Assessing Autophagy in Muscle Stem Cells.

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity's relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.Work in the authors’ laboratory has been supported by MINECO-Spain (RTI2018-096068), ERC-2016-AdG- 741966, LaCaixa-HEALTH-HR17-00040, MDA, Fundació LaMarató/TV3, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain, as well as María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370), Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). SC was supported by a Predoctoral Fellowship from Ayudas para la formación y contratación de personal investigador novel (FI) – AGAUR (Barcelona, Spain), IR-P was supported by a Predoctoral Fellowship from Programa de Formación de Personal Investigador (Spain), and XH was supported by a Severo-Ochoa Pre-doctoral Fellowship (Spain).S

Stem Cells and Aging:What's Next?

We asked 12 leaders in the stem cell and aging fields to share their personal perspectives on the future of the field and the unanswered questions that drive them to work in this exciting area

Attenuated epigenetic suppression of muscle stem cell necroptosis is required for efficient regeneration of dystrophic muscles

Somatic stem cells expand massively during tissue regeneration, which might require control of cell fitness, allowing elimination of non-competitive, potentially harmful cells. How or if such cells are removed to restore organ function is not fully understood. Here, we show that a substantial fraction of muscle stem cells (MuSCs) undergo necroptosis because of epigenetic rewiring during chronic skeletal muscle regeneration, which is required for efficient regeneration of dystrophic muscles. Inhibition of necroptosis strongly enhances suppression of MuSC expansion in a non-cell-autonomous manner. Prevention of necroptosis in MuSCs of healthy muscles is mediated by the chromatin remodeler CHD4, which directly represses the necroptotic effector Ripk3, while CHD4-dependent Ripk3 repression is dramatically attenuated in dystrophic muscles. Loss of Ripk3 repression by inactivation of Chd4 causes massive necroptosis of MuSCs, abolishing regeneration. Our study demonstrates how programmed cell death in MuSCs is tightly controlled to achieve optimal tissue regeneration

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.We thank M. Jardí, A. Navarro, J. M. Ballestero, K. Slobodnyuk, M. González, J. López and M. Raya for their technical contributions; A. Harada and K. Tanaka for assistance in ATAC-seq; all of the members of the P.M.-C. laboratory for discussions; J. Campisi for p16-3MR mice; J. A. Fernández-Blanco (PRBB Animal Facility); O. Fornas (UPF/CRG FACS Facility); E. Rebollo (IBMB Molecular Imaging Platform); V. A. Raker for manuscript editing; and the members of the Myoage network (A. Maier) for human material. We acknowledge funding from MINECO-Spain (RTI2018-096068, to P.M.-C. and E.P.); ERC-2016-AdG-741966, LaCaixa-HEALTHHR17-00040, MDA, UPGRADE-H2020-825825, AFM, DPP-Spain, Fundació La MaratóTV3-80/19- 202021 and MWRF to P.M.-C.; Fundació La MaratóTV3-137/38-202033 to A.L.S.; Maria-de-Maeztu ́ Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa Program for Centers of Excellence to CNIC (SEV-2015-0505). This work was also supported by JST-CREST JPMJCR16G1 and MEXT/JSPS JP20H00456/18H05527 to Y.O.; the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502) to M.A.E.; V.M. and A.C. were supported by FPI and Maria-de-Maeztu predoctoral fellowships, respectively, and V.S. by a Marie Skłodowska-Curie individual fellowship. Parts of the figures were drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licences/by/3.0/).S

Definitions for adult stem cells debated.

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Aging, metabolism and stem cells: Spotlight on muscle stem cells.

All tissues and organs undergo a progressive regenerative decline as they age. This decline has been mainly attributed to loss of stem cell number and/or function, and both stem cell-intrinsic changes and alterations in local niches and/or systemic environment over time are known to contribute to the stem cell aging phenotype. Advancing in the molecular understanding of the deterioration of stem cell cells with aging is key for targeting the specific causes of tissue regenerative dysfunction at advanced stages of life. Here, we revise exciting recent findings on why stem cells age and the consequences on tissue regeneration, with a special focus on regeneration of skeletal muscle. We also highlight newly identified common molecular pathways affecting diverse types of aging stem cells, such as altered proteostasis, metabolism, or senescence entry, and discuss the questions raised by these findings. Finally, we comment on emerging stem cell rejuvenation strategies, principally emanating from studies on muscle stem cells, which will surely burst tissue regeneration research for future benefit of the increasing human aging population.Work in the authors' laboratories was supported by SAF201567369-R (MINECO, Spain), Convenio UPF-CNIC, AFM, E-Rare/Eranet, MDA, Fundacio Marato-TV3, DPP-NLS

Fibrosis development in early-onset muscular dystrophies: Mechanisms and translational implications.

