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Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.
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Formaldehído , ARN , Ratones , Animales , Fijación del Tejido/métodos , Transcriptoma , Encéfalo , Adhesión en Parafina/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Onset and rates of sarcopenia, a disease characterized by a loss of muscle mass and function with age, vary greatly between sexes. Currently, no clinical interventions successfully arrest age-related muscle impairments since the decline is frequently multifactorial. Previously, we found that systemic transplantation of our unique adult multipotent muscle-derived stem/progenitor cells (MDSPCs) isolated from young mice-but not old-extends the health-span in DNA damage mouse models of progeria, a disease of accelerated aging. Additionally, induced neovascularization in the muscles and brain-where no transplanted cells were detected-strongly suggests a systemic therapeutic mechanism, possibly activated through circulating secreted factors. Herein, we used ZMPSTE24-deficient mice, a lamin A defect progeria model, to investigate the ability of young MDSPCs to preserve neuromuscular tissue structure and function. We show that progeroid ZMPST24-deficient mice faithfully exhibit sarcopenia and age-related metabolic dysfunction. However, systemic transplantation of young MDSPCs into ZMPSTE24-deficient progeroid mice sustained healthy function and histopathology of muscular tissues throughout their 6-month life span in a sex-specific manner. Indeed, female-but not male-mice systemically transplanted with young MDSPCs demonstrated significant preservation of muscle endurance, muscle fiber size, mitochondrial respirometry, and neuromuscular junction morphometrics. These novel findings strongly suggest that young MDSPCs modulate the systemic environment of aged animals by secreted rejuvenating factors to maintain a healthy homeostasis in a sex-specific manner and that the female muscle microenvironment remains responsive to exogenous regenerative cues in older age. This work highlights the age- and sex-related differences in neuromuscular tissue degeneration and the future prospect of preserving health in older adults with systemic regenerative treatments.
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Células Madre Adultas , Progeria , Sarcopenia , Masculino , Ratones , Femenino , Animales , Progeria/genética , Modelos Animales de Enfermedad , Células Madre Adultas/metabolismo , Músculos/metabolismoRESUMEN
Osteoarthritis (OA) is the most common and debilitating joint disease of advanced age and has no universally effective therapy. Here, we demonstrate that systemic transplantation of adult multipotent muscle-derived stem/progenitor cells (MDSPCs)-isolated from young mice-rejuvenates the knee articular cartilage (AC) of naturally aged mice. This intervention reduced expression of pro-inflammatory cytokines (Tnf and Il1a) and catabolic matrix-degrading proteinases (Mmp3 and Mmp13) in aged cartilage. Treatment with young MDSPCs also increased expression of pro-regenerative (Col2a1 and Acan) and prolongevity genes (Pot1b), including those associated with chondrocyte proliferation and differentiation, cartilage growth, and telomere protection. Indeed, the AC of MDSPC-treated mice exhibited reduced age-related histological pathologies. Importantly, the reduced mobility and arthritis-related gait dysfunctions of aged mice were also ameliorated by this treatment. Together, our findings demonstrate the rejuvenating effects of systemic transplantation of young MDSPCs on aging AC-at the molecular, tissue, and functional levels. This suggests that MDSPCs, or their secreted factors, may represent a novel therapy that can increase mobility and function in aged or OA patients.
RESUMEN
BACKGROUND: Osteoarthritis (OA) is one of the most prevalent joint diseases of advanced age and is a leading cause of disability worldwide. Ageing is a major risk factor for the articular cartilage (AC) degeneration that leads to OA, and the age-related decline in regenerative capacity accelerates OA progression. Here we demonstrate that systemic transplantation of a unique population of adult multipotent muscle-derived stem/progenitor cells (MDSPCs), isolated from young wild-type mice, into Zmpste24-/- mice (a model of Hutchinson-Gilford progeria syndrome, a condition marked by accelerated ageing), prevents ageing-related homeostatic decline of AC. RESULTS: MDSPC treatment inhibited expression of cartilage-degrading factors such as pro-inflammatory cytokines and extracellular matrix-proteinases, whereas pro-regenerative markers associated with cartilage mechanical support and tensile strength, cartilage resilience, chondrocyte proliferation and differentiation, and cartilage growth, were increased. Notably, MDSPC transplantation also increased the expression level of genes known for their key roles in immunomodulation, autophagy, stress resistance, pro-longevity, and telomere protection. Our findings also indicate that MDSPC transplantation increased proteoglycan content by regulating chondrocyte proliferation. CONCLUSIONS: Together, these findings demonstrate the ability of systemically transplanted young MDSPCs to preserve a healthy homeostasis and promote tissue regeneration at the molecular and tissue level in progeroid AC. These results highlight the therapeutic potential of systemically delivered multipotent adult stem cells to prevent age-associated AC degeneration.
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Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here, we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone, while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration, which led to functional recovery as measured by sustained gait improvement. Furthermore, no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy, gastrocnemius muscles from mice exhibited substantially less muscle atrophy, an increase in muscle mass after denervation, and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies.
