ABSTRACT
Insight into the expanding themes of regenerative medicine is provided by the American Society for Neural Therapy and Repair's annual meeting. The 17th meeting covered a wide range of neurodegenerative disorders, exploring methods to elucidate the currently unknown mechanisms behind the disorders, as well as possible treatments ranging from the use of growth factors, gene therapy to cell transplantation. The importance of growth factors, both as a contributing factor to a disease and as a possible treatment either solo, or as a consequence of, or in conjunction with, stem cell therapy, was highlighted. The potential for viral vectors was also explored either for cells prior to transplantation or as a direct treatment regime into the brain itself. Identification of biomarkers that would allow early detection of a disease is an important factor in our fight against disease. The ability to now perform whole genome analysis and biomolecular profiling provides hope that such markers could be identified which not only could identify this likely to suffer from a disorder but also could allow its progress to be monitored. A few preclinical and clinical cell transplantation trials were also introduced as potential areas of followup in the years to come.
1. INTRODUCTION
The field of regenerative medicine is rapidly expanding with new information on the causes of diseases and possible treatments occurring all the time. This is reflected in the diversity of the presentations that occur annually at the American Society for Neural Therapy and Repair (ASNTR). Many of the presentations are subsequently expanded on at future meetings or in high profile publications. We therefore feel that a review of the topics presented at the 17th ASNTR meeting held in Clearwater, Fla, 2010, can provide insight into the everchanging and evolving field of regenerative medicine for neurological disorders.
As shown in Table 1, there is a wide diversity of topics presented at the annual meeting, which can be split into two camps; specific disease-related studies and general research on mechanisms, treatments, and their characterization. Related publications in the TheScientificWorldJournal over the last two years will be used to help demonstrate their relevance and provide some perspectives for the readers of this journal.
2. PARKINSON’S DISEASE
As in previous years [1, 2], the most popular disease topic was Parkinson’s disease (PD; Table 1), which made up approximately 30% of the presentations. These can be further subdivided into studies that explore the characteristics of the disease in both humans and disease models and those that look at different treatments ranging from the currently in use deep brain stimulation to new potential therapies such as stem cell transplantation. Several of the other disease-related topics can be divided in a similar fashion.
Several presentations looked at different models for PD and how they are compared to Parkinson’s disease. For instance, the rotenone model [3] demonstrates many similarities to PD, and there is some epidemiological evidence that rotenone is a risk factor for PD. Green et al. [4] compared numerous models (including the rotenone model reported on by Greenamyre et al. [3]) and demonstrated that similar gastrointestinal pathology such as alpha-synuclein accumulation is seen in PD and a variety of animal models. Carvey et al. [5] reported on the dysfunction of the blood-brain barrier (BBB) that has been observed in several animal models and in PD. Treatment with an inhibitor of angiogenesis has been shown to reduce BBB impairment, loss of tyrosine hydroxylase (TH) positive neurons, and inflammation in a 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine-(MPTP) treated mouse model of PD [6]. Different models of PD have also been explored in TheScientificWorldJournal, such as the use of organotypic slice cultures of ventral mesencephalon with unilateral tissue surface application via microelectrode of 6-hydroxydopamine (6-OHDA) by Stahl et al. [7]. They observed localized and specific cell death that resembled the effects of 6-OHDA observed in vivo, thus providing another potential model system for exploring PD.
Two studies examined the effects of inflammation on the survival of dopaminergic neurons that are normally lost in PD. Monahan et al. [8] demonstrated that prenatal exposure to lipopolysaccharide caused increased microglial activation and reduced dopaminergic cell survival, whereas Boger et al. [9] demonstrated a similar effect on dopaminergic neurons in adult rats. Both authors suggested and Monahan et al. provided evidence that these effects may result from increased proinflammatory cytokine release. Interestingly, Pabón et al. [10] showed data suggesting that the chemokine fractalkine, which is involved in neuronal-microglial signaling, could be neuroprotective in the 6-OHDA-treated rat due to its ability to reduce microglial activation. Further studies also explored how alpha-synuclein, the accumulation of which in the brain is a pathological hallmark of PD, can mediate oxidative stress and microglial activation [11] whereas another study looked at how dopamine itself could be toxic to dopaminergic neurons [12]. The possible protective role of uncoupling proteins and mitochondrial homeostasis in young primates that is observed after MPTP treatment was investigated in another study [13].
In the human alpha-synuclein-overexpressing rat model, silencing of the overexpressing gene was found to partially rescue forelimb use, but also reduced TH expression, suggesting that silencing of the overexpressing gene does not mean that the animal reverts back to a wild-type state [14]. Gene transduction can also be used as a neuroprotectant since overexpression of the growth factor pleiotrophin was found to promote survival in the 6-OHDA-treated rat model of Parkinson’s disease [15].
Growth factors are known to play an important role in the development and survival of cells. An increased number of striatal neurons expressing the epidermal growth factor superfamily protein fetal-antigen-1, which is believed to be a growth and/or differentiation factor, was observed following 6-OHDA treatment in rats, suggesting that this protein may play a role in PD [16]. Strömberg et al. [17] demonstrated that glial-derived neurotrophic factor (GDNF) was crucial for the maintenance but not the original development of the dopaminergic nigrostriatal system. This was achieved by transplantation of a lateral ganglionic eminence (LGE) and ventral mesencephalon (VM) graft into a host animal and demonstrating “normal” development with GDNF-deficient tissue. However, this graft did not survive for six months, whereas GDNF-expressing tissue did. Blanchard et al. [18] used a similar transplant, except in some cases the orientation of the LGE was reversed, to attempt to demonstrate the existence of a developmental gradient of growth factors within the striatum. Andressoo et al. [19] presented further data on how GDNF (or the lack of it) affects the development of the dopaminergic system, whereas Saarma et al. [20] discussed how cerebral dopamine neurotrophic factor (CDNF) may have a potential role in the treatment of PD and suggested that it may be a better factor than GDNF in this regard due to its ready ability to diffuse through brain tissue.
