Together, targeting Foxa2 lineage for lung generation is a novel paradigm, holding a grand promise for future human whole lung generation in large animals using human iPSCs.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation

“…We utilized the Foxa2 Cre/+ ; Fgfr2 flox/flox ; Rosa LSL-tdTomato/+ mice. This mouse model, which we previously reported, exhibits an absence of lung formation 32 . In the Fgfr2 heterozygous knockout mice (Foxa2 Cre/+ ; Fgfr2 flox/+ ; Rosa LSL-tdTomato/+ , hereafter, Fgfr2 hetero ), tdTomato-positive salivary gland primordia were detected at the base of the oral cavity at E13.5 (Figure 2A).…”

“…Interestingly, the Shh-driven Fgfr2 knockout phenotype did not show gross morphological changes in E18.5 salivary glands compared to the littermate controls, which is contradictory evidence of the Fgfr2's effect in ex vivo culture 14 . Based on our data, we speculate that Fgfr2 depletion requires specific time windows to induce salivary gland agenesis phenotype, most likely in the salivary gland precursors around the boundary regions, as the lung agenesis model requires the depletion of the genes before the lung primordia formation 13,42 . For the exact requirement of Fgfr2 during salivary gland branching morphogenesis in vivo, spatiotemporal Fgfr2 depletion using Shh CreERT2/+ ; Fgfr2 flox/flox or Foxa2 CreERT2/+ ; Fgfr2 flox/flox during salivary gland development is required in future experiments.…”

“…Since the Shh-driven Fgfr2 knockout phenotype showed lung agenesis phenotype, the Cre driver activity and genotyping cannot be mistaken in our experiments 13,32 . Interestingly, the Shh-driven Fgfr2 knockout phenotype did not show gross morphological changes in E18.5 salivary glands compared to the littermate controls, which is contradictory evidence of the Fgfr2's effect in ex vivo culture 14 .…”

“…To address this issue, we further investigated another potential lineage involved in the salivary gland precursor niche around the boundary region of endoderm and ectoderm by analyzing deposited single-cell RNA-seq (scRNAseq) data from E8.5 mouse 30 and E12.0 OE 31 because the organ agenesis phenotype caused by Fgfr2 deficiency in other organs, like lungs, requires to target the precursor niche but not the progenitors to obtain after the organ initiation 13,32 . In the E12.0 OE scRNAseq analysis, we identified high-expression genes in the posterior-lateral epithelium (PL) and tongue epithelium (T) adjacent to the salivary gland primordium (Figure S1D, S1E, and S1F).…”

Ephrin Forward Signaling Controls Interspecies Cell Competition in Pluripotent Stem Cells

“…We established mouse iPSC lines (CD1 iPSC or ntdTomato iPSC) using Sendai virus-mediated reprogramming kit: CytoTune2.0 (Thermo Fisher, A16517) and followed manufactured protocol as described before 24 . Briefly, we harvested E14.5 lung tissues of Rosa26 nt-ng/nt-ng mice (Jax, 023035) or CD1 mice (Charles River, 022).…”