Tom H. Cheung

Molecular regulation of stem cell quiescence

Subsets of mammalian adult stem cells reside in the quiescent state for prolonged periods of time. This state, which is reversible, has long been viewed as dormant and with minimal basal activity. Recent advances in adult stem cell isolation have provided insights into the epigenetic, transcriptional and post-transcriptional control of quiescence and suggest that quiescence is an actively maintained state in which signalling pathways are involved in maintaining a poised state that allows rapid activation. Deciphering the molecular mechanisms regulating adult stem cell quiescence will increase our understanding of tissue regeneration mechanisms and how they are dysregulated in pathological conditions and in ageing.

Maintenance of muscle stem-cell quiescence by microRNA-489

Among the key properties that distinguish adult mammalian stem cells from their more differentiated progeny is the ability of stem cells to remain in a quiescent state for prolonged periods of time. However, the molecular pathways for the maintenance of stem-cell quiescence remain elusive. Here we use adult mouse muscle stem cells (satellite cells) as a model system and show that the microRNA (miRNA) pathway is essential for the maintenance of the quiescent state. Satellite cells that lack a functional miRNA pathway spontaneously exit quiescence and enter the cell cycle. We identified quiescence-specific miRNAs in the satellite-cell lineage by microarray analysis. Among these, miRNA-489 (miR-489) is highly expressed in quiescent satellite cells and is quickly downregulated during satellite-cell activation. Further analysis revealed that miR-489 functions as a regulator of satellite-cell quiescence, as it post-transcriptionally suppresses the oncogene Dek, the protein product of which localizes to the more differentiated daughter cell during asymmetric division of satellite cells and promotes the transient proliferative expansion of myogenic progenitors. Our results provide evidence of the miRNA pathway in general, and of a specific miRNA, miR-489, in actively maintaining the quiescent state of an adult stem-cell population.

Notch Signaling Is Necessary to Maintain Quiescence in Adult Muscle Stem Cells

Satellite cells (SCs) are myogenic stem cells found in skeletal muscle that function to repair tissue damaged by injury or disease. SCs are quiescent at rest, although the signaling pathways required to maintain quiescence are unknown. Using a transgenic Notch reporter mouse and quantitative reverse‐transcription polymerase chain reaction analysis of Notch target genes, we determined that Notch signaling is active in quiescent SCs. SC‐specific deletion of recombining binding protein‐Jκ (RBP‐Jκ), a nuclear factor required for Notch signaling, resulted in the depletion of the SC pool and muscles that lacked any ability to regenerate in response to injury. SC depletion was not due to apoptosis. Rather, RBP‐Jκ‐deficient SCs spontaneously activate, fail to self‐renew, and undergo terminal differentiation. Intriguingly, most of the cells differentiate without first dividing. They then fuse with adjacent myofibers, leading to the gradual disappearance of SCs from the muscle. These results demonstrate the requirement of Notch signaling for the maintenance of the quiescent state and for muscle stem cell homeostasis by the regulation of self‐renewal and differentiation, processes that are all critical for normal postnatal myogenesis. STEM CELLS 2012; 30:232–242.

Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging

Summary The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.

IL-33 ameliorates Alzheimer’s disease-like pathology and cognitive decline

Significance Dysfunction of the innate immune system is involved in the pathogenesis of Alzheimer’s disease (AD); however, the pathophysiological mechanisms underlying these dysfunctions are unclear. Here we report that stimulation of IL-33/ST2 signaling rescues memory deficits and reduces the accumulation of β-amyloid in APP/PS1 mice that exhibit select pathologies associated with AD. Although impaired IL-33/ST2 signaling is associated with early progression of AD, IL-33 injection rescues contextual memory deficits and reduces the accumulation of β-amyloid in APP/PS1 mice. IL-33 skews the microglia toward an alternative activation state with enhanced Aβ phagocytic capacity and elevated antiinflammatory gene expression, which results in a decreased proinflammatory response in the brain. Thus, this study suggests that IL-33 can be developed as a new therapeutic intervention for AD. Alzheimer’s disease (AD) is a devastating condition with no known effective treatment. AD is characterized by memory loss as well as impaired locomotor ability, reasoning, and judgment. Emerging evidence suggests that the innate immune response plays a major role in the pathogenesis of AD. In AD, the accumulation of β-amyloid (Aβ) in the brain perturbs physiological functions of the brain, including synaptic and neuronal dysfunction, microglial activation, and neuronal loss. Serum levels of soluble ST2 (sST2), a decoy receptor for interleukin (IL)-33, increase in patients with mild cognitive impairment, suggesting that impaired IL-33/ST2 signaling may contribute to the pathogenesis of AD. Therefore, we investigated the potential therapeutic role of IL-33 in AD, using transgenic mouse models. Here we report that IL-33 administration reverses synaptic plasticity impairment and memory deficits in APP/PS1 mice. IL-33 administration reduces soluble Aβ levels and amyloid plaque deposition by promoting the recruitment and Aβ phagocytic activity of microglia; this is mediated by ST2/p38 signaling activation. Furthermore, IL-33 injection modulates the innate immune response by polarizing microglia/macrophages toward an antiinflammatory phenotype and reducing the expression of proinflammatory genes, including IL-1β, IL-6, and NLRP3, in the cortices of APP/PS1 mice. Collectively, our results demonstrate a potential therapeutic role for IL-33 in AD.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM+CD31−CD45−Sca1−). The isolation process takes ∼5–6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells

