Extruded alginate tubes with myogenic potential.

BACKGROUND: There are currently no proven methods to reverse muscle loss in humans, which is caused by trauma (e.g., volumetric muscle loss, VML), genetic neuromuscular diseases (e.g., muscular dystrophies, MDs), and accelerated senescence (e.g., sarcopenia). Since muscle tissue is capable of regeneration through muscle satellite cells (MuSCs), the implantation of autologous (or other) donor MuSCs and MuSC-derived myoblasts into host muscles can promote donor-cell-derived myogenesis. Direct injection or implantation of MuSCs or MuSC-derived myoblasts into host muscles only promotes minimal donor-cell-derived myogenesis, whereas implantation of MuSCs/myoblasts along with associated muscle tissue (muscle fibers, extracellular matrix, neurovascular pathways, etc.) gives better results. METHODS: We aim to leverage the benefits of constraining donor myogenic cells within a template that resembles muscle tissue. In this paper, we present a workflow for basic and translational studies aimed at promoting donor-cell-derived myogenesis to increase functional muscle mass in mice. Our workflow involves preparing a slurry of 10% sodium alginate mixed with myogenic cells in cell culture media, extruding the cell-containing slurry into 10% calcium lactate to form tubes, and implanting the cellularized alginate tubes into host muscle. RESULTS: Our data suggest that, the extruded alginate tubes can tolerate a peak stress of 1892 ± 527 mN, that the elastic range is at ~75-125% strain beyond initial length, and that the Young's modulus (stiffness) is 14.17 ± 1.68 %/mm2. Importantly, these mechanical properties render the alginate tubes suitable for a published technique known as minimally-invasive muscle embedding (MIME) that was developed by us to implant myogenic material into host muscle. MIME involves threading donor myogenic tissue into a needle track created within a host muscle. Cellularized alginate tubes implanted into the tibialis anterior muscle of previously euthanized mice had numerous hematoxylin-stained structures similar to nuclear staining, supporting the idea that our alginate tubes can support cell seeding. Alginate tubes that were seeded with MuSCs, incubated in MuSC/myoblast growth (i.e., proliferation) media for two days, incubated in myotube differentiation media for six days, and then minced and reseeded in new dishes, were able to promote in vitro myoblast outgrowth over several days. DISCUSSION: This pilot study is limited in its translational scope because it was performed in vitro and with previously euthanized mice. Additional studies are needed to confirm that cellularized alginate tubes can promote the de novo development of donor-cell-derived muscle fibers, which can contribute to contractile force production. CONCLUSION: Alginate tubes with MuSC/myoblasts can be generated by a simple extrusion method. The alginate tubes have sufficient mechanical strength to tolerate insertion into a host muscle, in a minimally-invasive manner, through a needle track. The cellularized alginate tubes demonstrate myogenic potential since they are capable of being maintained in culture conditions for several days, after which they can still facilitate myoblast outgrowth in a dish.

Division-Independent Differentiation of Muscle Stem Cells During a Growth Stimulus.

Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory.

Regulation of adult stem cell function by ketone bodies.

Adult stem cells play key roles in tissue homeostasis and regeneration. Recent evidence suggests that dietary interventions can significantly impact adult stem cell function. Some of these effects depend on ketone bodies. Adult stem cells could therefore potentially be manipulated through dietary regimens or exogenous ketone body supplementation, a possibility with significant implications for regenerative medicine. In this review we discuss recent findings of the mechanisms by which ketone bodies could influence adult stem cells, including ketogenesis in adult stem cells, uptake and transport of circulating ketone bodies, receptor-mediated signaling, and changes to cellular metabolism. We also discuss the potential effects of ketone bodies on intracellular processes such as protein acetylation and post-transcriptional control of gene expression. The exploration of mechanisms underlying the effects of ketone bodies on stem cell function reveals potential therapeutic targets for tissue regeneration and age-related diseases and suggests future research directions in the field of ketone bodies and stem cells.

Absolute Quantification of mRNA Isoforms in Adult Stem Cells Using Microfluidic Digital PCR.

