Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Development and Cell Biology of the Blood-Brain Barrier.

The vertebrate vasculature displays high organotypic specialization, with the structure and function of blood vessels catering to the specific needs of each tissue. A unique feature of the central nervous system (CNS) vasculature is the blood-brain barrier (BBB). The BBB regulates substance influx and efflux to maintain a homeostatic environment for proper brain function. Here, we review the development and cell biology of the BBB, focusing on the cellular and molecular regulation of barrier formation and the maintenance of the BBB through adulthood. We summarize unique features of CNS endothelial cells and highlight recent progress in and general principles of barrier regulation. Finally, we illustrate why a mechanistic understanding of the development and maintenance of the BBB could provide novel therapeutic opportunities for CNS drug delivery.

Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation.

The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-ß) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-ß activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Pericytes and the Control of Blood Flow in Brain and Heart.

Pericytes, attached to the surface of capillaries, play an important role in regulating local blood flow. Using optogenetic tools and genetically encoded reporters in conjunction with confocal and multiphoton imaging techniques, the 3D structure, anatomical organization, and physiology of pericytes have recently been the subject of detailed examination. This work has revealed novel functions of pericytes and morphological features such as tunneling nanotubes in brain and tunneling microtubes in heart. Here, we discuss the state of our current understanding of the roles of pericytes in blood flow control in brain and heart, where functions may differ due to the distinct spatiotemporal metabolic requirements of these tissues. We also outline the novel concept of electro-metabolic signaling, a universal mechanistic framework that links tissue metabolic state with blood flow regulation by pericytes and vascular smooth muscle cells, with capillary KATP and Kir2.1 channels as primary sensors. Finally, we present major unresolved questions and outline how they can be addressed.

Predicting prognosis and immunotherapy response in colorectal cancer by pericytes insights from single-cell RNA sequencing.

Immunotherapy has revolutionized the treatment of tumors, but there are still a large number of patients who do not benefit from immunotherapy. Pericytes play an important role in remodeling the immune microenvironment. However, how pericytes affect the prognosis and treatment resistance of tumors is still unknown. This study jointly analyzed single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing data of multiple cancers to reveal pericyte function in the colorectal cancer microenvironment. Analyzing over 800 000 cells, it was found that colorectal cancer had more pericyte enrichment in tumor tissues than other cancers. We then combined the TCGA database with multiple public datasets and enrolled more than 1000 samples, finding that pericyte may be closely related to poor prognosis due to the higher epithelial-mesenchymal transition (EMT) and hypoxic characteristics. At the same time, patients with more pericytes have higher immune checkpoint molecule expressions and lower immune cell infiltration. Finally, the contributions of pericyte in poor treatment response have been demonstrated in multiple immunotherapy datasets (n = 453). All of these observations suggest that pericyte can be used as a potential biomarker to predict patient disease progression and immunotherapy response.

Biology of vascular mural cells.

The vasculature consists of vessels of different sizes that are arranged in a hierarchical pattern. Two cell populations work in concert to establish this pattern during embryonic development and adopt it to changes in blood flow demand later in life: endothelial cells that line the inner surface of blood vessels, and adjacent vascular mural cells, including smooth muscle cells and pericytes. Despite recent progress in elucidating the signalling pathways controlling their crosstalk, much debate remains with regard to how mural cells influence endothelial cell biology and thereby contribute to the regulation of blood vessel formation and diameters. In this Review, I discuss mural cell functions and their interactions with endothelial cells, focusing on how these interactions ensure optimal blood flow patterns. Subsequently, I introduce the signalling pathways controlling mural cell development followed by an overview of mural cell ontogeny with an emphasis on the distinguishing features of mural cells located on different types of blood vessels. Ultimately, I explore therapeutic strategies involving mural cells to alleviate tissue ischemia and improve vascular efficiency in a variety of diseases.

Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

Pericyte-Specific Secretome Profiling in Hypoxia Using TurboID in a Multicellular in Vitro Spheroid Model.