Two studies looked at oral administration of possible neuroprotective agents and found some degree of success with allantoin, the end product of purine metabolism, in the 6-OHDA-treated rat model [21], and a peroxisome proliferator activated receptor- (PPAR-) agonist in the MPTP-treated mouse model [22]. The possible involvement of this receptor as a therapeutic target was reviewed in an article published in TheScientificWorldJournal [23]. Of particular interest are the receptor’s anti-inflammatory, proapoptotic, and cell cycle arresting properties and how they could interact with disease processes. Subcutaneous administration of a full dopamine D1 receptor agonist was also shown to exert behavioral benefits in the 6-OHDA-treated rat [24]. These studies are the roots for further evaluation of these compounds as potential treatments for PD.
Dodiya et al. [25] looked at human postmortem tissue and provided evidence that striatal dopamine innervation by nigral neurons dropped off rapidly in patients with more than 5 years disease duration, which may mean that trophic factor therapies that rely on retrograde transport mechanisms are likely to be ineffective due to the absence of surviving dopaminergic projections. PD patients that had only recently been diagnosed who are also on antidepressants have been shown to be less behaviorally impaired than those who are not, suggesting that antidepressant therapy might provide some benefit [26]. Data from two clinical studies were presented that explore the potential of deep brain stimulation (DBS) in early PD patients [27, 28]. A third study of deep brain stimulation in PD touched on the possible beneficial effects of this treatment on speech problems and whether an algorithm could be created which could monitor this as previously reported results are mixed [29]. By comparison, a few studies published in TheScientificWorldJournal looked at whether complementary treatments such as active theater or mental and physical exercise could be beneficial in PD, cognitive impairment, and aging [30–32]. Modugno et al. [30] observed a significant improvement in the clinical scale of PD patients after 3 years of an active theater program compared with patients on physiotherapy. Frick and Benoit [31] reviewed animal models that used environmental enrichment, focusing primarily on the effects on aging, where cognitive improvement (or stabilization) has been seen, while Asha Devi [32] focused on the ability of exercise and a vitamin E regimen to slow cognitive decline.
One possible treatment for PD that has previously been explored is transplantation. Early studies focused on tissues and two studies presented at the ASNTR meeting also elaborated on this type of treatment. Rao et al. [33] showed that cografts of human retinal pigment epithelial cells and striatal mouse VM in 6-OHDA-treated rats survived better than VM alone and also produced behavioral improvements. Soderstrom et al. [34] reported that graft survival and dyskinesia incidence improved when embryonic dopaminergic tissue that had been pretreated with calcium channel inhibitors to promote medium spiny neuron dendritic spine survival was grafted, suggesting that spines are important in the derivation of grafts and in reducing side effects. Surmeier [35] reviewed the possible contribution of calcium channels in dopaminergic neurons to the generation of oxidative stress and the selective vulnerability of these cells in PD.
As well as transplanting tissue, studies are progressing towards the transplantation of specific cell types, such as stem cells. Three studies looked at the potential benefits of transplanting human-derived neural progenitor cells into the 6-OHDA-treated rat [36, 37] or MPTP-lesioned primate [38]. The cells used by Collier et al. [36] included cells from NeuralStem, Inc. Endogenous neurogenesis was shown to be promoted by Madhavan et al. [37], whereas growth factors were also shown to play a role in cell survival and demonstrated some benefit in each study. Wakeman et al. [38] actually pretreated the animals with adeno-associated virus-GDNF to explore the effects of this factor on transplantation. Interestingly, Kelly et al. [39] used a similar approach to “enrich the environment” prior to human umbilical cord blood transplantation in MPTP-treated primates, and neuroprotective effects were observed in all of these studies. The final reported cell transplantation study for PD used human mesenchymal stem cells (hMSCs) transplanted into the 6-OHDA-treated rat and again showed that growth factors played a role in the cells’ survival and benefits [40].
These studies are summarized in Table 2 and show that PD research is progressing with research expanding our understanding of what goes wrong in Parkinson’s disease and how current treatments, such as deep brain stimulation, may prove to be beneficial in the early stages of the disease. Potential treatments are likely to focus on ways to promote growth factor expression which can provide an environment more favorable to cell survival and promote endogenous neuronal survival, possibly by stem cell transplantation or other means.
3. BRAIN ISCHEMIA
The second largest disease-related category is that of brain ischemia; the majority of the studies focused on stroke. Since tissue-plasminogen activator (t-PA) is currently the only FDA-approved treatment for stroke and this has a small window of effectiveness, it is imperative that other therapies can be identified. This group highlights this, since in contrast to the PD group, these studies exclusively explored possible treatment regimes including the use of diet, growth factors, or cell transplantation. Borlongan et al. [41] demonstrated that proprietary herbal extracts of cacao and red sage could confer neuroprotection in both the in vitro oxygen-glucose deprivation (OGD) primary neuronal culture and in vivo middle cerebral artery occlusion rodent models (MCAo) of stroke. Interestingly, the extracts were only effective in vivo when given prophylactically. Three studies looked at using adeno-associated viral vectors (AAVs) for trophic factors such as mesencephalic astrocyte-derived neurotrophic factor (MANF) and vascular endothelial growth factor (VEGF), or the glutamate transporter (GLT1) in an MCAo (or focal cerebral ischemia) rat model of stroke or ischemia [42–44]. In each case, a reduced infarct size was observed, with the mechanism of action being proposed as modulation of endoplasmic reticulum stress, activation of Akt, and reduced extracellular glutamate, respectively. Interestingly, the combined use of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) was shown to promote neuronal network formation after cortical ischemia [45], as well as promote blood flow, and reduce cerebral amyloid angiopathy in an animal model of inherited stroke and vascular dementia called cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [46]. These studies provide support for the use of combination therapies to treat disorders.
