ENDOTHELIAL CELLS PROMOTE DISTAL AIRWAY PATTERNING OF HUMAN IPSC-DERIVED LUNG PROGENITORS

Abstract

Induced pluripotent stem cells (iPSCs) offer unprecedented potential for patient-specific regeneration of tissues and organs. The lung is a vital organ that mediates gas exchange across the alveolar-capillary barrier. Thus, the regeneration of functional lung tissue from iPSCs requires the ability to generate both lung endothelial cells (ECs) and distal type I and II alveolar epithelial cells. Multiple protocols have been developed to specify proximal lung epithelial differentiation from iPSCs; however, the differentiation to a distal lung epithelial phenotype has remained elusive. In light of the importance that endothelial-epithelial cell juxtaposition forms the essential alveolar-capillary barrier, we examined the contribution of iPSC-derived endothelial cells (iECs) in influencing cell fate of de novo lung progenitors (LPs). Human (h) iPSCs were cultured on Matrigel during their differentiation into ECs or LPs. EC differentiation was achieved using combinatorial treatment of hiPSCs with BIO and VEGF. VE-cadherin+ iECs, exhibiting typical endothelial cobblestone morphology, were selected by immnomagnetic isolation followed by expansion, during which conditioned medium (iEC-CM) was collected. Flow cytometric analyses demonstrated CD34, PECAM-1, and VEGFR-2 expression in the VE-cadherin+ population, signifying successful EC differentiation. In vitro lung embryogenesis was recapitulated by generating a definitive endoderm intermediate followed by CXCR4+ immunomagnetic selection. CXCR4+ cells were sequentially exposed to hedgehog, bone morphogenetic proteins, fibroblast growth factors to yield flat NKX2.1+Sox9+ LPs exhibiting a high cytoplasmic to nuclear ratio. Subsequently, LPs were cultured at an air-liquid interface in 50% iEC-CM with daily media changes for 21 days. Quantitative RT-PCR analyses revealed highly significant (#p < 0.01) loss of pluripotency markers (NANOG, OCT4, SOX2) upon differentiation into iECs or LPs. NKX2.1 and SOX9 expressions were ≥15-fold (*p < 0.05) in the LP pool compared to earlier stages of lung development, and notably absent in iECs. Interestingly, LPs cultured in iEC-CM showed a strong bias towards distal rather proximal airway patterning as evidenced by augmented expression (≥3-fold, *p < 0.05) of mature alveolar pneumocyte I and II markers (AQP5, SFTPB, SFTPC); concomitant with reduced (∼60%, *p < 0.05) cilia or goblet cell markers (FOXJ1 and MUC5AC). For the first time we show that ECs play a key role in promoting the differentiation of LP cells to a distal epithelial cell fate that is essential for the formation of the alveolar air-blood barrier. Moreover, this effect is mediated through paracrine mechanisms, likely involving exosome secretion. These findings have profound implications for the development of efficient approaches for the regeneration of functional alveoli.