Summary Adult skeletal muscle stem cells, or satellite cells (SCs), regenerate functional muscle following transplantation into injured or diseased tissue. To gain insight into human SC (huSC) biology, we analyzed transcriptome dynamics by RNA sequencing of prospectively isolated quiescent and activated huSCs. This analysis indicated that huSCs differentiate and lose proliferative potential when maintained in high-mitogen conditions ex vivo. Further analysis of gene expression revealed that p38 MAPK acts in a transcriptional network underlying huSC self-renewal. Activation of p38 signaling correlated with huSC differentiation, while inhibition of p38 reversibly prevented differentiation, enabling expansion of huSCs. When transplanted, expanded huSCs differentiated to generate chimeric muscle and engrafted as SCs in the sublaminar niche with a greater frequency than freshly isolated cells or cells cultured without p38 inhibition. These studies indicate characteristics of the huSC transcriptome that promote expansion ex vivo to allow enhanced functional engraftment of a defined population of self-renewing huSCs.

Global gene expression analysis of ERK5 and ERK1/2 signaling reveals a role for HIF-1 in ERK5-mediated responses.

ERK5 is a recently characterized MAPK, which is most similar to the well studied ERK1/2 subfamily but uses distinct mechanisms to elicit responses. To understand the specificity of signaling through ERK5 versus ERK1/2, we examined global gene expression changes in response to each pathway. Microarray measurements in retinal pigment epithelial cells revealed 36 genes regulated by ERK5, all which were novel targets for this pathway. 39 genes were regulated by ERK1/2, which included 11 known genes. Of these genes, 19 were regulated by both pathways. Inspection of the 17 genes uniquely regulated by ERK5 revealed that 14 genes (82%) were previously associated with hypoxia via regulation by HIF-1. In contrast, 16 genes (84%) regulated by either ERK5 or ERK1/2 were implicated in hypoxia, most through mechanisms independent of HIF-1. Of the 20 genes regulated by ERK1/2, only 9 were implicated in hypoxia and were not well characterized hypoxia targets. Thus, unlike ERK5, a mechanistic link between ERK1/2 and HIF-1/HRE could not be established on the basis of gene regulation. Activation of both pathways enhanced transcription from a hypoxia-response element and increased HIF-1α protein expression. In contrast, ERK5 but not ERK1/2 elevated transcription through GAL4-HIF-1. Most interestingly, ERK5 is not significantly activated by hypoxia in retinal pigment epithelial cells, indicating that ERK5 regulation of these genes is relevant in normoxia rather than hypoxia. Thus, ERK5 and ERK1/2 differ in their mechanisms of gene regulation, and indicate that ERK5 may control hypoxia-responsive genes by a mechanism independent of HIF-1α expression control.

All's well that ends well: alternative polyadenylation and its implications for stem cell biology.

Stem cell quiescence, activation, and differentiation are governed by a complex network of molecular pathways. There has been a growing recognition that posttranscriptional modifications, such as alternative polyadenylation (APA) of transcripts, play an important role in regulating gene expression and function. Recent analyses of stem cell populations have suggested that APA controls stem cell fate and behavior. Here, we review recent developments that have shaped our understanding of the control of stem cell behavior by APA and we highlight promising areas for future investigation.

Control of cell cycle-dependent degradation of c-Ski proto-oncoprotein by Cdc34

It is known that excess amounts of Ski, or any member of its proto-oncoprotein family, causes disruption of the transforming growth factor beta signal transduction pathway, thus causing oncogenic transformation of cells. Previous studies indicate that Ski is a relatively unstable protein whose expression levels can be regulated by ubiquitin-mediated proteolysis. Here, we investigate the mechanism by which the stability of Ski is regulated. We show that the steady-state levels of Ski protein are controlled post-translationally by cell cycle-dependent proteolysis, wherein Ski is degraded during the interphase of the cell cycle but is relatively stable during mitosis. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme Cdc34 mediates cell cycle-dependent Ski degradation both in vitro and in vivo. Overexpression of dominant-negative Cdc34 stabilizes Ski and enhances its ability to antagonize TGF-β signaling. Our data suggest that regulated proteolysis of Ski is one of the key mechanisms that control the threshold levels of this proto-oncoprotein, and thus prevents epithelial cells from becoming TGF-β resistant.