Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice

Abstract

Durable reconstitution of the injured distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising potential therapy for diseases that result from alveolar damage. Here we differentiate murine PSCs in vitro into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. Emulating the roadmap of the developing embryo, we generate transplantable PSC-derived Nkx2-1+/Sox9+ lung epithelial progenitors that are highly similar to cultured primary embryonic distal lung bud tip progenitors. These cells display a stable phenotype after frozen archiving or extensive expansion in culture, providing a nearly inexhaustible source of cells that can be engrafted into syngeneic injured mouse lungs without the need for immunosuppression. After transplantation PSC-derived tip-like progenitors downregulate Sox9 and mature in the distal lung, upregulating alveolar type 2 cell markers or assuming the flat morphology and molecular phenotype of terminally differentiated alveolar type 1 cells. After months in vivo, donor-derived cells retain their alveolar epithelial type 2-like and type 1-like phenotypes, as characterized by single cell RNA sequencing, ultrastructural analyses, in vivo histologic profiling, and ex vivo organoid assays that demonstrate continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung′s facultative progenitor and differentiated epithelial cell compartments in vivo with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy which can be utilized to better understand the mechanisms and utility of engraftment prior to future clinical studies.

scTOP: physics-inspired order parameters for cellular identification and visualization

“…To demonstrate the efficacy of scTOP in the case where the reference basis and query data come from different sources, we projected mouse lung data from Herriges et al [35] onto the mouse cell atlas. The Herriges et al dataset measures gene expression levels of six different specialized lung types from healthy mice: AT1, AT2, basal, ciliated, secretory, and neuroendocrine (See figure 2).…”

“…To compare two distinct pulmonary cell transplant protocols, we used scTOP to analyze the results of scRNA-seq from two recent publications featuring murine pulmonary cell transplantation [35, 47]. In Louie et al, adult mouse AT2 cells were maintained in culture with support cells for 3 weeks to generate a donor cell population.…”

“…scTOP is especially suited for tasks where the cell types of interests are well characterized. For example, we have found in our own work that scTOP is an extremely sensitive measure for assessing the fidelity of engineered cells from directed differentiation protocols and reprogramming [35]. Since scTOP does not require data harmonization, joint embeddings, or make use of cellular neighborhood information, scTOP is able to distinguish between technical noise and biologically meaningful differences.…”

Engraftment of wild-type alveolar type II epithelial cells in surfactant protein C deficient mice

“…Multipotent Sox9 + lung progenitors have been isolated from fetal lungs, expanded in 3D culture, and showed to engraft in immunocompromised mice after injury and differentiate in airway and alveolar cells (76). Recently, another group has been able to derive and expand in vitro Nkx2-1+/Sox9+ lung epithelial progenitors from murine embryonic stem cell line and showed differentiation into ATII-like and ATI-like cells when transplanted into the lungs of syngeneic immunocompetent recipients (71). While these results are excited and promising, they have not yet been applied to a disease model nor evaluated for therapeutic effect on disease progression.…”

“…Our study has several limitations. While bleomycin is commonly used as pre-conditioning strategies (34,71–73), this conditioning approach is not applicable to humans. Another limitation is that it is challenging to accurately determine the total number of engrafted Sftpc +/+ ATII cells in the whole mouse lung.…”