Pretreatment with pyruvate was also shown to confer some protection in an OGD hippocampal slice model, which may relate to the ability of pyruvate to increase glycogen stores [47]. OGD and the MCAo rat model were used to investigate the ability of human umbilical cord blood cells (hUCBCs) to modify the neuronal gene expression profile both in vitro and in vivo following ischemia and saw changes in transcription factors that could promote neuronal survival [48].
Bible et al. [49] characterized a 19F-contrast agent for the labeling of different human neural stem cell (NSC) lines so that they could be followed after transplanting them into stroke-lesioned animal brains. On the other hand, Daadi et al. [50] used superparamagnetic iron oxide particles (SPIOs) to label human embryonic-stem-cell-(ESC) derived NSCs that could be monitored by magnetic resonance imaging following transplantation into the MCAo-treated rodent brain. They observed a marked reduction in the size of the infarct over time and monitored the survival and location of the transplanted cells.
Two studies investigated ReNeuron’s immortalized neural stem cell line that is currently undergoing clinical trials for the treatment of stroke in the United Kingdom. The first study looked at i.v. administration of the cells within 2 days of MCAo in rats [51], whereas the second study transplanted cells either directly into surviving brain tissue or into the ventricles 3 weeks after MCAo [52]. The acute treatment showed significant improvement in the elevated body swing test within 3 days. A similar recovery in this and other behavioral tests was observed over a 3-month period following parenchymal but not ventricular transplantation.
One study combined the use of stem cells and growth factors by transplanting human NSCs within a VEGF-releasing microparticle scaffold into the stroke-induced lesion of rats [43, 53]. They observed some indication of increased vascularization within the lesion. Several papers published in TheScientificWorldJournal [54–58] looked at the potential benefits of the angiogenic protein, erythropoietin for the treatment of stroke. These studies included those demonstrating benefit when erythropoietin was administered intranasally or by other routes [54, 55, 58], and Hermann [56] explored possible variants of the molecule that maintain the neuroprotective but not the hematopoietic actions of the molecule. While this compound was not explored at this meeting, it was a topic of interest at previous meetings and could tie in with the proangiogenic properties of VEGF (e.g., [43, 53]).
One clinical study using intracerebral transplantation of autologous peripheral blood stem cells in old stroke patients was also reported [59]. They observed behavioral improvements only when the cells were combined with G-CSF, which can aid the mobilization of the transplanted cells.
Using dog placenta as a source of embryonic-like stem cells, Yu et al. [60] demonstrated increased cell survival of primary rat neurons/astrocytes that were cocultured with these cells after OGD treatment. The authors demonstrated that the beneficial effect may result from an upregulation of heat shock protein 27.
These studies are summarized in Table 3 and show that there are several potential cell transplantation procedures currently being studied that could confer some degree of recovery from a stroke. A number of different cell types have so far been explored, and the optimal type and route of delivery awaits identification. Alternative remedies such as growth factors are also being tested to modify the microenvironment and possibly promote neurogenesis, although combined studies may prove to be the most effective.
4. ALZHEIMER’S DISEASE, AGING, AND COGNITION
Alzheimer’s disease (AD) is the most common neurodegenerative disease observed in man, and it is always a popular topic at the ASNTR meeting. In this group, we are also including aging and cognitive impairment as they can relate to AD. As with PD, this section includes articles on furthering our understanding of the disease, as well as possible treatments such as diet or natural supplements, neurotrophic factors, or cell transplantation for improving cognition or treating the disease.
Mervis et al. [61] studied how cortical neuroplasticity and neurodegeneration change from the suspected precursor to AD, mild cognitive impairment (MCI), to full blown AD. They observed frontal cortical increases compared with parietal and temporal decreases in synaptic plasticity in MCI, whereas all regions were decreased in AD. Spine density was also found to be reduced in both MCI and AD patients.
The use of dietary or natural supplements included epigallocatechin-3-gallate (EGCG), an extract from green tea. This has previously shown promising preclinical results but has limited potential due to the molecule’s poor bioavailability. Using a nanoparticle assembly to stabilize EGCG was shown to potentiate its beneficial effects in vitro and its bioavailability in vivo [62]. In addition, the supplement NT-020, derived from blueberries and other natural products, was shown to promote spatial memory, increase neurogenesis, and decrease inflammation in aged rats, suggesting it could have beneficial effects in treating the cognition-related effects of aging and possible AD [63]. Freeman and Granholm [64] continued their investigations of the effects of a high-fat/high-cholesterol diet and showed that prolonged treatment compromised the BBB and promoted inflammation and hippocampal damage. Dietary effects on AD were also explored in TheScientificWorldJournal where two reports looked at dietary fatty acids [65, 66]. Interestingly, Mohagheghi et al. [67] reported in TheScientificWorldJournal that intake of virgin olive oil appeared to reduce the BBB permeability and other effects observed in rats subjected to ischemia, providing further evidence of dietary impacts on disease and the involvement of the BBB.
