Understanding the Bud Tip Progenitor Cell Niche During Human Lung Development

Abstract

Human development is a complex but fascinating process by which a single fertilized egg grows into three tissue layers, giving rise to diverse organs and tissues that each have unique developmental requirements. Stem and progenitor cells are at the heart of development, whereby they self-renew and make cell fate decisions to create the array of cells that make up an entire organism. The mechanisms that control cell fate are critical for understanding how normal development takes place and how genetic abnormalities, premature birth, and environmental factors influence development. A subset of stem and progenitor cells persist into adulthood and often use regulatory mechanisms that are similar to those used during fetal development, so understanding how stem and progenitor cells are controlled is also important for adult tissue repair following injury and for developing cellular therapies for diseases.

The respiratory epithelium is specified from the ventral-anterior aspect of a common endoderm-derived gut tube, which gives rise to a specialized lung progenitor. These ‘bud tip progenitors’ are maintained through lung development and give rise to all cell types lining the lungs. Bud tip progenitors are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung is poorly understood. The first portion of research in this thesis explores how bud tip progenitors are regulated by mesenchymal cells in their niche. To address this, we interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present that is highly enriched for expression of the WNT agonist RSPO2. We found that adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that acts through LGR5 on bud tip progenitors to potentiate WNT signaling in bud tips, which is required for their maintenance and multipotency.

Animal models and human tissue have been instrumental in addressing questions pertaining to lung development; however, animal models limit the understanding of huma-specific biology and access to human tissue is limited and burdened by regulatory and ethical considerations. Overcoming many of these limitations are recent advances in iPSC-derived models of human development. The second portion of research in this thesis was aimed at developing an iPSC organoid model enriched with bud tip progenitor-like cells that faithfully recapitulates the bud tip progenitors found in the developing human lung. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed bud tip progenitor-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced bud tip progenitors (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Overall, the work in this thesis has increased our understanding in the bud tip progenitor niche influence cell fate decisions and has created an iPSC-derived bud tip progenitor organoid model that can be used to study human lung development.