Engineering and Modeling the Lung Mesenchyme.

The structure of the mammalian lung controls the flow of air through the airways and into the distal alveolar region where gas exchange occurs. Specialized cells in the lung mesenchyme produce the extracellular matrix (ECM) and growth factors required for lung structure. Historically, characterizing the mesenchymal cell subtypes was challenging due to their ambiguous morphology, overlapping expression of protein markers, and limited cell-surface molecules needed for isolation. The recent development of single-cell RNA sequencing (scRNA-seq) complemented with genetic mouse models demonstrated that the lung mesenchyme comprises transcriptionally and functionally heterogeneous cell-types. Bioengineering approaches that model tissue structure clarify the function and regulation of mesenchymal cell types. These experimental approaches demonstrate the unique abilities of fibroblasts in mechanosignaling, mechanical force generation, ECM production, and tissue regeneration. This chapter will review the cell biology of the lung mesenchyme and experimental approaches to study their function.

Ezh2 restricts the smooth muscle lineage during mouse lung mesothelial development.

During development, the lung mesoderm generates a variety of cell lineages, including airway and vascular smooth muscle. Epigenetic changes in adult lung mesodermal lineages are thought to contribute towards diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, although the factors that regulate early lung mesoderm development are unknown. We show in mouse that the PRC2 component Ezh2 is required to restrict smooth muscle differentiation in the developing lung mesothelium. Mesodermal loss of Ezh2 leads to the formation of ectopic smooth muscle in the submesothelial region of the developing lung mesoderm. Loss of Ezh2 specifically in the developing mesothelium reveals a mesothelial cell-autonomous role for Ezh2 in repression of the smooth muscle differentiation program. Loss of Ezh2 derepresses expression of myocardin and Tbx18, which are important regulators of smooth muscle differentiation from the mesothelium and related cell lineages. Together, these findings uncover an Ezh2-dependent mechanism to restrict the smooth muscle gene expression program in the developing mesothelium and allow appropriate cell fate decisions to occur in this multipotent mesoderm lineage.

Ezh2 represses the basal cell lineage during lung endoderm development.

The development of the lung epithelium is regulated in a stepwise fashion to generate numerous differentiated and stem cell lineages in the adult lung. How these different lineages are generated in a spatially and temporally restricted fashion remains poorly understood, although epigenetic regulation probably plays an important role. We show that the Polycomb repressive complex 2 component Ezh2 is highly expressed in early lung development but is gradually downregulated by late gestation. Deletion of Ezh2 in early lung endoderm progenitors leads to the ectopic and premature appearance of Trp63+ basal cells that extend the entire length of the airway. Loss of Ezh2 also leads to reduced secretory cell differentiation. In their place, morphologically similar cells develop that express a subset of basal cell genes, including keratin 5, but no longer express high levels of either Trp63 or of standard secretory cell markers. This suggests that Ezh2 regulates the phenotypic switch between basal cells and secretory cells. Together, these findings show that Ezh2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation.

Expression of histone deacetylase 3 instructs alveolar type I cell differentiation by regulating a Wnt signaling niche in the lung.

The commitment and differentiation of the alveolar type I (AT1) cell lineage is a critical step for the formation of distal lung saccules, which are the primitive alveolar units required for postnatal respiration. How AT1 cells arise from the distal lung epithelial progenitor cells prior to birth and whether this process depends on a developmental niche instructed by mesenchymal cells is poorly understood. We show that mice lacking histone deacetylase 3 specifically in the developing lung mesenchyme display lung hypoplasia including decreased mesenchymal proliferation and a severe impairment of AT1 cell differentiation. This is correlated with a decrease in Wnt/ß-catenin signaling in the lung epithelium. We demonstrate that inhibition of Wnt signaling causes defective AT1 cell lineage differentiation ex vivo. Importantly, systemic activation of Wnt signaling at specific stages of lung development can partially rescue the AT1 cell differentiation defect in vivo. These studies show that histone deacetylase 3 expression generates an important developmental niche in the lung mesenchyme through regulation of Wnt signaling, which is required for proper AT1 cell differentiation and lung sacculation.

How microRNAs facilitate reprogramming to pluripotency.

The ability to generate pluripotent stem cells from a variety of cell and tissue sources through the ectopic expression of a specific set of transcription factors has revolutionized regenerative biology. The development of this reprogramming technology not only makes it possible to perform basic research on human stem cells that do not have to be derived from embryos, but also allows patient-specific cells and tissues to be generated for therapeutic use. Optimizing this process will probably lead to a better and more efficient means of generating pluripotent stem cells. Here, we discuss recent findings that show that, in addition to transcription factors, microRNAs can promote pluripotent reprogramming and can even substitute for these pluripotency transcription factors in some cases. Taking into consideration that microRNAs have the potential to be used as small-molecule therapeutics, such findings open new possibilities for both pluripotent stem cell reprogramming and the reprogramming of cells into other cell lineages.

Generation of pluripotent stem cells from patients with type 1 diabetes.

Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic beta cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy.

Lithium and Therapeutic Targeting of GSK-3.

Lithium salts have been in the therapeutic toolbox for better or worse since the 19th century, with purported benefit in gout, hangover, insomnia, and early suggestions that lithium improved psychiatric disorders. However, the remarkable effects of lithium reported by John Cade and subsequently by Mogens Schou revolutionized the treatment of bipolar disorder. The known molecular targets of lithium are surprisingly few and include the signaling kinase glycogen synthase kinase-3 (GSK-3), a group of structurally related phosphomonoesterases that includes inositol monophosphatases, and phosphoglucomutase. Here we present a brief history of the therapeutic uses of lithium and then focus on GSK-3 as a therapeutic target in diverse diseases, including bipolar disorder, cancer, and coronavirus infections.

Adult hippocampal neurogenesis is not necessary for the response to lithium in the forced swim test.

Chronic lithium treatment stimulates adult hippocampal neurogenesis, but whether increased neurogenesis contributes to its therapeutic mechanism remains unclear. We use a genetic model of neural progenitor cell (NPC) ablation to test whether a lithium-sensitive behavior requires hippocampal neurogenesis. NPC-ablated mice were treated with lithium and assessed in the forced swim test (FST). Lithium reduced time immobile in the FST in NPC-ablated and control mice but had no effect on activity in the open field, a control for the locomotion-based FST. These findings show that hippocampal NPCs that proliferate in response to chronic lithium are not necessary for the behavioral response to lithium in the FST. We further show that 4-6 week old immature hippocampal neurons are not required for this response. These data suggest that increased hippocampal neurogenesis does not contribute to the response to lithium in the forced swim test and may not be an essential component of its therapeutic mechanism.

WNT10A mutation causes ectodermal dysplasia by impairing progenitor cell proliferation and KLF4-mediated differentiation.

Human WNT10A mutations are associated with developmental tooth abnormalities and adolescent onset of a broad range of ectodermal defects. Here we show that ß-catenin pathway activity and adult epithelial progenitor proliferation are reduced in the absence of WNT10A, and identify Wnt-active self-renewing stem cells in affected tissues including hair follicles, sebaceous glands, taste buds, nails and sweat ducts. Human and mouse WNT10A mutant palmoplantar and tongue epithelia also display specific differentiation defects that are mimicked by loss of the transcription factor KLF4. We find that ß-catenin interacts directly with region-specific LEF/TCF factors, and with KLF4 in differentiating, but not proliferating, cells to promote expression of specialized keratins required for normal tissue structure and integrity. Our data identify WNT10A as a critical ligand controlling adult epithelial proliferation and region-specific differentiation, and suggest downstream ß-catenin pathway activation as a potential approach to ameliorate regenerative defects in WNT10A patients.

Emergence of a Wave of Wnt Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation.

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, in vivo clonal analysis, and ex vivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice.

In contrast to lower vertebrates, the mammalian heart has limited capacity to regenerate after injury in part due to ineffective reactivation of cardiomyocyte proliferation. We show that the microRNA cluster miR302-367 is important for cardiomyocyte proliferation during development and is sufficient to induce cardiomyocyte proliferation in the adult and promote cardiac regeneration. In mice, loss of miR302-367 led to decreased cardiomyocyte proliferation during development. In contrast, increased miR302-367 expression led to a profound increase in cardiomyocyte proliferation, in part through repression of the Hippo signal transduction pathway. Postnatal reexpression of miR302-367 reactivated the cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction. However, long-term expression of miR302-367 induced cardiomyocyte dedifferentiation and dysfunction, suggesting that persistent reactivation of the cell cycle in postnatal cardiomyocytes is not desirable. This limitation can be overcome by transient systemic application of miR302-367 mimics, leading to increased cardiomyocyte proliferation and mass, decreased fibrosis, and improved function after injury. Our data demonstrate the ability of microRNA-based therapeutic approaches to promote mammalian cardiac repair and regeneration through the transient activation of cardiomyocyte proliferation.

Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.

Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis.

Macrophages show endoplasmic reticulum (ER) stress when exposed to lipotoxic signals associated with atherosclerosis, although the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. Here we show that mitigation of ER stress with a chemical chaperone results in marked protection against lipotoxic death in macrophages and prevents macrophage fatty acid-binding protein-4 (aP2) expression. Using genetic and chemical models, we show that aP2 is the predominant regulator of lipid-induced macrophage ER stress. The absence of lipid chaperones incites an increase in the production of phospholipids rich in monounsaturated fatty acids and bioactive lipids that render macrophages resistant to lipid-induced ER stress. Furthermore, the impact of aP2 on macrophage lipid metabolism and the ER stress response is mediated by upregulation of key lipogenic enzymes by the liver X receptor. Our results demonstrate the central role for lipid chaperones in regulating ER homeostasis in macrophages in atherosclerosis and show that ER responses can be modified, genetically or chemically, to protect the organism against the deleterious effects of hyperlipidemia.

Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.

Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.