There are two major pathological hallmarks of AD, the presence of amyloid plaques, due to the accumulation of β-amyloid (Aβ) and neurofibrillary tangles, due to excess tau phosphorylation and aggregation. Some of the studies of the disease model at the ASNTR meeting focused on one or the other of these pathologies. Borysov et al. [68] report that Aβ inhibits the microtubule-based motors that are crucial for the organization and function of the microtubule cytoskeleton, which could lead to aneuploidy and impaired synaptic activity. Conversely two studies focused on tauopathies exploring the importance of heat shock protein 27 and inflammation in the generation of hyperphosphorylation and aggregation of tau [69, 70]. A review of heat shock proteins was also recently published in TheScientificWorldJournal [71] which explored how they may act to promote the removal of damaged or misfolded proteins (such as hyperphosphorylated tau).
Zhu et al. [72] demonstrated what they believe is an improved AD model; presenilin-amyloid precursor protein (PSAPP) mice which do not express CD45. These mice were found to exhibit greater neurodegeneration as well as higher levels of intra- and extracellular Aβ, but decreased plasma Aβ compared with PSAPP mice expressing CD45.
Brownlow et al. [73] used an AAV to overexpress neprilysin, an enzyme that breaks down amyloid, in the PSAPP mouse and showed that amyloid deposits were reduced. By comparison, Li et al. [74] showed that amyloid deposition in the PSAPP mouse could also be reduced by treating with the neurotrophic factors SCF and G-CSF. The authors also observed an increased presence of microglia around plaques, and they proposed that these factors may promote the targeting of microglia to the plaques which can then clear them. The interaction between microglia and neurons was also explored further by studies looking at the protein fractalkine which is found on neurons and its receptor CX3CR1 which is found on microglia. The absence of the receptor in knockout (KO) mice was shown to reduce hippocampal synaptic plasticity and neurogenesis [75], whereas the different roles of the soluble and membrane-bound forms of fractalkine were explored using AAV vectors [76]. The potential role of microglia and inflammation in aging and AD has been previously reported in TheScientificWorldJournal [77–79]. For instance, Solomon [79] reviewed the current state of immunotherapies such as Aβ antibodies for the treatment of AD discussing their success in animal models and the problems with translating to the clinic, while Brown [78] presented an overview of how subtle age-related changes in microglia could lead to a gradual neuronal loss.
Cognitive impairment has also been observed with sufferers of gulf war illness. A rat model of this disorder, that involves exposing the animals to a cocktail of chemicals similar to those the veterans may have been exposed to, showed an impairment in learning and memory that is related to a prolonged decline in hippocampal neurogenesis [80]. This could be a possible cause of at least some of the neurological symptoms observed.
Unfortunately, cognitive deficits are also frequently a side effect of irradiation therapy for the treatment of brain tumors. Acharya et al. [81] transplanted human embryonic stem cells into rats after head only irradiation and found that the transplanted cells that survived had differentiated into neurons, astrocytes, and oligodendrocytes and improved the cognitive deficits that are frequently seen. The authors did not report whether the cells had resulted in tumors, but further studies could show that this may be a potentially interesting treatment for some of the side effects of irradiation therapy.
Cognitive impairment is also a major characteristic of Down’s syndrome which is caused by the trisomy of chromosome 21 (including the APP gene). Rachubinski et al. [82] investigated whether neonatal transplantation of mouse NPCs into a transgenic mouse model of Down’s syndrome resulted in cognitive improvements in adulthood. Unfortunately, they observed no significant beneficial effects with the tests that they used.
These studies are summarized in Table 4 and show that there are a number of ways that are effective in animal models that can be applied to remove amyloid deposition and try and improve Alzheimer’s disease. However, Bartfai [83] presented an article in TheScientificWorldJournal which discusses how the majority of studies are focused on removing amyloid deposition and how these studies in animals have not translated well to the human condition. This goes hand in hand with the fact that our understanding of the disease is still unfolding, and it would appear that this route (i.e., removal of amyloid deposition) may not be sufficient by itself to treat AD. Further studies are required to determine if these treatments will also affect the tauopathy observed in AD. Some of the data above suggests that progress is being made in our understanding of how we can alter the underlying causes of the pathology, though in all likelihood there is still some way to go before a suitable therapy reaches the clinic. Combined therapies that also target neurogenesis and inflammation are likely to prove to be more beneficial than those that focus exclusively on removal of amyloid deposition. Dietary manipulation and improved animal models of the disease as presented at the meeting are two possible avenues that may prove fruitful in the years to come as our understanding of the disorder progresses.
5. SPINAL CORD INJURY, PAIN, AND AMYOTROPHIC LATERAL SCLEROSIS
Spinal cord injury (SCI) is a frequent side effect of car accidents and other injuries to the back, which frequently target the young and can result in many years of disability. There are no real therapies that effectively cure the injury, but research is ongoing to find potential treatments. A review of the current research for SCI performed in mice was recently published in TheScientificWorldJournal [132] in which the authors discussed a variety of approaches including genetic and molecular engineering and how they could lead to advances in potential methods for repair and regeneration of the injured spinal cord.
For instance, three studies from the same researchers presented at the ASNTR meeting further explored how the location of the injury, that is, white matter versus combined white and grey matter injury, can dictate the degree of neuroplasticity with respect to the respiratory drive that is observed. Lane et al. [84] showed compensatory improvements following a white matter injury, while Mercier et al. [85] demonstrated compensatory contralateral phrenic motor activity following a similar injury. In contrast, Salazar [86] presented data on combined grey and white matter SCI which did not induce significant changes in ventilatory function.
Hentall et al. [92] reported that early electrical stimulation of the raphe nucleus after an SCI speeds up recovery, and they propose that this is likely to result from the action of 5-hydroxytryptamine (5-HT) or other peptides released from the axon terminals in the spinal cord.
