Open AccessHeterogeneity in Human Induced Pluripotent Stem Cell–derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters
Author(s) -
Yuliang Sun,
Killian Hurley,
Carlos Villacorta-Martín,
Jessie Huang,
Anne Hinds,
Krithi Gopalan,
Ignacio Caballero,
Scott J. Russo,
Joseph A. Kitzmiller,
Jeffrey A. Whitsett,
Michael F. Beers,
Darrell N. Kotton
Publication year - 2021
Publication title -
american journal of respiratory cell and molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.469
H-Index - 161
eISSN - 1535-4989
pISSN - 1044-1549
DOI - 10.1165/rcmb.2020-0259oc
Subject(s) - surfactant protein c , biology , green fluorescent protein , induced pluripotent stem cell , microbiology and biotechnology , population , progenitor cell , mesenchymal stem cell , lamellar granule , stem cell , embryonic stem cell , anatomy , genetics , gene , medicine , environmental health , ultrastructure
Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPC tdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPC tdTomato expression. Comparing ABCA3:GFP/SFTPC tdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.