Cellular communication within the brain is imperative for maintaining homeostasis and mounting effective responses to pathological triggers like hypoxia. However, a comprehensive understanding of the precise composition and dynamic release of secreted molecules has remained elusive, confined primarily to investigations using isolated monocultures. To overcome these limitations, we utilized the potential of TurboID, a non-toxic biotin ligation enzyme, to capture and enrich secreted proteins specifically originating from human brain pericytes in spheroid cocultures with human endothelial cells and astrocytes. This approach allowed us to characterize the pericyte secretome within a more physiologically relevant multicellular setting encompassing the constituents of the blood-brain barrier. Through a combination of mass spectrometry and multiplex immunoassays, we identified a wide spectrum of different secreted proteins by pericytes. Our findings demonstrate that the pericytes secretome is profoundly shaped by their intercellular communication with other blood-brain barrier-residing cells. Moreover, we identified substantial differences in the secretory profiles between hypoxic and normoxic pericytes. Mass spectrometry analysis showed that hypoxic pericytes in coculture increase their release of signals related to protein secretion, mTOR signaling, and the complement system, while hypoxic pericytes in monocultures showed an upregulation in proliferative pathways including G2M checkpoints, E2F-, and Myc-targets. In addition, hypoxic pericytes show an upregulation of proangiogenic proteins such as VEGFA but display downregulation of canonical proinflammatory cytokines such as CXCL1, MCP-1, and CXCL6. Understanding the specific composition of secreted proteins in the multicellular brain microvasculature is crucial for advancing our knowledge of brain homeostasis and the mechanisms underlying pathology. This study has implications for the identification of targeted therapeutic strategies aimed at modulating microvascular signaling in brain pathologies associated with hypoxia.

Intraluminal pressure elevates intracellular calcium and contracts CNS pericytes: Role of voltage-dependent calcium channels.

Arteriolar smooth muscle cells (SMCs) and capillary pericytes dynamically regulate blood flow in the central nervous system in the face of fluctuating perfusion pressures. Pressure-induced depolarization and Ca2+ elevation provide a mechanism for regulation of SMC contraction, but whether pericytes participate in pressure-induced changes in blood flow remains unknown. Here, utilizing a pressurized whole-retina preparation, we found that increases in intraluminal pressure in the physiological range induce contraction of both dynamically contractile pericytes in the arteriole-proximate transition zone and distal pericytes of the capillary bed. We found that the contractile response to pressure elevation was slower in distal pericytes than in transition zone pericytes and arteriolar SMCs. Pressure-evoked elevation of cytosolic Ca2+ and contractile responses in SMCs were dependent on voltage-dependent Ca2+ channel (VDCC) activity. In contrast, Ca2+ elevation and contractile responses were partially dependent on VDCC activity in transition zone pericytes and independent of VDCC activity in distal pericytes. In both transition zone and distal pericytes, membrane potential at low inlet pressure (20 mmHg) was approximately -40 mV and was depolarized to approximately -30 mV by an increase in pressure to 80 mmHg. The magnitude of whole-cell VDCC currents in freshly isolated pericytes was approximately half that measured in isolated SMCs. Collectively, these results indicate a loss of VDCC involvement in pressure-induced constriction along the arteriole-capillary continuum. They further suggest that alternative mechanisms and kinetics of Ca2+ elevation, contractility, and blood flow regulation exist in central nervous system capillary networks, distinguishing them from neighboring arterioles.

The emergence of the calvarial hematopoietic niche in health and disease.

The diploë region of skull has recently been discovered to act as a myeloid cell reservoir to the underlying meninges. The presence of ossified vascular channels traversing the inner skull of cortex provides a passageway for the cells to traffic from the niche, and CNS-derived antigens traveling through cerebrospinal fluid in a perivascular manner reaches the niche to signal myeloid cell egress. This review will highlight the recent findings establishing this burgeoning field along with the known role this niche plays in CNS aging and disease. It will further highlight the anatomical routes and physiological properties of the vascular structures these cells use for trafficking, spanning from skull to brain parenchyma.

Functional and clinical roles of stromal PDGF receptors in tumor biology.