One of the biggest problems with spinal cord injury is the formation of a glial scar zone which is not conducive for repair. Therefore, one possible treatment is to provide a scaffold that can offer a more favorable repair environment. Khaing et al. [87] reported on using hyaluronan and laminin hydrogels as scaffolds that can prevent or modify the scar tissue to promote recovery. This scaffold could also be used to support a cell transplant. For instance, Sykova et al. [88] reported on using a hydrogel seeded with MSCs to treat acute and chronic SCI. They found that the transplant would reduce the size of the scar and tissue atrophy (if given acutely), help bridge the lesion, and increase functional activity (though not without the stem cells). This scaffold for regeneration was shown to integrate into the cavity and also be infiltrated by blood vessels and axons.
Busch et al. [96] transplanted rat multipotent adult progenitor cells at the time of injury and found that these cells reduced axonal dieback and promoted neurite outgrowth in a rat model of SCI and this may result from factors secreted by the cells. By comparison, Yu et al. [89] transplanted human neural stem cells within 3 days of SCI and observed restoration of respiratory function, as well as showing evidence of tissue reconstitution and neurite reorganization.
One of the symptoms of spinal cord injury can be severe pain, and there is currently an FDA-approved drug available which is a synthetic version of a conotoxin. One study demonstrated that gene therapy could be used to consistently transduce expression of conopeptides within the spinal cord [93]. However, no information on their antinociceptive activity was provided. Lee et al. [94] using a similar adenovirus-conotoxin construct, which they injected into the left sciatic nerve of rats and mice, demonstrated a reduced response to formalin injection, suggesting that conotoxin gene therapy could be useful for treating peripheral neuropathic pain.
Jergova et al. [90] assessed whether transplantation of a gamma aminobutyric acid (GABA)ergic neural progenitor cell could help alleviate peripheral neuropathic pain in a rodent model. They observed decreased hyperalgesia and allodynia and demonstrated that the antinociceptive effect was GABA mediated.
A recent report in TheScientificWorldJournal [133] focuses on the recently announced termination of many studies by a number of major pharmaceutical companies as they streamline their research. One of the avenues of research that was apparently dropped is the development of drugs for neuropathic pain, which is unfortunately considering the studies mentioned above and their potential promise.
Amyotrophic lateral sclerosis (ALS) results from the progressive loss of spinal motor neurons by an as yet unknown mechanism. Two studies reported on cell transplants for this disorder. Human umbilical cord blood cells were administered intravenously into the G93A ALS mouse model and were observed to delay disease onset and increase lifespan. Immunohistochemical analysis revealed a reduced presence of activated microglia supporting an anti-inflammatory benefit for these cells [95]. The second study reported on the start of a Phase I clinical trial using NeuralStem Inc.’s human fetal spinal cord-derived NSCs for the treatment of this disease [91]. This trial will include immunosuppression and initially involve lumbar transplants, which once proven safe will progress to cervical transplants. The animal studies this trial is based on showed that these cells were capable of forming synaptic connections and secreting a number of neurotrophic factors that could facilitate recovery.
There is some promising research for the treatment of SCIs and pain that could prove to be effective, and the studies from the ASNTR meeting are summarized in Table 5. Combined treatments such as scaffold and cells may turn out to be the most effective as they have potential in promoting the restoration of connectivity across the glial scar.
6. HUNTINGTON’S DISEASE
Huntington’s disease (HD) is an autosomal dominantly inherited progressive neurodegenerative disorder that involves severe brain atrophy and deficits in motor ability and cognition. The cause of the disease is multiple glutamine repeats in the huntingtin protein, and there are several animal models available. At the ASNTR meeting, one study provided a detailed analysis of the CAG140 knockin mouse model, that has 140 glutamine repeats [99]. They observed an increasing incidence of neuronal intranuclear inclusions over time as well as a progressive decrease in cortical thickness. A second presentation studied the R6/2 transgenic mouse model of HD, which expresses exon 1 of the human mutant huntingtin gene, providing a detailed analysis of the functional and neuropathological changes that occur over time [100]. Motor deficits developed between 4 and 8 weeks of age, and magnetic resonance imaging (MRI) analysis was used to correlate any changes in regional brain volume.
Two additional studies using the R6/2 mouse model involved cell transplantation as a possible treatment. El-Akabawy et al. [103] transplanted human striatal NSCs directly into the striatum and found no improvement of motor deficits, but did observe an improvement in the swimming T-maze. This suggests that with some refinement of the technique, this treatment may prove to be useful in treating HD. By comparison, Rossignol et al. [104] transplanted low-and-high passage MSCs into the striatum and observed greater behavioral improvement with the lower passage than the higher passage cells supporting the idea that “younger” cells may be better. A possible mode of action may be their ability to secrete growth factors such as brain-derived growth factor (BDNF). Tebano et al. [134] published a manuscript in TheScientificWorldJournal that discussed how adenosine receptors and BDNF levels could contribute to the pathogenesis of HD since an impairment of both appears to be involved. A normal level of adenosine receptor activation is required for the maintenance of BDNF levels, and so reduced adenosine signaling will also lead to reduced BDNF.
These studies are summarized in Table 6 and show that cell transplantation could be beneficial as a treatment for HD, based on their effects in the R6/2 model of HD. This model exhibits a number of the characteristics of HD, making it a potentially effective model.
