Glycogen Synthase Kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture

Abstract

Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in Collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells arranged in pseudostratified epithelia, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal at the expense of a proximal and ciliated cell fate, while 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.

Current strategies and opportunities to manufacture cells for modeling human lungs

“…Different groups have adhered to their own methods of generating definitive endoderm, which primarily involves exposing PSCs to high concentrations of Activin A. Slight variations such as introducing WNT agonism (through WNT3a or CHIR99021) prior to [ 25 ] or alongside [ 11 , 30 , 32 , 33 ] Activin A, or additional exposure to BMP4 and FGF2 [ 23 , 25 , 29 ] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing definitive endoderm. In addition, the use of embryoid bodies, which are limited by user experience and technique, has resulted in a wide range of production efficiencies for achieving this stage: from 45% CKIT + CXCR4 + EPCAM + cells [ 33 ] to >90% CKIT + CXCR4 + cells [ 23 , 25 ].…”

“…A comparison of the latter two strategies demonstrated that SHH and FGF2 are insufficient in producing reliable NKX2.1 + lung progenitors [ 25 ], possibly because FGF2 is involved in promoting thyroid lineages [ 120 ]. In general, TGFβ and BMP inhibition [ 23 ] is the basis for currently applied endoderm anteriorization strategies [ 27 , [29] , [30] , [31] , 36 , 121 ].…”

“…As such, CHIR99021 (CHIR; WNT agonist), BMP4, and RA are necessary and sufficient for producing lung progenitors from anterior foregut endoderm derived from both mouse and human PSCs [ 25 , 30 , 32 , 119 ]. Despite finding that FGF7 and FGF10 are non-essential for inducing NKX2.1 expression, they continue to be used for ventralization in some protocols [ 29 ]. Although each protocol differs in terms of the duration of each phase, NKX2.1 + lung progenitors are generally achieved by 15 days, with the exception of a study by de Carvalho et al ., in which they maintained their cultures for an additional 10 days in FGF7, FGF10, and CHIR99021 to attain 90-98% NKX2.1 + FOXA2 + lung progenitors.…”

“…Older lung directed differentiation protocols from the late 2000s have been proven inefficient due to the non-standardized methods through which they derive lung endoderm from embryoid bodies [ [19] , [20] , [21] ]. A series of more standardized stepwise protocols have since emerged in the last decade that provide avenues for developing airway and lung epithelia, albeit with variable efficiencies [ 11 , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] ]. The first influential directed differentiation protocol to produce lung epithelia used human PSCs in 2011 [ 23 ], which was further supported by two prominent studies conducted using mouse PSCs in 2012 [ 22 , 24 ].…”

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC‐derived air‐liquid interface model

“…Limited access to primary human ATII cells, particularly from diseased patients, and the rapid loss of ATII marker expression and function when cultured in vitro, make it challenging to study the role of human ATII cells in IPF 19 . Recent advances have led to the successful derivation of ATII‐like cells from human pluripotent stem cells (hPSCs, including both embryonic stem cells and induced pluripotent stem cells (iPSCs)), providing a promising alternative cell source for in vitro disease modeling 20‐26 . Previous reports on hPSC‐derived lung cells showed that despite their phenotypic similarities to mature lung epithelium, these models are equivalent to human fetal lung cells, rather than representing an adult state 27,28 .…”