Hongmei Mou

Dedifferentiation of committed epithelial cells into stem cells in vivo

Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.

Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells

Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.

Yap Tunes Airway Epithelial Size and Architecture by Regulating the Identity, Maintenance, and Self-Renewal of Stem Cells

Our understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. We show that Yap (Yes-associated protein 1) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. We then show that Yap functionally interacts with p63, the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, we show that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.

cGMP Binding to Noncatalytic Sites on Mammalian Rod Photoreceptor Phosphodiesterase Is Regulated by Binding of Its γ and δ Subunits

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, αβγ2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, αβγ2δ) can release and bind cGMP at both noncatalytic sites; the δ subunit is likely responsible for this difference in cGMP exchange rates. Removal of the δ and/or γ subunits yields a catalytic αβ dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (K D >1 μm); cGMP binding affinity at the second site (K D ∼60 nm) is reduced 3–4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of γ subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the γ subunit with the αβ dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.

Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations.

Following injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63(+) basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63(+) airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell-autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity.

Regulation of Photoreceptor Phosphodiesterase (PDE6) by Phosphorylation of Its Inhibitory γ Subunit Re-evaluated

Phosphorylation of the inhibitory γ subunit (Pγ) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pγ phosphorylation for PDE6 regulation by preparing Pγ stoichiometrically phosphorylated at Thr22 or at Thr35. Phosphorylation of Pγ at either residue caused a minor decrease—not the previously reported increase—in the ability of Pγ to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pγ phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pγ subunits. The strength of Pγ interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pγ phosphorylation at Thr22 (but not Thr35), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527–27534). In contrast, the effects of Pγ phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pγ at either Thr22 or Thr35 greatly diminished its ability to bind activated transducin, consistent with earlier work. In situphosphorylation of Pγ by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only ∼10% of the endogenous Pγ was phosphorylated. This is attributed to Pγ being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pγ phosphorylation at either Thr22 or Thr35modestly weakens its direct interactions with PDE6. However, Pγ phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pγ available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802–37808) in photoreceptor cells.

ACTL6A Is Co-Amplified with p63 in Squamous Cell Carcinoma to Drive YAP Activation, Regenerative Proliferation, and Poor Prognosis

Loss-of-function mutations in SWI/SNF chromatin-remodeling subunit genes are observed in many cancers, but an oncogenic role for SWI/SNF is not well established. Here, we reveal that ACTL6A, encoding an SWI/SNF subunit linked to stem cell and progenitor cell function, is frequently co-amplified and highly expressed together with the p53 family member p63 in head and neck squamous cell carcinoma (HNSCC). ACTL6A and p63 physically interact, cooperatively controlling a transcriptional program that promotes proliferation and suppresses differentiation, in part through activation of the Hippo-YAP pathway via regulators including WWC1. Ectopic ACTL6A/p63 expression promotes tumorigenesis, while ACTL6A expression and YAP activation are highly correlated in primary HNSCC and predict poor patient survival. Thus, ACTL6A and p63 collaborate as oncogenic drivers in HNSCC.

Personalized medicine for cystic fibrosis: Establishing human model systems

With over 1,500 identifiable mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that result in distinct functional and phenotypical abnormalities, it is virtually impossible to perform randomized clinical trials to identify the best therapeutics for all patients. Therefore, a personalized medicine approach is essential. The only way to realistically accomplish this is through the development of improved in vitro human model systems. The lack of a readily available and infinite supply of human CFTR‐expressing airway epithelial cells is a key bottleneck. We propose that a concerted two‐pronged approach is necessary for patient‐specific cystic fibrosis research to continue to prosper and realize its potential: (1) more effective culture and differentiation conditions for growing primary human airway and nasal epithelial cells and (2) the development of collective protocols for efficiently differentiating disease‐ and patient‐specific induced pluripotent stem cells (iPSC) into pure populations of adult epithelial cells. Ultimately, we need a personalized human model system for cystic fibrosis with the capacity for uncomplicated bankability, widespread availability, and universal applicability for patient‐specific disease modeling, novel pharmacotherapy investigation and screening, and readily executable genetic modification. Pediatr Pulmonol. 2015; 50:S14–S23.

Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold

A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer.