[Show abstract][Hide abstract] ABSTRACT: There is a growing demand for long-term in vivo stem cell imaging for assessing cell therapy techniques and guiding therapeutic decisions. This work develops the production of 52Mn and establishes proof of concept for the use of divalent metal transporter 1 (DMT1) as a positron emission tomography (PET) and magnetic resonance imaging (MRI) reporter gene for stem cell tracking in the rat brain. 52Mn was produced via proton irradiation of a natural chromium target. In a comparison of two 52Mn separation methods, solvent-solvent extraction was preferred over ion exchange chromatography because of reduced chromium impurities and higher 52Mn recovery. In vitro uptake of Mn-based PET and MRI contrast agents (52Mn2+ and Mn2+, respectively) was enhanced in DMT1 over-expressing human neural progenitor cells (hNPC-DMT1) compared to wild-type control cells (hNPC-WT). After cell transplantation in the rat striatum, increased uptake of Mn-based contrast agents in grafted hNPC-DMT1 was detected in in vivo manganese-enhanced MRI (MEMRI) and ex vivo PET and autoradiography. These initial studies indicate that this approach holds promise for dual-modality PET/MR tracking of transplanted stem cells in the central nervous system and prompt further investigation into the clinical applicability of this technique.
[Show abstract][Hide abstract] ABSTRACT: Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity.
In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long term.
We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration.
Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact.
These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.
Journal of neuroscience methods 03/2014; · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using stem cells to replace degenerating muscle cells and restore lost skeletal muscle function is an attractive therapeutic strategy for treating neuromuscular diseases. Myogenic progenitors are a valuable cell type for cell-based therapy and also provide a platform for studying normal muscle development and disease mechanisms in vitro. Human pluripotent stem cells represent a valuable source of tissue for generating myogenic progenitors. Here, we present a novel protocol for deriving myogenic progenitors from human embryonic stem (hES) and induced pluripotent stem (iPS) cells using free-floating spherical culture (EZ spheres) in a defined culture medium. hES cell colonies and human iPS cell colonies were expanded in medium supplemented with high concentrations (100 ng/ml) of fibroblast growth factor-2 (FGF-2) and epidermal growth factor in which they formed EZ spheres and were passaged using a mechanical chopping method. We found myogenic progenitors in the spheres after 6 weeks of culture and multinucleated myotubes following sphere dissociation and 2 weeks of terminal differentiation. A high concentration of FGF-2 plays a critical role for myogenic differentiation and is necessary for generating myogenic progenitors from pluripotent cells cultured as EZ spheres. Importantly, EZ sphere culture produced myogenic progenitors from human iPS cells generated from both healthy donors and patients with neuromuscular disorders (including Becker's muscular dystrophy, spinal muscular atrophy, and familial amyotrophic lateral sclerosis). Taken together, this study demonstrates a simple method for generating myogenic cells from pluripotent sources under defined conditions for potential use in disease modeling or cell-based therapies targeting skeletal muscle.
STEM CELLS TRANSLATIONAL MEDICINE 03/2014; · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the neuromuscular system and does not have a known singular cause. Genetic mutations, extracellular factors, non-neuronal support cells, and the immune system have all been shown to play varied roles in clinical and pathological disease progression. The therapeutic plasticity of mesenchymal stem cells (MSCs) may be well matched to this complex disease pathology, making MSCs strong candidates for cellular therapy in ALS. In this review, we summarize a variety of explored mechanisms by which MSCs play a role in ALS progression, including neuronal and non-neuronal cell replacement, trophic factor delivery, and modulation of the immune system. Currently relevant techniques for applying MSC therapy in ALS are discussed, focusing in particular on delivery route and cell source. We include examples from in vitro, preclinical, and clinical investigations to elucidate the remaining progress that must be made to understand and apply MSCs as a treatment for ALS.
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.Molecular Therapy (2013); doi:10.1038/mt.2013.108.
[Show abstract][Hide abstract] ABSTRACT: Recently, we reported that low reward dependence, and to a lesser extent, low cooperativeness in the Temperature and Character Inventory (TCI) may be risk factors for treatment-resistant depression. Here, we analyzed additional psychological traits in these patients.
We administered Costa and McCrae's five-factor model personality inventory, NEO Personality Inventory-Revised (NEO-PI-R), to antidepressant-treatment resistant depressed patients (n = 35), remitted depressed patients (n = 27), and healthy controls (n = 66). We also evaluated the relationships between scores on NEO and TCI, using the same cohort of patients with treatment-resistant depression, as our previous study.
Patients with treatment-resistant depression showed high scores for neuroticism, low scores for extraversion, openness and conscientiousness, without changes in agreeableness, on the NEO. However, patients in remitted depression showed no significant scores on NEO. Patients with treatment-resistant depression and low openness on NEO showed positive relationships with reward dependence and cooperativeness on the TCI.
Many studies have reported that depressed patients show high neuroticism, low extraversion and low conscientiousness on the NEO. Our study highlights low openness on the NEO, as a risk mediator in treatment-resistant depression. This newly identified trait should be included as a risk factor in treatment-resistant depression.
PLoS ONE 01/2013; 8(9):e71964. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The clinical outcome of antidepressant treatment in patients with major depressive disorder (MDD) is thought to be associated with personality traits. A number of studies suggest that depressed patients show high harm avoidance, low self-directedness and cooperativeness, as measured on the Temperament and Character Inventory (TCI). However, the psychology of these patients is not well documented.
Psychological evaluation using Cloninger's TCI, was performed on treatment-resistant MDD patients (n = 35), remission MDD patients (n = 31), and age- and gender-matched healthy controls (n = 174).
Treatment-resistant patients demonstrated high scores for harm avoidance, and low scores for reward dependence, self-directedness, and cooperativeness using the TCI, compared with healthy controls and remission patients. Interestingly, patients in remission continued to show significantly high scores for harm avoidance, but not other traits in the TCI compared with controls. Moreover, there was a significant negative correlation between reward dependence and harm avoidance in the treatment-resistant depression cohort, which was absent in the control and remitted depression groups.
This study suggests that low reward dependence and to a lesser extent, low cooperativeness in the TCI may be risk factors for treatment-resistant depression.
PLoS ONE 01/2013; 8(5):e63756. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle progenitor cells (SMPCs) are considered one of the most valuable cells for cell-based therapy targeting skeletal muscle. However, an efficient protocol for isolating and maintaining human myogenic progenitors in vitro has not been fully established. In this study, we demonstrate that human myogenic progenitors can be expanded and proliferated from human fetal muscles. Human SMPCs were prepared from fetal hind limb muscles and induced to proliferate as free-floating spheres termed myospheres in the medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Both myogenic progenitors and myoblast populations from human fetal muscles were effectively propagated in myospheres and passaged by a mechanical chopping. After expanding these spheres in culture, we tested whether myogenic progenitor cells can differentiate into multinucleated myotubes. The myospheres were dissociated, plated down on coverslips and cultured in the medium for terminal differentiation. We could confirm that the plated cells formed well-developed, multinucleated myotubes. This culture method using myospheres is an effective protocol to isolate and maintain SMPCs from human fetal skeletal muscles in culture.
Cell Biology International 12/2012; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: Preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end-stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPC) bracketing the phrenic motor nucleus prior to disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end-stage. MEASUREMENTS AND RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end-stage was: 1) transiently restored by a single presentation of AIH; and 2) preserved ipsilateral to hNPC transplants made prior to disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in ALS patients.
American Journal of Respiratory and Critical Care Medicine 12/2012; · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies have shown a higher incidence of amyotrophic lateral sclerosis (ALS) in men than women. Interestingly, there are clear gender differences in disease onset and progression in rodent models of familial ALS overexpressing mutated human superoxide dismutase-1 (SOD1-G93A). In the present study we sought to determine whether the alterations of serum steroid levels by gonadectomy or chronic treatment of neuroprotective neurosteroids can modulate disease onset and progression in a rat model of ALS (SOD1-G93A transgenic rats). Presymptomatic SOD1-G93A rats were gonadectomized or treated with a neurosteroid dehydroepiandrosterone (DHEA) using silastic tubing implants. Disease onset and progression of the animals were determined by the routine analyses of locomotor testing using the Basso-Beattie-Bresnahan (BBB) score. Although sexual dimorphism was observed in intact and gonadectomized SOD1-G93A rats, there was no significant effect of gonadectomy on disease onset and progression. DHEA treatment did not alter disease progression or survival in SOD1-G93A rats. Our results indicate that gonadal steroids or neurosteroids are not one of the possible modulators for the occurrence or disease progression in a rat model of ALS. Further analysis will be necessary to understand how sexual dimorphism is involved in ALS disease progression.
