Pluripotent Stem Cells Models for Huntington＇s Disease： Prospects and Challenges

Pluripotent cellular models have shown great promise in the study of a number of neurological disorders.Several advantages of using a stem cell model include the potential for cells to derive disease relevant neuronal cell types,providing a system for researchers to monitor disease progression during neurogenesis,along with serving as a platform for drug discovery.A number of stem cell derived models have been employed to establish in vitro research models of Huntington’s disease that can be used to investigate cellular pathology and screen for drug and cell-based therapies.Although some progress has been made,there are a number of challenges and limitations that must be overcome before the true potential of this research strategy is achieved.In this article we review current stem cell models that have been reported,as well as discuss the issues that impair these studies.We also highlight the prospective application of Huntington’s disease stem cell models in the development of novel therapeutic strategies and advancement of personalized medicine.     

Exosomes in Mesenchymal Stem Cells,a New Therapeutic Strategy for Cardiovascular Diseases?

Cardiovascular diseases (CVDs) are still a major cause of people deaths worldwide, and mesenchymal stem cells (MSCs) transplantation holds great promise due to its capacity to differentiate into cardiovascular cells and secrete protective cytokines, which presents an important mechanism of MSCs therapy for CVDs. Although the capability of MSCs to differentiate into cardiomyocytes (CMCs), endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) has been well recognized in massive previous experiments both in vitro and in vivo, low survival rate of transplanted MSCs in recipient hearts suggests that therapeutic effects of MSCs transplantation might be also correlated with other underlying mechanisms. Notably, recent studies uncovered that MSCs were able to secret cholesterol-rich, phospholipid exosomes which were enriched with microRNAs (miRNAs). The released exosomes from MSCs acted on hearts and vessels, and then exerted anti-apoptosis, cardiac regeneration, anti-cardiac remodeling, anti-inflammatory effects, neovascularization and anti-vascular remodeling, which are considered as novel molecular mechanisms of therapeutic potential of MSCs transplantation. Here we summarized recent advances about the role of exosomes in MSCs therapy for CVDs, and discussed exosomes as a novel approach in the treatment of CVDs in the future.     

β-Arrestins as Potential Therapeutic Targets for Alzheimer’s Disease

β-arrestins represent a small family of G protein-coupled receptors (GPCRs) regulators, which provide modulating effects by facilitating desensitization and internalization of GPCRs as well as initiating their own signalings. Recent reports have demonstrated that β-arrestins levels were correlated with amyloid-β peptide (Aβ) pathology in brains of Alzheimer’s disease (AD) patients and animal models. β-arrestins could enhance the activity of γ-secretase via interacting with anterior pharynx defective 1 subunit, which increased Aβ production and contributed to the pathogenesis of AD. In addition, Aβ-induced internalization of β2-adrenergic receptor internalization and loss of dendritic spine in neurons were proven to be mediated by β-arrestins, further establishing their pathogenic role in AD. More importantly, deletion of β-arrestins markedly attenuated AD pathology, without causing any gross abnormality. Here, we review the evidence about the roles of β-arrestins in the progression of AD. In addition, the established and postulated mechanisms by which β-arrestins mediated in AD pathogenesis are also discussed. Based on the role of β-arrestins in AD pathogenesis, genetically or pharmacologically targeting β-arrestins might provide new opportunities for AD treatment.     

Therapeutic Strategies for Alzheimer’s Disease

Therapeutic approaches for Alzheimer's disease (AD) are guided by four disease characteristics: amyloid plaques, neurofibrillar tangles (NFT), neurodegeneration, and dementia. Amyloid plaques are composed largely of 4 kDa beta-amyloid (Abeta) peptides, with the more amyloidogenic, 42 amino acid form (Abeta42) as the primary species. Because multiple, rare mutations that cause early-onset, familial AD lead to increased production or aggregation of Abeta42, amyloid therapeutics aim to reduce the amount of toxic Abeta42 aggregates. Amyloid-based therapies include gamma-secretase inhibitors and modulators, BACE inhibitors, aggregation blockers, catabolism inducers, and anti-Abeta biologics. Tangles are composed of paired helical filaments of hyperphosphorylated tau protein. Tau-based therapeutics include kinase inhibitors, microtubule stabilizers, and catabolism inducers. Therapeutic strategies for neurodegeneration target multiple mechanisms, including excitotoxicity, mitochondrial dysfunction, oxidative damage, and inflammation or stimulation of neuronal viability. Although not disease modifying, cognition enhancers are important to treat the symptom of dementia. Strategies for cognition enhancement include cholinesterase inhibitors, and other approaches to enhance the signaling of cholinergic and glutamatergic neurons. In summary, plaques, tangles, neurodegeneration and dementia guide the development of multiple therapeutic approaches for AD and are the subject of this review.     