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Células Madre Adultas/trasplante , Músculo Esquelético/citología , Regeneración Nerviosa/fisiología , Células-Madre Neurales/trasplante , Adulto , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Ratones , Atrofia Muscular/patología , Atrofia Muscular/terapia , Regeneración Nerviosa/genética , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Células de Schwann/citología , Células de Schwann/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Transcriptoma , Adulto JovenRESUMEN
Here, we demonstrated the differentiation potential of murine muscle-derived stem/progenitor cells (MDSPCs) toward myogenic, neuronal, and glial lineages. MDSPCs, following transplantation into a critical-sized sciatic nerve defect in mice, showed full regeneration with complete functional recovery of the injured peripheral nerve at 6 weeks post-implantation. However, several weeks after regeneration of the sciatic nerve, neoplastic growths were observed. The resulting tumors were malignant peripheral nerve sheath tumors (MPNSTs) with rhabdomyoblastic differentiation, expressing myogenic, neurogenic, and glial markers, common markers of human malignant triton tumors (MTTs). No signs of tumorigenesis were observed 17 weeks post-implantation of MDSPCs into the gastrocnemius muscles of dystrophic/mdx mice, or 1 year following subcutaneous or intravenous injection. While MDSPCs were not oncogenic in nature, the neoplasias were composed almost entirely of donor cells. Furthermore, cells isolated from the tumors were serially transplantable, generating tumors when reimplanted into mice. However, this transformation could be abrogated by differentiation of the cells toward the neurogenic lineage prior to implantation. These results establish that MDSPCs participated in the regeneration of the injured peripheral nerve but transformed in a microenvironment- and time-dependent manner, when they likely received concomitant neurogenic and myogenic differentiation signals. This microenvironment-specific transformation provides a useful mouse model for human MTTs and potentially some insight into the origins of this disease.
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Células Madre Adultas/patología , Transformación Celular Neoplásica/patología , Microambiente Celular , Neurilemoma/patología , Adulto , Animales , Diferenciación Celular , Linaje de la Célula , Separación Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos , Músculo Esquelético/patología , Regeneración Nerviosa , Neurilemoma/fisiopatología , Neurogénesis , Neuroglía/citología , Recuperación de la Función , Células de Schwann/citología , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Nicho de Células Madre , Trasplante de Células MadreRESUMEN
INTRODUCTION: Loss of adult stem cell function during aging contributes to impaired tissue regeneration. Here, we tested the aging-related decline in regeneration potential of adult stem cells residing in the skeletal muscle. METHODS: We isolated muscle-derived stem/progenitor cells (MDSPCs) from progeroid Zmpste24-deficient mice (Zmpste24(-/-)) with accelerated aging phenotypes to investigate whether mutation in lamin A has an adverse effect on muscle stem/progenitor cell function. RESULTS: Our results indicate that MDSPCs isolated from Zmpste24(-/-) mice show reduced proliferation and myogenic differentiation. In addition, Zmpste24(-/-) MDSPCs showed impaired muscle regeneration, with a limited engraftment potential when transplanted into dystrophic muscle, compared with wild-type (WT) MDSPCs. Exposure of progeroid Zmpste24(-/-) MDSPCs to WT MDSPCs rescued the myogenic differentiation defect in vitro. CONCLUSIONS: These results demonstrate that adult stem/progenitor cell dysfunction contributes to impairment of tissue regeneration and suggest that factors secreted by functional cells are indeed important for the therapeutic effect of adult stem cells.
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Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Músculo Esquelético/fisiología , Células Madre/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Lamina Tipo A/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Músculo Esquelético/citología , Fenotipo , Regeneración , Trasplante de Células Madre , Células Madre/citología , Trasplante HomólogoRESUMEN
Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to a collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appears to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1-5) are likely populations of more committed (differentiated) cells, including fibroblast- and myoblast-like cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle, and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend life span and healthspan in mouse models of accelerated aging through an apparent paracrine/endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs.
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Adhesión Celular/fisiología , Separación Celular/métodos , Colágeno/metabolismo , Músculo Esquelético/citología , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Ratones , Músculo Esquelético/fisiología , Células Madre/fisiologíaRESUMEN
We demonstrate that subpopulations of adult human skeletal muscle-derived stem cells, myogenic endothelial cells (MECs), and perivascular stem cells (PSCs) can be simultaneously purified by fluorescence-activated cell sorting (FACS) from cryopreserved human primary skeletal muscle cell cultures (cryo-hPSMCs). For FACS isolation, we utilized a combination of cell lineage markers: the myogenic cell marker CD56, the endothelial cell marker UEA-1 receptor (UEA-1R), and the perivascular cell marker CD146. MECs expressing all three cell lineage markers (CD56(+)UEA-1R(+)CD146(+)/CD45(-)) and PSCs expressing only CD146 (CD146(+)/CD45(-)CD56(-)UEA-1R(-)) were isolated by FACS. To evaluate their myogenic capacities, the sorted cells, with and without expansion in culture, were transplanted into the cardiotoxin-injured skeletal muscles of immunodeficient mice. The purified MECs exhibited the highest regenerative capacity in the injured mouse muscles among all cell fractions tested, while PSCs remained superior to myoblasts and the unpurified primary skeletal muscle cells. Our findings show that both MECs and PSCs retain their high myogenic potentials after in vitro expansion, cryopreservation, and FACS sorting. The current study demonstrates that myogenic stem cells are prospectively isolatable from long-term cryopreserved primary skeletal muscle cell cultures. We emphasize the potential application of this new approach to extract therapeutic stem cells from human muscle cells cryogenically banked for clinical purposes.
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Músculo Esquelético/citología , Células Madre/citología , Adolescente , Adulto , Anciano , Animales , Antígeno CD146/metabolismo , Antígeno CD56/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Niño , Preescolar , Criopreservación , Células Endoteliales/citología , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Regeneración/fisiología , Trasplante de Células Madre , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity. METHODOLOGY/PRINCIPAL FINDINGS: Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDH(hi) subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDH(lo) counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDH(hi) murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDH(lo) myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDH(hi) hMDCs demonstrated superior muscle regenerative capacity compared to ALDH(lo) hMDCs. CONCLUSIONS: The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.