The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues

Opportunities for organoids as new models of aging.

The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research

A Computational Approach to Understand In Vitro Alveolar Morphogenesis

Primary human alveolar type II (AT II) epithelial cells maintained in Matrigel cultures form alveolar-like cysts (ALCs) using a cytogenesis mechanism that is different from that of other studied epithelial cell types: neither proliferation nor death is involved. During ALC formation, AT II cells engage simultaneously in fundamentally different, but not fully characterized activities. Mechanisms enabling these activities and the roles they play during different process stages are virtually unknown. Identifying, characterizing, and understanding the activities and mechanisms are essential to achieving deeper insight into this fundamental feature of morphogenesis. That deeper insight is needed to answer important questions. When and how does an AT cell choose to switch from one activity to another? Why does it choose one action rather than another? We report obtaining plausible answers using a rigorous, multi-attribute modeling and simulation approach that leveraged earlier efforts by using new, agent and object-oriented capabilities. We discovered a set of cell-level operating principles that enabled in silico cells to self-organize and generate systemic cystogenesis phenomena that are quantitatively indistinguishable from those observed in vitro. Success required that the cell components be quasi-autonomous. As simulation time advances, each in silico cell autonomously updates its environment information to reclassify its condition. It then uses the axiomatic operating principles to execute just one action for each possible condition. The quasi-autonomous actions of individual in silico cells were sufficient for developing stable cyst-like structures. The results strengthen in silico to in vitro mappings at three levels: mechanisms, behaviors, and operating principles, thereby achieving a degree of validation and enabling answering the questions posed. We suggest that the in silico operating principles presented may have a biological counterpart and that a semiquantitative mapping exists between in silico causal events and in vitro causal events

Leveraging mechanobiology and biophysical cues in lung organoids for studying lung development and disease

Lung organoids have emerged as powerful tools for studying lung distal diseases by recapitulating the cellular diversity and microenvironment of the lung tissue. This review article highlights the advancements in leveraging mechanobiology and biophysical cues in lung organoid engineering to improve their physiological relevance and disease modelling capabilities. We discuss the role of mechanobiology in lung development and homeostasis, as well as the integration of biophysical cues in the design and culture of lung organoids. Furthermore, we explore how these advancements have contributed to the understanding of lung distal diseases pathogenesis. We also discuss the challenges and future directions in harnessing mechanobiology and biophysical cues in lung organoid research. This review showcases the potential of lung organoids as a platform to investigate the underappreciated impacts of biophysical and biomechanical properties in enhancing lung organoids complexity and functionality, and ultimately provide new insight into embryonic lung development and pulmonary distal diseases pathogenesis

Pluripotent stem cell modeling of airway epithelial fate

Although severe lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD), represent a significant global disease burden, little is known about the molecular pathways by which the cells of the lung develop, respond to damage, or become diseased. Consequently, there are few treatment options for patients. Improving lung disease outcomes therefore relies on both refining the current understanding of the normal development of the lung epithelium and developing new model systems to provide mechanistic insight into disease biology. In this thesis, I describe a multifaceted approach using both in vivo models and novel mouse and human pluripotent stem cell reporter systems to explore this important topic, focusing primarily on the hypothesis that canonical Wnt signaling is a key stage-dependent inhibitor of proximal lung development. To address this hypothesis, I developed new tools allowing for the precise manipulation of developmental pathways and access to rare cell populations in vitro. This toolkit included both mouse and human pluripotent stem cell (mPSC/hPSC) lines with reporters for specific airway lineages. In parallel, I built on our lab’s previous work in directed differentiation of hPSCs to lung progenitors. I found that canonical Wnt signaling regulates proximodistal epithelial patterning in human NKX2-1+ lung progenitors. While canonical Wnt activation is required for lung specification, withdrawal of Wnt activation leads to emergence of a proximal airway program and loss of distal identity. This finding culminated in the development of a novel protocol to differentiate epithelial-only airway organoids from hPSCs. These organoids are derived from purified NKX2-1+ lung progenitors, contain functional airway cell types including secretory, goblet, and basal cells, and can be further expanded and differentiated to multiciliated epithelia in air-liquid interface culture. To provide a proof of principle for the clinical utility of this platform, I generated airway organoids from cystic fibrosis patient-derived hPSC lines pre- and post-correction of the dF508 mutation in the CFTR gene. These organoids respond in a CFTR-dependent manner to epithelial forskolin swelling assays, highlighting the potential utility of this approach for disease modeling and drug screening for a variety of genetic and acquired airway disorders

Multiscale dynamics of branching morphogenesis.