7. OTHER NEUROLOGICAL DISORDERS
Traumatic brain injury (TBI) is a frequent consequence of war time that results in immediate extensive brain damage and severe neurological impairment. Recent studies suggest that there may also be a secondary progressive damage which maybe easier to treat. Shojo et al. [106] using the moderate fluid percussion model of TBI demonstrated a rapid increase in interleukin-1 (IL-1) and tumor necrosis factor (TNF) which peaked three hrs after injury. Apoptosis was seen to progressively increase from 3 hrs onwards to plateau at 48 hrs by which time IL-1 and TNF had returned to normal levels. Using the same model, another group showed that early intermittent stimulation of the rat dorsal or median raphe for a week restored some of the cognitive and motor deficits caused by TBI [105].
The symptoms of temporal lobe epilepsy (TLE) consist of spontaneous recurrent motor seizures (SRMS) and cognitive deficits and can be induced in rats by the use of graded intraperitoneal injections of kainic acid. Grier et al. [97] demonstrated that there is significant loss of reelin+ interneurons that correlates with impaired learning and memory, but not with SRMS frequency.
Sanfilippo type B is a progressive disorder that results in the accumulation of cerebral and systemic organ abnormalities due to glycosaminoglycan accumulation within cells as a consequence of a deficiency in the catabolic enzyme, alpha-N-acetylglucosaminidase. Impairment of the BBB has been shown in a number of disorders (see the previously referred to reports by Carvey et al., Freeman and Granholm at ASNTR [5, 64] and Mohagheghi et al., Thal in TheScientificWorldJournal [67, 135]. Thal proposes that altered drainage of extracellular fluid due to impairments in the BBB competes with perivascular drainage and could result in the accumulation of Aβ and other proteins within the brain). Garbuzova-Davis et al. [98] established that BBB impairment can also be seen in animal models of Sanfilippo type B. This shows that deficiencies in the BBB may have an important part to play in many diseases and disorders, and therefore ways to repair the BBB could prove to be therapeutic.
Many brain disorders could potentially be treated by increased neurogenesis, but in many cases this has been reported to be reduced in the disease state. Song et al. [101] demonstrated that stereotaxic microlesions caused by a microneedle into specific brain regions could induce new neurons and glia in the damaged nonneurogenic regions, by the migration of NSCs or even bone-marrow-derived stem cells. Elucidation of the pathways which trigger neurogenesis in response to microlesions could be valuable in the development of neurodegenerative disease therapies, and, according to the authors, may also provide a mode of action for deep brain stimulation. Recent evidence suggests that neurogenesis may also affect other processes since sexual activity was elevated in male rats that had been treated with a drug that has been shown to promote new neuron formation [102]. This demonstrates that control of neurogenesis not only may be useful for the repair of neurodegenerative disorders but also could impact more social disorders such as sexual dysfunction.
These studies are summarized in Table 6 and show that there are a number of different models available for the study of disease which can be used to investigate the effects of transplantation and treatments such as neuronal stimulation and the role of growth factors is again important.
8. DISEASE BIOMARKERS AND GENOME ANALYSIS
Many neurodegenerative disorders involve the progressive loss of specific areas of the central nervous system (CNS). Frequently, by the time symptoms are observed, the damage is substantial, so the search for biomarkers that could indicate the likelihood to develop a disease is a potentially important weapon in treating these disorders early while the damage is not too severe. Mhyre et al. [107] reported on a pilot study to see if biomolecular profiling of peripheral leukocytes could be used to distinguish between different forms of dementia. They observed disease-specific alterations in gene and protein expression, which on repetition in larger studies could lead to specific biomarkers for dementia-related disorders. Lanari and Parnetti [136] report in TheScientificWorldJournal on the presence of biomarkers in MCI patients that could allow one to predict whether they will progress to AD. They observed that baseline CSF levels of Aβ42, total tau [T-tau], and phosphorylated tau [P-tau] were altered in MCI patients who progressed to exhibit dementia over a three-year period. An additional paper by Farnaud et al. [137] reviewed whether saliva could be an effective source of biomarkers for cancers and other disorders.
Different clustering methods of nuclear magnetic resonance (NMR) were shown to be able to distinguish between R6/2 HD mice of different age groups and wild-type mice with 100% accuracy [108]. The authors believed that this technique could be refined further to be able to distinguish between other disorders also.
The presidential symposium proposed the question of paleoneurology, the concept that neurodegenerative disorders are evolutionary new as a consequence of the development of the relatively recent complexity of the brain [112]. The high plasticity of the brain may be a sign of rapid evolution. Hardy [113] discussed how whole genome analysis has now progressed to the point where genetic risk factors for numerous diseases are being identified. The microtubule-associated protein tau has been shown to be a risk loci for a number of disorders. Its evolutionary history and unusual European loci structure may suggest that its disease association could relate to changes in the gene expression of different haplotypes.
Lineage mapping of glioblastomas suggested that the cells which comprise a glioma are polyclonal nonhierarchical neural stem cells in origin [114]. These cells were found to be steered towards inappropriate gliogenesis rather than a neuronal fate, which was shown to be reversible.
These studies are summarized in Table 7 and show that current advances in genetic analysis and biomolecular profiling are leading to the identification of potential biomarkers for disease, which need to be evaluated further to determine their specificity.
9. ANIMAL MODELS OF DISEASE
New animal models of disease are being generated by altered expression of specific genes. For instance, knockout of the inducible form of nitric oxide synthase (NOS2) results in an animal model that exhibits excess neuroinflammation, which could lead to elucidation of the role of NOS2 in inflammatory and neurodegenerative disorders [115]. The role of inflammation in neurodegeneration has been directly explored at previous ASNTR meetings as well as a number of reports presented herein (e.g., [6, 9, 63, 64, 70, 115, 131]). The topic of inflammation has also been heavily featured in TheScientificWorldJournal (e.g., [77, 138–141]) with Catania et al. [138] exploring the possible role of melanocortins in inflammation. Claria et al. [140] and Bannenberg [77] discussed the role of lipid mediators, while Díez-Dacal and Pérez-Sala [141] expanded on the contribution of cyclopentanone prostaglandins. El-Kebir and Filep [139] studied ways to terminate inflammation, such as the use of cyclin-dependent kinase inhibitors to trigger the apoptosis of neutrophils and thus remove their contribution to the inflammatory response.
