Single-cell time-series mapping of cell fate trajectories reveals an expanded developmental potential for human PSC-derived distal lung progenitors

Abstract

Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life, yet are difficult to access from patients for biomedical research or lung regeneration applications. Here we engineer AEC2s from human induced pluripotent stem cells (iPSCs) in vitro and use single cell RNA sequencing (scRNA-seq) to profile the detailed kinetics of their differentiation over time. We focus on both the desired target cells as well as those that appear to diverge to alternative endodermal fates. By combining scRNA-seq with lentiviral barcoding to trace differentiating clones, we reveal the bifurcating cell fate trajectories followed as primordial lung progenitors differentiate into mature AEC2s. We define the global transcriptomic signatures of primary developing human AEC2s from fetal through adult stages in order to identify the subset of in vitro differentiating cells that appear to recapitulate the path of in vivo development. In addition, we develop computational methods based on Continuous State Hidden Markov Models (CSHMM) to identify the precise timing and type of signals, such as over-exuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate as they clonally diverge into a variety of non-lung endodermal lineages. Finally, we find that this initial developmental plasticity is regulatable via Wnt modulation, and subsides over time, ultimately resulting in iPSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity in vitro. Our methods and computational approaches can be widely applied to study and control directed differentiation, producing an inexhaustible supply of mature lineages, exemplified here by the generation of AEC2s.

SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

“…Because the directed differentiation of human iPSCs in vitro is designed to recapitulate the sequence of developmental milestones that accompanies in vivo human fetal organogenesis, we first sought to understand the ontogeny of expression of the coronavirus receptor, ACE2, during human fetal development from early endodermal progenitors through increasingly more mature stages of alveolar development. By analyzing our previously published transcriptomic time series profiles of developing human fetal and adult primary AT2s (Hurley et al, 2020), we found that ACE2 expression increases from early to late canalicular stages of distal human lung development, and expression levels were similar to adult AT2 levels present by week 21 of gestation in developing alveolar epithelial cells (Figure 1E). We found the directed differentiation of human pluripotent stem cells (RUES2 embryonic stem cells and SPC2 iPSCs) in vitro into purified distal lung AT2-like cells resulted in cells expressing similar levels of ACE2 to those of adult primary cell controls in head-to-head comparisons (Figures 1E and S1,respectively).…”

“…iAT2s in ALI Culture Are Permissive to SARS-CoV-2 Infection and Replication In order to develop a human model system, we used the technique of directed differentiation (Jacob et al, 2017(Jacob et al, , 2019 to generate iAT2s from either human embryonic stem cells or iPSCs engineered to carry a tdTomato reporter targeted to the endogenous SFTPC locus (Hurley et al, 2020;Jacob et al, 2017). In 3D Matrigel cultures, we established self-renewing epithelial spheres composed of purified iAT2s, >90% of which expressed surfactant protein-C (SFTPC), the canonical AT2 marker, as monitored by flow cytometry assessment of the SFTPC tdTomato reporter at each passage in culture (Figures 1A and 1B).…”

“…In 3D Matrigel cultures, we established self-renewing epithelial spheres composed of purified iAT2s, >90% of which expressed surfactant protein-C (SFTPC), the canonical AT2 marker, as monitored by flow cytometry assessment of the SFTPC tdTomato reporter at each passage in culture (Figures 1A and 1B). Serially passaging these epithelial spheres generated >10 30 iAT2s per starting sorted tdTomato+ cell over 225 days in culture (Hurley et al, 2020), generating cells that maintained expression of AT2 marker genes, including surfactants as shown by single cell RNA sequencing (scRNA-seq) (Figures 1C, 1D, and S1), and providing a stable source of human primary-like AT2 cells for viral infection disease modeling.…”

“…To provide alternative sources of self-renewing human lung epithelial lineages, our group and others have recently developed human lung epithelial organoids and 2D ALI lung cultures through the directed differentiation of induced pluripotent stem cells (iPSCs) in vitro (Abo et al, 2020;Hawkins et al, 2017;Huang et al, 2014;Hurley et al, 2020;Jacob et al, 2017;Longmire et al, 2012;McCauley et al, 2017McCauley et al, , 2018aMcCauley et al, , 2018bSerra et al, 2017;Yamamoto et al, 2017). Here, we report the successful infection of a pure population of human iPSC-derived AT2-like cells (iAT2s) with SARS-CoV-2, providing a reductionist model that reveals the cell-intrinsic distal lung epithelial global transcriptomic responses to infection.…”

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

“…As expected NGFR was only expressed in the basal-like population cultured in BC medium (cluster 1; Figures 2F). Notably, we have previously demonstrated that plasticity of iPSC cells undergoing lung directed differentiation can sometimes result in reversion to non-lung endodermal lineages (McCauley et al 2018;Hurley et al 2019), however the day 30-32 sorting step for GFP+/TOM+ cells significantly reduced the percentage of non-lung endoderm cells in both conditions to less than 0.05% of all cells (Figure S3B).…”