Branching morphogenesis is a prototypical example of complex three-dimensional organ sculpting, required in multiple developmental settings to maximize the area of exchange surfaces. It requires, in particular, the coordinated growth of different cell types together with complex patterning to lead to robust macroscopic outputs. In recent years, novel multiscale quantitative biology approaches, together with biophysical modelling, have begun to shed new light of this topic. Here, we wish to review some of these recent developments, highlighting the generic design principles that can be abstracted across different branched organs, as well as the implications for the broader fields of stem cell, developmental and systems biology.wellcome trust royal societ

Generation of Lung Epithelium from Pluripotent Stem Cells

The understanding of key processes and signaling mechanisms in lung development has been mainly demonstrated through gain and loss of function studies in mice, while human lung development remains largely unexplored due to inaccessibility. Several recent reports have exploited the identification of key signaling mechanisms that regulate lineage commitment and restriction in mouse lung development, to direct differentiation of both mouse and human pluripotent stem cells towards lung epithelial cells. In this review, we discuss the recent advances in the generation of respiratory epithelia from pluripotent stem cells and the potential of these engineered cells for novel scientific discoveries in lung diseases and future translation into regenerative therapies

Airway progenitor cell development and function : mimicking in vivo behavior in vitro

This thesis presents novel in vitro models to study the differentiation of airway progenitor cells. These models were subsequently used to study the function of SOX2 and SOX21 in the development and regeneration of the airways

Doctor of Philosophy

dissertationThrough the production of milk, the mammary gland provides nutritional and immunological protection for newborns. However, lactation is required for only short periods of time in the adult animal, which allows the mammary gland to develop through a series of distinct stages. During embryogenesis and puberty, the gland establishes an extensive network of epithelial ducts, which then undergo widespread branching and differentiation during pregnancy to maximize milk production. Throughout this process, the mammary gland relies on coordination of major cellular processes, including proliferation, invasion, and differentiation. As many of these pathways become aberrantly regulated during breast cancer, understanding mechanisms that regulate mammary gland development has important disease implications. Although previous studies have characterized several systemic hormones and local factors central to mammary development, little is known about the downstream mediators of these pathways. To identify new factors, we established a three-dimensional model of mammary branching morphogenesis using primary mammary epithelial cells (MECs) stimulated with fibroblast growth factor-2 (FGF2). We performed a forward chemical genetic screen to identify compounds that modulate FGF2-induced branching and discovered a novel bis-aryloxadiazole, called 1023, which completely blocks branching through activation of the aryl hydrocarbon receptor (AHR). iv Using 1023 as a molecular probe, we found AHR activation blocks mammary branching through upregulation of desmosomal adhesion. These results identified desmosomes as a novel target of AHR signaling and suggested desmosomes are downregulated to facilitate mammary branching. Supporting this hypothesis, we found desmosomes absent in the mammary glands of pregnant mice in a cell-type specific manner. These results suggest desmosomes control initiation of mammary branching, and may also be targeted during breast cancer to promote cellular invasion. We also investigated mechanisms of AHR activation and the impact of AHR on mammary differentiation. We performed a structure activity relationship study of 1023 and defined moieties of the molecule critical for AHR stimulation. Moreover, we investigated the effect of AHR on mammary differentiation and elucidated a transcriptional mechanism through which the AHR pathway directly blocks lactation in MECs. Since several environmental pollutants stimulate AHR, these studies provide mechanistic insight for how toxins impair mammary function