Use of a viral vector to express melanopsin, a light-sensitive photopigment, in the surviving ganglions of mice with deficient photoreceptors (rd/rd) was found to restore some of the visual function [111].
A Down’s syndrome mouse model (TS65Dn), which exhibits some of the neuropathological hallmarks of AD over time, can be used to investigate the development of these features. For instance, this model develops loss of the basal forebrain cholinergic neurons (BFCNs). This may result from reduced levels of nerve growth factor (NGF) binding to their receptors and thus decreased survival signals (retrograde transport of the NGF/receptor complex), leading to cell death [109].
One of the less common symptoms of PD includes nonmotor symptoms including neurodegeneration of the autonomic nervous system. The resulting dysautonomias have not been well modeled. Joers et al. [110] proposed using systemic 6-OHDA to induce cardiac dysautonomia in primates to generate a model that could be used to investigate potential treatments.
New animal models are summarized in Table 7 and can be used to mimic specific aspects of a disease such as dysautonomia or excessive inflammation to help reveal new treatments for symptoms that result from the disorder and provide further information on the possible causes of these “side effects” in the larger picture of the disease or disorder.
10. STEM CELL CHARACTERIZATION AND VIRAL VECTORS
As well as studying specific diseases, ASNTR also focuses on the development and characterization of stem cells for future applications as possible treatments. For instance, elucidation of the pathways involved in the differentiation of stem cells to a specific phenotype is of utmost importance. El-Akabawy et al. [116] reported on the use of purmorphamine, a sonic hedgehog signaling molecule to produce more neurons that are DARPP-32+ (dopamine and cyclic AMP-regulated phosphoprotein 32 KDa) from a human striatal neural cell line in preference to astrocytes. These cells can be potentially useful for the treatment of HD, since DARPP-32+ neurons are the cell type predominantly lost in HD. Gene delivery by retrovirus of specific lineage instruction factors (e.g., dominant-negative Olig2 to suppress glial fate, or Pax6 to promote the neuronal phenotype) into NSCs was also shown to result in differentiation to specific neuronal phenotypes by Klempin et al. [126].
Nash et al. [127] provided the proof of principle that recombinant AAV (rAAV) could be used to insert transgenes into murine NSCs with serotype 1 being most effective and serotype 5 the least. This was expanded on by the work of Manfredsson et al. [129] who showed that use of an rAAV type 2 with mutated tyrosine residues (to reduce ubiquitination and hence clearance of the plasmid from the body) promoted the transduction of specific genes into cells within the striatum of rats. Using what Nash et al. [127] reported was the least effective serotype, Nan et al. [130] successfully transduced green fluorescent protein using rAAV5 into the brains of mice following either a neonatal or adult ventricular injection that persisted for at least 4 months. Neonatal delivery was shown to be more effective. These three studies demonstrate the ability to insert specific genes into neural cells either in vivo or in culture.
Plasmid transduction can also result in immortalization. Using either a respiratory syncytial virus or elongation factor 1 promotor, the N-terminal SV40 large T antigen fragment was transfected into rat primary mesencephalic cultures to induce immortalization [128]. Interestingly, only partial incorporation of the plasmid was observed, suggesting that immortalization may have been spontaneous, when linearized plasmids are used.
Solanky et al. [119] reported on the use of magnetic resonance spectroscopy of in vitro cell extracts to distinguish between the more metabolically active undifferentiated NSCs and differentiated cells. This could prove to be useful in tracking transplanted cells and following their fate if it can be translated to an in vivo setting.
Song et al. [118, 120] presented two studies on UCB-derived MSCs. In the first, they used trophogel (a placental-derived matrix) as a substrate for the culturing of the cells and demonstrated that this helped maintain the adhesion and proliferative capacity of these cells [118]. Their ability to differentiate into bone and fat cells was also studied and found to be increased while growing on this substrate. In their second study, they reported on a scoring system to distinguish between active and senescent UCB-MSCs [120]. The scoring system looks at multilineage differentiations, growth rates, morphologies, passages, immunophenotypic analysis, free radical production, and senescence-associated gene expression. Cells that score highly on this scale are more likely to possess regenerative properties, instead of being senescent.
These studies are summarized in Table 8 and show some of the ways that stem cells can be manipulated to optimize their therapeutic potential. While the use of viral vectors are unlikely to reach the clinic (unless the viral component can be excised), they could still be a valuable tool for determining and directing the differentiation of stem cells. Methods for the sorting or tracking of a specific cell population are also important concerns when considering the use of cells.
11. TRANSPLANTATION AND MIGRATION
In the clinical setting, it is likely that stem cells will eventually be transplanted for a number of neurodegenerative disorders, though animal studies are required to confirm safety and mode of action. This section will cover the presentations which study different aspects of this process such as the need for immunosuppression and the targeting of the cells to the site of interest and integration.
Bjugstad and Rachubinski [122] followed up on previous studies of neonatal transplantation of mouse NSCs into a trisomic mouse model of Down’s syndrome. Early data had suggested some benefit, but, at 12 months, the cell survival was negligible and little behavioral improvement compared with saline-treated animals was observed in contrast to studies when the cells were transplanted into adults. Interestingly, the authors found that transplantation (of either saline or cells) in normal mice may have caused some behavioral changes, suggesting that this should be carefully considered when deciding what controls should be performed.
