A promising therapeutic strategy for diverse genetic disorders involves transplantation of autolo... more 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.
Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current tre... more Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML.
Adult muscle stem cells, or satellite cells have essential roles in homeostasis and regeneration ... more Adult muscle stem cells, or satellite cells have essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here, we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1(-/-) mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1(-/-) muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases.
The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog t... more The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca 2þ ] i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca 2þ ] i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.
In recent years, the continuous development of nanotechnology manufacturing, like Nano Imprinting... more In recent years, the continuous development of nanotechnology manufacturing, like Nano Imprinting Lithography (N.I.L.), have permitted the design and the implementation of innovative devices based on chemical-physical properties of nano-structured surfaces. These nano-structured electronic devices have shown great ability to detect and quantify micro- and nano-molar concentrations of molecules, of great interest in biological, medical and chemical areas, by using
Muscle stem cells (MuSCs) have long been considered to be potential therapeutic vehicles for dise... more Muscle stem cells (MuSCs) have long been considered to be potential therapeutic vehicles for diseases of muscle such as muscular dystrophies. A recent study published in Cell Research by Fu et al. reveals that recapitulating in vitro the in vivo microenvironment of MuSCs that occurs during muscle regeneration might be a major step towards translation.
In this work we present the preliminary resulting measurements of an enzyme-based biosensor for t... more In this work we present the preliminary resulting measurements of an enzyme-based biosensor for the amperometric detection of lactic acid (LA). The sensor is based on low-cost gold electrodes on polymeric substrate. The redox catalytic enzyme used for analyte amperometric detection is lactate oxidase (LOx) from Pediococcus sp. This enzyme has been immobilized over electrodes surfaces by direct adsorption methodologies. Analysis of the enzyme-modified electrodes have been carried out by means of Electrochemical Impedance Spectroscopy (EIS) and with the development of an equivalent electrical model, in order to improve the adsorption process. Biosensors performance have been evaluated with Cyclic Voltammetry (CVM) measurements in different lactic acid solutions with concentrations from 1 μM up to 300 mM. The lactate sensitivity of this disposable biosensor results in about 6.24 μA mM-1 cm-2.
American journal of physiology. Cell physiology, Jan 15, 2015
Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is t... more Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contr...
1: N Biotechnol. 2009 Jan 31. [Epub ahead of print]. Wetware Concepts The wave: 1970 the hardware... more 1: N Biotechnol. 2009 Jan 31. [Epub ahead of print]. Wetware Concepts The wave: 1970 the hardware, 1990 the software, 2010 the wetware. Quarta M. Department of Neurology and Neurological Sciences, Stanford University ...
Background: Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca ... more Background: Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca 2+ ) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca 2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat-and anesthetic-induced hypermetabolic episodes that resemble MH events in humans. Methods: We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.
A promising therapeutic strategy for diverse genetic disorders involves transplantation of autolo... more 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.
Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current tre... more Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML.
Adult muscle stem cells, or satellite cells have essential roles in homeostasis and regeneration ... more Adult muscle stem cells, or satellite cells have essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here, we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1(-/-) mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1(-/-) muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases.
The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog t... more The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca 2þ ] i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca 2þ ] i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.
In recent years, the continuous development of nanotechnology manufacturing, like Nano Imprinting... more In recent years, the continuous development of nanotechnology manufacturing, like Nano Imprinting Lithography (N.I.L.), have permitted the design and the implementation of innovative devices based on chemical-physical properties of nano-structured surfaces. These nano-structured electronic devices have shown great ability to detect and quantify micro- and nano-molar concentrations of molecules, of great interest in biological, medical and chemical areas, by using
Muscle stem cells (MuSCs) have long been considered to be potential therapeutic vehicles for dise... more Muscle stem cells (MuSCs) have long been considered to be potential therapeutic vehicles for diseases of muscle such as muscular dystrophies. A recent study published in Cell Research by Fu et al. reveals that recapitulating in vitro the in vivo microenvironment of MuSCs that occurs during muscle regeneration might be a major step towards translation.
In this work we present the preliminary resulting measurements of an enzyme-based biosensor for t... more In this work we present the preliminary resulting measurements of an enzyme-based biosensor for the amperometric detection of lactic acid (LA). The sensor is based on low-cost gold electrodes on polymeric substrate. The redox catalytic enzyme used for analyte amperometric detection is lactate oxidase (LOx) from Pediococcus sp. This enzyme has been immobilized over electrodes surfaces by direct adsorption methodologies. Analysis of the enzyme-modified electrodes have been carried out by means of Electrochemical Impedance Spectroscopy (EIS) and with the development of an equivalent electrical model, in order to improve the adsorption process. Biosensors performance have been evaluated with Cyclic Voltammetry (CVM) measurements in different lactic acid solutions with concentrations from 1 μM up to 300 mM. The lactate sensitivity of this disposable biosensor results in about 6.24 μA mM-1 cm-2.
American journal of physiology. Cell physiology, Jan 15, 2015
Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is t... more Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contr...
1: N Biotechnol. 2009 Jan 31. [Epub ahead of print]. Wetware Concepts The wave: 1970 the hardware... more 1: N Biotechnol. 2009 Jan 31. [Epub ahead of print]. Wetware Concepts The wave: 1970 the hardware, 1990 the software, 2010 the wetware. Quarta M. Department of Neurology and Neurological Sciences, Stanford University ...
Background: Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca ... more Background: Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca 2+ ) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca 2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat-and anesthetic-induced hypermetabolic episodes that resemble MH events in humans. Methods: We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.
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Papers by Marco Quarta