PDGF receptors play pivotal roles in both developmental and physiological processes through the regulation of mesenchymal cells involved in paracrine instructive interactions with epithelial or endothelial cells. Tumor biology studies, alongside analyses of patient tissue samples, provide strong indications that the PDGF signaling pathways are also critical in various types of human cancer. This review summarizes experimental findings and correlative studies, which have explored the biological mechanisms and clinical relevance of PDGFRs in mesenchymal cells of the tumor microenvironment. Collectively, these studies support the overall concept that the PDGF system is a critical regulator of tumor growth, metastasis, and drug efficacy, suggesting yet unexploited targeting opportunities. The inter-patient variability in stromal PDGFR expression, as being linked to prognosis and treatment responses, not only indicates the need for stratified approaches in upcoming therapeutic investigations but also implies the potential for the development of PDGFRs as biomarkers of clinical utility, interestingly also in settings outside PDGFR-directed treatments.

Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration.

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.

Cell-to-Cell Crosstalk: A New Insight into Pulmonary Hypertension.

Pulmonary hypertension (PH) is a disease with high pulmonary arterial pressure, pulmonary vasoconstriction, pulmonary vascular remodeling, and microthrombosis in complex plexiform lesions, but it has been unclear of the exact mechanism of PH. A new understanding of the pathogenesis of PH is occurred and focused on the role of crosstalk between the cells on pulmonary vessels and pulmonary alveoli. It was found that the crosstalks among the endothelial cells, smooth muscle cells, fibroblasts, pericytes, alveolar epithelial cells, and macrophages play important roles in cell proliferation, migration, inflammation, and so on. Therefore, the heterogeneity of multiple pulmonary blood vessels and alveolar cells and tracking the transmitters of cell communication could be conducive to the further insights into the pathogenesis of PH to discover the potential therapeutic targets for PH.

Cell-Specific Mechanisms in the Heart of COVID-19 Patients.

From the onset of the pandemic, evidence of cardiac involvement in acute COVID-19 abounded. Cardiac presentations ranged from arrhythmias to ischemia, myopericarditis/myocarditis, ventricular dysfunction to acute heart failure, and even cardiogenic shock. Elevated serum cardiac troponin levels were prevalent among hospitalized patients with COVID-19; the higher the magnitude of troponin elevation, the greater the COVID-19 illness severity and in-hospital death risk. Whether these consequences were due to direct SARS-CoV-2 infection of cardiac cells or secondary to inflammatory responses steered early cardiac autopsy studies. SARS-CoV-2 was reportedly detected in endothelial cells, cardiac myocytes, and within the extracellular space. However, findings were inconsistent and different methodologies had their limitations. Initial autopsy reports suggested that SARS-CoV-2 myocarditis was common, setting off studies to find and phenotype inflammatory infiltrates in the heart. Nonetheless, subsequent studies rarely detected myocarditis. Microthrombi, cardiomyocyte necrosis, and inflammatory infiltrates without cardiomyocyte damage were much more common. In vitro and ex vivo experimental platforms have assessed the cellular tropism of SARS-CoV-2 and elucidated mechanisms of viral entry into and replication within cardiac cells. Data point to pericytes as the primary target of SARS-CoV-2 in the heart. Infection of pericytes can account for the observed pericyte and endothelial cell death, innate immune response, and immunothrombosis commonly observed in COVID-19 hearts. These processes are bidirectional and synergistic, rendering a definitive order of events elusive. Single-cell/nucleus analyses of COVID-19 myocardial tissue and isolated cardiac cells have provided granular data about the cellular composition and cell type-specific transcriptomic signatures of COVID-19 and microthrombi-positive COVID-19 hearts. Still, much remains unknown and more in vivo studies are needed. This review seeks to provide an overview of the current understanding of COVID-19 cardiac pathophysiology. Cell type-specific mechanisms and the studies that provided such insights will be highlighted. Given the unprecedented pace of COVID-19 research, more mechanistic details are sure to emerge since the writing of this review. Importantly, our current knowledge offers significant clues about the cardiac pathophysiology of long COVID-19, the increased postrecovery risk of cardiac events, and thus, the future landscape of cardiovascular disease.

Imaging the Retinal Vascular Mural Cells In Vivo: Elucidating the Timeline of Their Loss in Diabetic Retinopathy.

