Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids.

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

Novel FOXF1-Stabilizing Compound TanFe Stimulates Lung Angiogenesis in Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the FOXF1 (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Objectives: Identify-small molecule compounds that stimulate FOXF1 signaling. Methods: We used mass spectrometry, immunoprecipitation, and the in vitro ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a small-molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. Measurements and Main Results: We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells in vitro. TanFe increased protein concentrations of FOXF1 and its target genes Flk1, Flt1, and Cdh5 in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of Foxf1+/- embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of Foxf1+/- mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with FOXF1 deletion. Conclusions: TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.