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the progressive degeneration of upper and lower motor neurons (MNs), leading to muscular atrophy and eventual respiratory failure. ALS research has primarily focused on mechanisms regarding MN cell death; however, degenerative processes in the skeletal muscle, particularly involving neuromuscular junctions (NMJs), are observed in the early stages of and throughout disease progression. According to the "dying-back" hypothesis, NMJ degeneration may not only precede, but actively cause upper and lower MN loss. The importance of NMJ pathology has relatively received little attention in ALS, possibly because compensatory mechanisms mask NMJ loss for prolonged periods. Many mechanisms explaining NMJ degeneration have been proposed such as the disruption of anterograde/retrograde axonal transport, irregular cellular metabolism, and changes in muscle gene and protein expression. Neurotrophic factors, which are known to have neuroprotective and regenerative properties, have been intensely investigated for their therapeutic potential in both the preclinical and clinical setting. Additional research should focus on the potential of preserving NMJs in order to delay or prevent disease progression.
Neurology research international. 01/2012; 2012:379657.
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1(G93A) transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1(G93A) transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1(G93A) over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1(G93A) over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1(G93A) transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1(G93A) over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.
PLoS ONE 01/2012; 7(11):e48581. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuromuscular diseases affect skeletal muscle and/or nervous control resulting in direct disruption of skeletal muscle and muscle pathology, or nervous system disruption which indirectly disrupts muscle function. Stem cell-based therapy is well-recognized as a promising approach for several types of diseases including those affecting the neuromuscular system. To design a successful therapeutic strategy, it is important to choose the most appropriate stem cell type. Skeletal muscle progenitor cells (SMPCs), also called myogenic progenitors, can contribute to muscle regeneration, differentiate into skeletal muscles, and are valuable cells for therapeutic application. Different types of stem/progenitor cells, including satellite cells, side population cells, muscle derived stem cells, mesenchymal stem cells, myogenic pericytes, and mesoangioblasts, have been identified as possible cell resources of SMPCs. Furthermore, recent advances in stem cell biology allow us to use embryonic stem cells and induced pluripotent stem cells for SMPC derivation. When skeletal muscle is chosen as a target of cell transplantation, the possible criteria for choosing the "best" progenitor/stem cell include preparation strategies, efficiency of intramuscular integration, method of cellular delivery, and functional improvement of the muscle after cell transplantation. Here, we discuss recent findings on various types of SMPCs and their promise for future clinical translation in neuromuscular diseases.
American journal of stem cells. 01/2012; 1(3):253-63.
[Show abstract][Hide abstract] ABSTRACT: The progranulin (PGRN) gene is involved in sexual differentiation of the brain during the perinatal period and estrogen-induced adult neurogenesis in the hippocampus. Mutations in the PGRN gene are also implicated in human frontotemporal lobar degeneration. Thus, while PGRN appears to play important roles as a growth factor in the brain, the localization of PGRN-expressing cells throughout the brain has not been fully established. In the present study, we examined the localization of PGRN proteins in the brain using adult male wild-type mice and PGRN-deficient mice we had generated previously. We also evaluated age-dependent changes in PGRN expression at the mRNA and protein levels. As expected, no immunoreactivity was observed in the brains of the PGRN-deficient mice. In the wild-type mice, intense immunoreactivity was observed in several brain regions including the cingulate and piriform cortices, the pyramidal cell layer and dentate gyrus of the hippocampus, the amygdala, the ventromedial and arcuate nuclei of the hypothalamus and the Purkinje cell layer in the cerebellum. Moreover, PGRN mRNA and protein expression decreased in the cortex, hippocampus and hypothalamus in an age-dependent manner. Since many of these brain regions are involved in emotion, memory and recognition, PGRN may play roles as a growth factor in these brain functions that decline with age.
Journal of Reproduction and Development 10/2010; 57(1):113-9. · 1.76 Impact Factor