Adult stem cells play key roles in homeostasis and tissue repair. These cells are regulated by a tight control of transcriptional programs. For example, muscle stem cells (MuSCs), located beneath the basal lamina, exist in the quiescent state but can transition to an activated, proliferative state upon injury. The control of MuSC state depends on the expression levels of myogenic transcription factors. Recent studies revealed the presence of different mRNA isoforms, with distinct biological regulation. Quantifying the exact expression levels of the mRNA isoforms encoding these myogenic transcription factors is therefore key to understanding how MuSCs switch between cell states. Previously, quantitative real-time polymerase chain reaction (qRT-PCR) has been used to quantify RNA expression levels. However, qRT-PCR depends on large amounts of RNA input and only measures relative abundance. Here, we present a protocol for the absolute quantification of mRNA isoforms using microfluidic digital PCR (mdPCR). Primary MuSCs isolated from individual skeletal muscles (gastrocnemius and masseter) are lysed, and their RNA is reverse-transcribed into cDNA and copied into double-stranded DNA. Following exonuclease I digestion to remove remaining single-stranded DNA, the samples are loaded onto a mdPCR chip with TaqMan probes targeting the mRNA isoforms of interest, whereupon target molecules are amplified in nanoliter chambers. We demonstrate that mdPCR can give exact molecule counts per cell for mRNA isoforms encoding the myogenic transcription factor Pax3. This protocol enables the absolute quantification of low abundant mRNA isoforms in a fast, precise, and reliable way.

Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

Multiomics reveals glutathione metabolism as a driver of bimodality during stem cell aging.

With age, skeletal muscle stem cells (MuSCs) activate out of quiescence more slowly and with increased death, leading to defective muscle repair. To explore the molecular underpinnings of these defects, we combined multiomics, single-cell measurements, and functional testing of MuSCs from young and old mice. The multiomics approach allowed us to assess which changes are causal, which are compensatory, and which are simply correlative. We identified glutathione (GSH) metabolism as perturbed in old MuSCs, with both causal and compensatory components. Contrary to young MuSCs, old MuSCs exhibit a population dichotomy composed of GSHhigh cells (comparable with young MuSCs) and GSHlow cells with impaired functionality. Mechanistically, we show that antagonism between NRF2 and NF-κB maintains this bimodality. Experimental manipulation of GSH levels altered the functional dichotomy of aged MuSCs. These findings identify a novel mechanism of stem cell aging and highlight glutathione metabolism as an accessible target for reversing MuSC aging.

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2´-deoxyuridine (EdU) incorporation assay.

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling.

Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically ß-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that ß-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.

ATR activity controls stem cell quiescence via the cyclin F-SCF complex.

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.

RNA splicing programs define tissue compartments and cell types at single-cell resolution.

The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10X Chromium data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes MYL6 and RPS24. These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in SAT1. Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.

Exercise rejuvenates quiescent skeletal muscle stem cells in old mice through restoration of Cyclin D1.

Aging impairs tissue repair. This is pronounced in skeletal muscle, whose regeneration by muscle stem cells (MuSCs) is robust in young adult animals but inefficient in older organisms. Despite this functional decline, old MuSCs are amenable to rejuvenation through strategies that improve the systemic milieu, such as heterochronic parabiosis. One such strategy, exercise, has long been appreciated for its benefits on healthspan, but its effects on aged stem cell function in the context of tissue regeneration are incompletely understood. Here we show that exercise in the form of voluntary wheel running accelerates muscle repair in old animals and improves old MuSC function. Through transcriptional profiling and genetic studies, we discovered that the restoration of old MuSC activation ability hinges on restoration of Cyclin D1, whose expression declines with age in MuSCs. Pharmacologic studies revealed that Cyclin D1 maintains MuSC activation capacity by repressing TGFß signaling. Taken together, these studies demonstrate that voluntary exercise is a practicable intervention for old MuSC rejuvenation. Furthermore, this work highlights the distinct role of Cyclin D1 in stem cell quiescence.

Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function.

Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3' untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.

Transcriptional Profiling of Quiescent Muscle Stem Cells In Vivo.

Muscle stem cells (MuSCs) persist in a quiescent state and activate in response to specific stimuli. The quiescent state is both actively maintained and dynamically regulated. However, analyses of quiescence have come primarily from cells removed from their niche. Although these cells are still quiescent, biochemical changes certainly occur during the isolation process. Here, we analyze the transcriptome of MuSCs in vivo utilizing MuSC-specific labeling of RNA. Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes. However, using the transcription inhibitor α-amanitin, we show that the ex vivo transcriptome remains largely reflective of the in vivo transcriptome. Together, these data provide perspective on the molecular regulation of the quiescent state at the transcriptional level, demonstrate the utility of these tools for probing transcriptional dynamics in vivo, and provide an invaluable resource for understanding stem cell state transitions.

Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence.

Tissue regeneration depends on the timely activation of adult stem cells. In skeletal muscle, the adult stem cells maintain a quiescent state and proliferate upon injury. We show that muscle stem cells (MuSCs) use direct translational repression to maintain the quiescent state. High-resolution single-molecule and single-cell analyses demonstrate that quiescent MuSCs express high levels of Myogenic Differentiation 1 (MyoD) transcript in vivo, whereas MyoD protein is absent. RNA pulldowns and costainings show that MyoD mRNA interacts with Staufen1, a potent regulator of mRNA localization, translation, and stability. Staufen1 prevents MyoD translation through its interaction with the MyoD 3'-UTR. MuSCs from Staufen1 heterozygous (Staufen1+/-) mice have increased MyoD protein expression, exit quiescence, and begin proliferating. Conversely, blocking MyoD translation maintains the quiescent phenotype. Collectively, our data show that MuSCs express MyoD mRNA and actively repress its translation to remain quiescent yet primed for activation.

Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c.

The myogenic regulatory factor MyoD has been implicated as a key regulator of myogenesis, and yet there is little information regarding its upstream regulators. We found that Deltex2 inhibits myogenic differentiation in vitro, and that skeletal muscle stem cells from Deltex2 knockout mice exhibit precocious myogenic differentiation and accelerated regeneration in response to injury. Intriguingly, Deltex2 inhibits myogenesis by suppressing MyoD transcription, and the Deltex2 knockout phenotype can be rescued by a loss-of-function allele for MyoD In addition, we obtained evidence that Deltex2 regulates MyoD expression by promoting the enrichment of histone 3 modified by dimethylation at lysine 9 at a key regulatory region of the MyoD locus. The enrichment is attributed to a Deltex2 interacting protein, Jmjd1c, whose activity is directly inhibited by Deltex2 and whose expression is required for MyoD expression in vivo and in vitro. Finally, we find that Deltex2 causes Jmjd1c monoubiquitination and inhibits its demethylase activity. Mutation of the monoubiquitination site in Jmjd1c abolishes the inhibitory effect of Deltex2 on Jmjd1c demethylase activity. These results reveal a mechanism by which a member of the Deltex family of proteins can inhibit cellular differentiation, and demonstrate a role of Deltex in the epigenetic regulation of myogenesis.

An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy.

A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during culture. Here we describe an artificial niche for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. Using a machine learning method, we identified a molecular signature of quiescence and used it to screen for factors that could maintain mouse MuSC quiescence, thus defining a quiescence medium (QM). We also engineered muscle fibers that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in QM on engineered fibers showed enhanced potential for engraftment, tissue regeneration and self-renewal after transplantation in mice. An artificial niche adapted to human cells similarly extended the quiescence of human MuSCs in vitro and enhanced their potency in vivo. Our approach for maintaining quiescence may be applicable to stem cells isolated from other tissues.

Dysferlin regulates cell adhesion in human monocytes.

Dysferlin is mutated in a group of muscular dystrophies commonly referred to as dysferlinopathies. It is highly expressed in skeletal muscle, where it is important for sarcolemmal maintenance. Recent studies show that dysferlin is also expressed in monocytes. Moreover, muscle of dysferlinopathy patients is characterized by massive immune cell infiltrates, and dysferlin-negative monocytes were shown to be more aggressive and phagocytose more particles. This suggests that dysferlin deregulation in monocytes might contribute to disease progression, but the molecular mechanism is unclear. Here we show that dysferlin expression is increased with differentiation in human monocytes and the THP1 monocyte cell model. Freshly isolated monocytes of dysferlinopathy patients show deregulated expression of fibronectin and fibronectin-binding integrins, which is recapitulated by transient knockdown of dysferlin in THP1 cells. Dysferlin forms a protein complex with these integrins at the cell membrane, and its depletion impairs cell adhesion. Moreover, patient macrophages show altered adhesion and motility. These findings suggest that dysferlin is involved in regulating cellular interactions and provide new insight into dysferlin function in inflammatory cells.