Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse–Rat Chimeras

Abstract

<b>Rationale:</b> Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.<b>Objectives:</b> To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.<b>Methods:</b> Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with <i>S52F Foxf1</i> mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.<b>Measurements and Main Results:</b> We identified a unique population of EPCs, FOXF1⁺cKIT⁺ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1⁺cKIT⁺ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1⁺cKIT⁺ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1⁺cKIT⁺ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.<b>Conclusions:</b> Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1⁺cKIT⁺ endothelial progenitors may be considered for treatment of human ACDMPV.