BACKGROUND: Vascular mural cells (VMCs) are integral components of the retinal vasculature with critical homeostatic functions such as maintaining the inner blood-retinal barrier and vascular tone, as well as supporting the endothelial cells. Histopathologic donor eye studies have shown widespread loss of pericytes and smooth muscle cells, the 2 main VMC types, suggesting these cells are critical to the pathogenesis of diabetic retinopathy (DR). There remain, however, critical gaps in our knowledge regarding the timeline of VMC demise in human DR. METHODS: In this study, we address this gap using adaptive optics scanning laser ophthalmoscopy to quantify retinal VMC density in eyes with no retinal disease (healthy), subjects with diabetes without diabetic retinopathy, and those with clinical DR and diabetic macular edema. We also used optical coherence tomography angiography to quantify capillary density of the superficial and deep capillary plexuses in these eyes. RESULTS: Our results indicate significant VMC loss in retinal arterioles before the appearance of classic clinical signs of DR (diabetes without diabetic retinopathy versus healthy, 5.0±2.0 versus 6.5±2.0 smooth muscle cells per 100 µm; P<0.05), while a significant reduction in capillary VMC density (5.1±2.3 in diabetic macular edema versus 14.9±6.0 pericytes per 100 µm in diabetes without diabetic retinopathy; P=0.01) and capillary density (superficial capillary plexus vessel density, 37.6±3.8 in diabetic macular edema versus 45.5±2.4 in diabetes without diabetic retinopathy; P<0.0001) is associated with more advanced stages of clinical DR, particularly diabetic macular edema. CONCLUSIONS: Our results offer a new framework for understanding the pathophysiologic course of VMC compromise in DR, which may facilitate the development and monitoring of therapeutic strategies aimed at VMC preservation and potentially the prevention of clinical DR and its associated morbidity. Imaging retinal VMCs provides an unparalleled opportunity to visualize these cells in vivo and may have wider implications in a range of diseases where these cells are disrupted.

Continued dysfunction of capillary pericytes promotes no-reflow after experimental stroke in vivo.

Incomplete reperfusion of the microvasculature ('no-reflow') after ischaemic stroke damages salvageable brain tissue. Previous ex vivo studies suggest pericytes are vulnerable to ischaemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains under-explored in vivo. Using longitudinal in vivo two-photon single-cell imaging over 7 days, we showed that 87% of pericytes constrict during cerebral ischaemia and remain constricted post reperfusion, and 50% of the pericyte population are acutely damaged. Moreover, we revealed ischaemic pericytes to be fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 h post stroke. Despite sustaining acute membrane damage, we observed that over half of all cortical pericytes survived ischaemia and responded to vasoactive stimuli, upregulated unique transcriptomic profiles and replicated. Finally, we demonstrated the delayed recovery of capillary diameter by ischaemic pericytes after reperfusion predicted vessel reconstriction in the subacute phase of stroke. Cumulatively, these findings demonstrate that surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischaemic stroke.

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions.

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)ß controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRß is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

MuSCs and IPCs: roles in skeletal muscle homeostasis, aging and injury.

Skeletal muscle is a highly specialized tissue composed of myofibres that performs crucial functions in movement and metabolism. In response to external stimuli and injuries, a range of stem/progenitor cells, with muscle stem cells or satellite cells (MuSCs) being the predominant cell type, are rapidly activated to repair and regenerate skeletal muscle within weeks. Under normal conditions, MuSCs remain in a quiescent state, but become proliferative and differentiate into new myofibres in response to injury. In addition to MuSCs, some interstitial progenitor cells (IPCs) such as fibro-adipogenic progenitors (FAPs), pericytes, interstitial stem cells expressing PW1 and negative for Pax7 (PICs), muscle side population cells (SPCs), CD133-positive cells and Twist2-positive cells have been identified as playing direct or indirect roles in regenerating muscle tissue. Here, we highlight the heterogeneity, molecular markers, and functional properties of these interstitial progenitor cells, and explore the role of muscle stem/progenitor cells in skeletal muscle homeostasis, aging, and muscle-related diseases. This review provides critical insights for future stem cell therapies aimed at treating muscle-related diseases.