Herriges, M. J. | bioRxiv Science

bioRxiv2024

Targeted pre-conditioning and cell transplantation in the murine lower respiratory tract

Transplantation of airway basal stem cells could achieve a durable cure for genetic diseases of the airway, such as cystic fibrosis and primary ciliary dyskinesia. Recent work demonstrated the potential of primary- and pluripotent stem cell (PSC)-derived basal cells to efficiently engrai into the mouse trachea aier injury. However, there are many hurdles to overcome in translating these approaches to humans including developing safe and efficient methods for delivery in larger animal models. We propose a model which targets preconditioning and cell-delivery to the intrapulmonary airways utilizing a micro- bronchoscope for delivery. The detergent polidocanol was adapted for distal lung pre-conditioning, inducing intrapulmonary airway epithelial denudation by 5 and 24-hours post-delivery. While initial re- epithelialization of airways occurred later than tracheas, complete repair was observed within 7-days. Both PSC-derived and primary basal cells delivered via micro-bronchoscope post-polidocanol injury engraied in tracheas and intrapulmonary airways, respectively. Transplanted cells differentiated into ciliated and secretory lineages while maintaining a population of basal cells. These findings demonstrate the utility of bronchoscopically targeted pre-conditioning and cell delivery to the conducting intra- pulmonary airways, providing an important framework for pre-clinical translation of approaches for engineered airway epithelial regeneration.

cell biology↗

bioRxiv2019

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

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.

developmental biology↗