Continuing with neonatal transplants, Dunnett et al. [123] found that transplantation of human progenitor cells during the first postnatal week of mice and rats, at a point of immune system immaturity, allowed subsequent transplantations in adulthood without the need for immunosuppression. This “neonatal tolerization” led to the grafted cells surviving for up to 40 weeks, whereas they were fully rejected within 5 weeks if tolerization was not performed. While this observation does not really advocate performing cell transplantations on newborns “just in case”, understanding the mechanisms behind this tolerance could prove very important in future transplantation therapies for maximizing the effectiveness of the cells, though paradoxically this would currently negate FDA approval for a study, as they are concerned about cell survival and the potential for tumorgenicity or other effects.
In several studies, the beneficial effects of cell transplantation do not appear to result from integration but rather the release of growth factors. However, when integration does occur, it is unclear exactly how this would proceed. Studying NSC transplantation into organotypic slice cultures, it has been shown that one of the first steps towards integration is the development of gap junctions between the transplanted and host cells [121]. This was observed to occur before the differentiation of the transplanted cells to electrophysiologically active neurons. Gap junction formation allows the transplanted cells to provide survival signals and synchronization of calcium currents, protecting the host cells from death both in culture and in murine models of purkinje cell degeneration. Prevention of gap junction formation was found to enhance cell death and prevent the behavioral recovery previously seen following cell transplantation.
Transplantation of hUCB-derived neurally-induced stem cells into a mouse model of glutaric aciduria type I (an autosomal recessive human disorder that results in the accumulation of organic acids) was shown to extend the lifespan and promote motor recovery of these mice [124]. The mode of action of this improvement is unclear and requires further elucidation. hUCBC transplants had previously been shown to be effective for another metabolic disorder sanfilippo type B, and it will be interesting to see whether their mode of action is similar once the mechanisms for both have been determined.
Many disorders involve increased inflammation, and this could act as a homing signal for transplanted cells. This was demonstrated in normal mice that had a cocktail of factors that mimic a classical pro-inflammatory response injected into their hippocampus followed two days later by an intracardiac injection of green-fluorescent-protein-(GFP-) labeled monocytes. After 24 hrs, cells were found within the brain to a larger extent than in animals not given the inflammatory cocktail [131].
Endogenous stem cell production and migration is another important potential source of cells that could be tapped for therapies. It is known that NSCs migrate from the subventricular zone towards the olfactory bulb along a pathway known as the rostral migratory stream. To investigate the origin and effects of endogenous stem cells, this pathway needs to be identified in a number of species to provide a more detailed picture of how the pathway could change with increased species complexity. Malik et al. [117] have identified its presence within cats and dogs, thus allowing for the characterization of the rostral migratory stream and endogenous NSCs in different species from the more traditional rodent. This could prove useful in the development of therapies for man as well as in cats and dogs.
Treatments for inner ear disorders need to be able to reach the inner ear without causing damage to the surrounding tissue. Syka et al. [125] demonstrated that a variety of different nanoparticles could traverse the round window membrane to allow entry into the cochlea within 24 hrs without causing any apparent damage or hearing impairment. This suggests that these nanoparticles could be used as transporters of drugs or other factors into the inner ear for the potential treatment of hearing or balance disorders.
These studies are summarized in Table 8.
12. CONCLUSIONS
The 2010 ASNTR meeting provides a window into the emerging avenues of research in regenerative medicine for the treatment of the central and peripheral nervous system.
The valuable use of models to characterize specific aspects of or provide further understanding of a pathogenesis of a disease is emphasized along with the potential use of stem cell transplantation as a therapy. Specific growth factors play an important role in the etiology of many disorders and could be utilized in treatments. One of the primary modes of action of stem cells is believed to be as a result of the secretion of these factors to modify the microenvironment to make it more conducive to the survival of endogenous cells (or the transplanted cells).
While it is true that the long-term contribution of these studies may prove to be minor, the path from bench to bedside is normally a long one with considerable knowledge of the mechanisms behind the therapy being necessary as well as the performance of extensive safety studies before the process is complete. This means that it is currently unclear which of these studies will eventually result in a therapy. Though different diseases frequently possess distinct anatomical and pathological characteristics, it is clear from some of the studies reported here that overlapping themes can be observed such as the contribution of inflammation or the impairment of the BBB to a number of diseases or disorders. This means that a therapy that will influence mutual characteristics of a disease such as BBB impairment could provide some degree of benefit to multiple disorders. Therapies for different disorders are going to need to provide variable improvements and ideally would function at any stage of a disorder. Whatever the outcome, the studies do help in piecing together likely future therapies which in all likelihood are likely to be multifactorial, working in a number of ways to combat or at least halt the disease process. These studies encompass methods to promote growth factor activity which can provide support to impaired cells, possibly by altering the hostile environment by reducing inflammation, as well as potentially enhancing the endogenous production of replacement cells. Alternatively transplanted cells could also integrate, though many studies suggest that this is not the case. Of particular note is that some of these reported studies do focus on therapies that are currently undergoing clinical trials (e.g. NeuralStem Inc.’s neural stem cells for SCI), while others are studies that help to explain the disease state better, a necessary step towards therapy. Many of the studies reported at the ASNTR are expanded further in future papers as exemplified by the overlap between topics at this year’s (and especially previous) meetings compared to those published in TheScientificWorldJournal. In this way, we feel that an overview of the topics presented at the ASNTR meeting can provide insight into the current progress of the field of regenerative medicine.