Duchenne muscular dystrophy (DMD) is one of the most devastating neuromuscular genetic diseases caused by the absence of dystrophin. The continuous episodes of muscle degeneration and regeneration in dystrophic muscle are accompanied by chronic inflammation and fibrosis deposition, which exacerbate disease progression. Thus, in addition of investigating strategies to cure the primary defect by gene/cell therapeutic strategies, increasing efforts are being placed on identifying the causes of the substitution of muscle by non-functional fibrotic tissue in DMD, aiming to attenuate its severity. Congenital muscular dystrophies (CMDs) are early-onset diseases in which muscle fibrosis is also present. Here we review the emerging findings on the mechanisms that underlie fibrogenesis in muscular dystrophies, and potential anti-fibrotic treatments.Work in the authors laboratory has been funded by ISCIII, Spain (FIS-P509/01267, FIS-PI13/02512) and MINECO (SAF2015-67369R; "Maria de Maeztu" Programme for Units of Excellence in R&D MDM-2014-0370), AFM, E-Rare/ERANET, Fundacio Marato TV3, MDA and DPP-Spain.S

Simultaneous Isolation of Stem and Niche Cells of Skeletal Muscle: Applicability for Aging Studies.

The maintenance of adult stem cells in their normal quiescent state depends on intrinsic factors and extrinsic signals originating from their microenvironment (also known as the stem cell niche). In skeletal muscle, its stem cells (satellite cells) lose their regenerative potential with aging, and this has been attributed, at least in part, to both age-associated changes in the satellite cells as in the niche cells, which include resident fibro-adipogenic progenitors (FAPs), macrophages, and endothelial cells, among others. To understand the regenerative decline of skeletal muscle with aging, there is a need for methods to specifically isolate stem and niche cells from resting muscle. Here we describe a fluorescence-activated cell sorting (FACS) protocol to simultaneously isolate discrete populations of satellite cells and niche cells from skeletal muscle of aging mice.Work in the authors’ laboratory has been supported by the Spanish Ministry of Science, Innovation and Universities, Spain (grant SAF2015-67369-R; and SAF 2015-70270-REDT, a María de Maeztu Unit of Excellence award to UPF [MDM-2014-0370], and a Severo Ochoa Center of Excellence award to the CNIC [SEV-2015-0505]), the UPF-CNIC collaboration agreement, ERC-2016-AdG-741966, La Caixa-HEALTH, AFM, MDA, and H2020-UPGRADE. V.M is recipient of a FPI predoctoral fellowship.S

Cilia Control Fat Deposition during Tissue Repair

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Understanding muscle regenerative decline with aging: new approaches to bring back youthfulness to aged stem cells.

Aging is characterized by the progressive dysfunction of most tissues and organs, which has been linked to the regenerative decline of their resident stem cells over time. Skeletal muscle provides a stark example of this decline. Its stem cells, also called satellite cells, sustain muscle regeneration throughout life, but at advanced age they fail for largely undefined reasons. Here, we discuss current understanding of the molecular processes regulating satellite cell maintenance throughout life and how age-related failure of these processes contributes to muscle aging. We also highlight the emerging field of rejuvenating biology to restore features of youthfulness in satellite cells, with the ultimate goal of slowing down or reversing the age-related decline in muscle regeneration.Work in the authors’ laboratories has been supported by the following funding sources: The Spanish Ministry of Science, Innovation and Universities, Spain (grants RTI2018-096068-B-I00 and SAF 2015-70270-REDT, a María de Maeztu Unit of Excellence award to UPF [MDM-2014-0370], and a Severo Ochoa Center of Excellence award to the CNIC [SEV-2015-0505]), ERC-2016-AdG-741966, La Caixa-HEALTH (HR17-00040), MDA, UPGRADE-H2020-825825, AFM and DPP-E to PMC; JN and PSV acknowledge support from iMM start-up funding and “la Caixa” Foundation for the Junior Leader Fellowship for PSV (LCF/BQ/PI19/11690006)S