We thank Dr Matthew Bacchetta for providing human adult lung samples

We thank Dr Matthew Bacchetta for providing human adult lung samples. GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling Arnt induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development. hPSC-based model offers a complementary and more malleable system where timing of addition and withdrawal of stimuli can be performed more precisely, and is directly relevant to human development. It was recently reported that canonical Wnt signaling induced by the GSK3 inhibitor CHIR9902 (CHIR) promoted specification of developmental lung progenitors (LPs) towards ATII cells, whereas its withdrawal induced a proximal fate. These studies used reporter lines to enrich for progenitor populations or identify desired differentiated lineages (Jacob et al., 2017; Longmire et al., 2012; McCauley et al., 2017), and are therefore not universally applicable. Several other reports also show the generation of ATII cells (Chen et al., 2017; Huang et al., 2014; Jacob et al., 2017; Yamamoto et al., 2017). However, neither mature NGFR+ basal cells (BCs) (Rock et al., 2009), the stem cells of the airways, nor ATI cells were ever generated, perhaps because both cell types arise late in development (Frank et al., 2016; Yang et al., 2018). To address these issues, a culture model that does not rely on reporter lines and is permissive for the specification of all lung and airway lineages, thus allowing investigation of conditions that favor specific lineages, is required. Here, we report a collagen I (Col I) 3D culture system that satisfies these criteria. We show that GSK3 inhibition, rather than favoring distal fates as reported previously (McCauley et al., 2017), promotes proliferation and inhibits differentiation, whereas withdrawal of Harmaline GSK3 inhibition induces multilineage proximal and distal maturation, including of NGFR+ basal cells, morphologically mature ATII cells and cells with the morphology and marker expression of ATI cells. Furthermore, a WNT ligand could not recapitulate the effect of GSK3 inhibition, suggesting that this effect is not primarily mediated by canonical WNT signaling. Generic cell cycle inhibition, on the other hand, partially recapitulated the effect of CHIR withdrawal, suggesting a role for GSK3-mediated cell cycle regulation in maturation of LPs. We next used this model to show that, after CHIR withdrawal, NOTCH inhibition promotes proximal and inhibits distal development, thus identifying NOTCH signaling as one of the signaling pathways involved in proximodistal specification. RESULTS Establishment of a 3D Col I model of human lung and airway lineage specification Our published 2D culture protocol recapitulates development (Huang et al., 2015, 2014). However, for further studies, the 2D model posed two problems. First, in large areas cell detachment occurred (Huang et al., 2015). Second, despite ample presence of cells expressing ATII markers, expression of the most specific ATII marker, SFTPC, was sporadic whereas ATI cells and BC-like cells were rare (Huang et al., 2015, 2014), and mature NGFR+ BCs were absent. We therefore proceeded to culture in a 3D matrix. We generated NKX2.1+FOXA2+ LPs, which lacked mature lung and mesenchymal markers, in 2D until day (d)25, when the purity of NKX2.1+FOXA2+ lung progenitors was maximal (90-98%), as described previously (Huang et al., 2015, 2014), and transferred these to Col I gels in the presence of factors used in 2D cultures (Huang et al., 2015, 2014) [CHIR, FGF10, KGF and dexamethasone, 8-bromo-cAMP and Harmaline isobutylmethylxanthine (DCI) (Gonzales et al., 2002)] (Fig.?1A, Harmaline top). The cells organized in strands enveloping empty lacunae (Fig.?1A, upper left) and almost uniformly expressed NKX2.1 (85.0817.54%) (Fig.?1A), FOXA2 (not shown) and the Harmaline surface mucin MUC1, the apical expression Harmaline of which indicated polarization (Fig.?1A). Most cells also co-expressed variable amounts of SOX9 and SOX2 (Fig.?1A), which, in contrast to the mouse (Rockich et al., 2013), are commonly co-expressed in distal tips during human lung development (Chen et al., 2017; Nikolic et al., 2017). SFTPC+ cells were abundant, indicating that a 3D environment is crucial for SFTPC expression (Fig.?1A). The ATII marker ABCA3 was also detected (Fig.?1A). Some cells co-expressed ATI (PDPN) and ATII (SFTPB, SFTPC) markers (Fig.?1A, arrows) (Desai et.