Efficient generation of lung and airway epithelial cells from human pluripotent stem cells

Abstract

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

Fig. 1. Establishment of a protocol of lung field induction

(a) Expression of NKX2.1 mRNA at d15 in the four culture conditions shown on top of the panel. *P<0.01, compared with IWP2/SB->DSM/SB group, triplicate experiments. ‘Liver’ represents DE cultured in ‘hepatic conditions’, data for definitive endoderm are samples analyzed at d4.5 of the differentiation protocol. (b) Expression of FOXA2, SOX2 and NKX2.1 in RUES2 cells cultured according to the protocol on top of the panel. (c) 10x tile scan of 25 (5×5) contiguous fields showing expression of FOXA2, SOX2 and NKX2.1 in RUES2 cells cultured according to the protocol on top of panel (b). (d) 20x tile scan of 4 (2×2) contiguous fields showing expression of TUJ1, NKX2.1 and FOXA2.1 in RUES2 cells cultured according to the protocol on top of panel (b). (e) 20x tile scan of 4 (2×2) contiguous fields showing expression of p63, NKX2.1 and FOXA2.1 in RUES2 cells cultured according to the protocol on top of panel (b). Immunofluorescence images from Fig. 2a–d represent reproducible results from 3 independent experiments.

Fig. 2. Requirement of morphogens for lung field induction from hPSCs

Effect of removing individual factors or blocking signaling pathways during the ‘ventralization’ stage (d6–d15) on the expression of FOXA2 and NKX2.1 in RUES2 cells cultured according to the protocol shown on the upper left of the figure. 10x tile scans of 9 (3×3) contiguous fields. Immunofluorescence images represent reproducible results from 3 independent experiments.

Fig. 3. In vivo potential of hPSC-derived lung and airway progenitors

(a) Representative examples of H&E stains of growths removed 6 months after transplantation of RUES2 cells differentiated according to the protocol shown in Fig. 1b, under the kidney capsule of NSG mice (BC: basal cell; Ca: cartilage; CC: Clara cell; Ci: ciliated cell; GC: goblet cell; Ki: mouse kidney; PSE: pseudostratified epithelium; SM: smooth muscle; SMG: submucosal glands). (b) Representative examples of the expression of markers of mature lung and airway epithelial cells in the growths from (a). Representative of 4 animals.

Fig. 4. Further differentiation of hPSCS-derived lung and airway progenitors

(a) Culture protocol of RUES2 cells shown in panels (b), (c) and (d). (b) and (c) 10x tile scans of the expression of p63, SOX2 and NKX2.1 in representative colonies obtained after culturing RUES2 cells according to the protocol shown in (a). (d) Expression of p63 and MUC5AC after culturing RUES2 cells according to the protocol shown in panel (a). Immunofluorescence images represent reproducible results from 4 independent experiments.

Fig. 5. Terminal differentiation of hPSCS-derived lung and airway progenitors

(a) Culture protocol of RUES2 cells shown in panels (b), (c) and (d). (b) Representative examples of the expression of markers of mature lung and airway epithelial cells after culturing RUES2 cells according to the protocol shown in panel (a). Immunofluorescence images represent reproducible results from 4 independent experiments. (c) Representative 10x whole culture tile scan of SP-B and SP-C expression in RUES2 cells cultured according to the protocol shown in (a), without (left) and with (right) addition of DCI at d25. (d) Cellular expansion of RUES2 cells during the culture according to the protocol shown on top of panel (a) (n=4).

Fig. 6. Morphology and function of hPSC-derived lung and airway epithelium

(a)Representative transmission electron micrographs of cultured FHL or RUES2 cells differentiated according to the protocol shown in Fig. 5a with DCI (LB: lamellar body; MVB: mulitvesicular body) (b). Fluorescence micrographs and flow cytometric analysis of uptake of BODIPY-SP-B by cells cultured according to the protocol shown in Fig. 5 a. Immunofluorescence images and flow cytometry represent reproducible results from 3 independent experiments. (c) Culture in the presence of decellularized human lung matrix. (i and ii) Expression p63 and NKX2.1 at d25 of cultures of RUES2 cells seeded on slices of decellularized human lung matrix at day 15 of the protocol in Fig. 5a. (iii and iv) Expression of endogenous SP-B at d48 of culture with DCI according to the protocol in Fig. 5a after seeded on decellularized human lung matrix (v) Confocal fluorescence micrograph of the uptake of BODIPY-SP-B at d48 in the same conditions (vi) Morphology of mouse ATII cells as observed by two-photon microscopy of live mouse lung after instillation of BODIPY-SPB. (d) qPCR of the expression of proximal and distal lung markers at days 15, 23 and 55 of culture in the conditions in Fig. 5a in the presence of DCI. Cultures in the presence or absence of BMP4 and RA (+BMP/RA vs. –BMP/RA) from d15 to d55 were compared. (ECM = human decellularized extracellular lung matrix) (representative triplicate experiment).