Hypoxia Promotes Dopaminergic Differentiation of Mesenchymal Stem Cells and Shows Benefits for Transplantation in a Rat Model of Parkinson’s Disease

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into dopaminergic (DAergic) neurons, which is one of the major cell types damaged in Parkinson’s disease (PD). For this reason, MSCs are considered a potential cell source for PD therapy. It has been proved that hypoxia is involved in the proliferation and differentiation of stem cells. In this study, we investigated the effect of hypoxia on MSC proliferation and DAergic neuronal differentiation. Our results demonstrate that 3% O₂ treatment can enhance rat MSC proliferation by upregulation of phosphorylated p38 MAPK and subsequent nuclear translocation of hypoxia inducible factor (HIF)-1α. During neural differentiation, 3% O₂ treatment increases the expression of HIF-1α, phosphorylated ERK and p38 MAPK. These changes are followed by promotion of neurosphere formation and further DAergic neuronal differentiation. Furthermore, we explored the physiological function of hypoxia-induced DAergic neurons from human fetal MSCs by transplanting them into parkinsonian rats. Grafts induced with hypoxia display more survival of DAergic neurons and greater amelioration of behavioral impairments. Altogether, these results suggest that hypoxia can promote MSC proliferation and DAergic neuronal differentiation, and benefit for intrastriatal transplantation. Therefore, this study may provide new perspectives in application of MSCs to clinical PD therapy.     

Pluripotent Stem Cells for Modelling and Cell Therapy of Parkinson’s Disease

Studying pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD), requires adequate disease models. The available patient’s material is limited to biological fluids and post mortem brain samples. Disease modeling and drug screening can be done in animal models, although this approach has its own limitations, since laboratory animals do not suffer from many neurodegenerative diseases, including PD. The use of neurons obtained by targeted differentiation from induced pluripotent stem cells (iPSCs) with known genetic mutations, as well as from carriers of sporadic forms of the disease, will allow to elucidate new components of the molecular mechanisms of neurodegeneration. Such neuronal cultures can also serve as unique models for testing neuroprotective compounds and monitoring neurodegenerative changes against a background of various therapeutic interventions. In the future, dopaminergic neurons differentiated from iPSCs can be used for cell therapy of PD.     

The Therapeutic Effects of Tyrosine Hydroxylase Gene Transfected Hematopoetic Stem Cells in a Rat Model of Parkinson’s Disease

Aims To investigate the therapeutic effects of tyrosine hydroxylase (TH)-transfected neuronal stem cells derived from bone marrow stem cells (NdSCs-D-BMSCs) on Parkinson’s disease (PD) through different transplantation protocols, including microinjection into the cerebral ventricles (CV) and the striatum (ST). Methods After identification by enzyme digestion, the constructed plasmid pEGFP-C2-TH was transfected into 8-day-cultured NdSCs-D-BMSCs by electroporation resulting in the coexpression of green fluorescent protein (GFP) and TH. The TH-transfected cells were injected into either the right ST or CV of PD rats. The changes in locomotor activity of PD rats and the migration of transplanted cells in cerebral tissue were monitored and cerebral DA levels were assayed by high performance liquid chromatography (HPLC). Results Five days after plasmid pEGFP-C2-TH transfection into NdSCs-D-BMSCs GFP was expressed in 62.1% of the cells and the rate of co-expression with TH was 83.5%. Ten weeks following transplantation, the symptoms of PD rats in both groups were significantly improved and DA levels were restored to 46.6% and 33% of control. The transferred cells showed excellent survival rates in PD rat brains and distant migration was observed. Conclusion Both CV and ST transplantation of TH-transfected NDSCs-D-BMSCs has obvious therapeutic effects on PD rats. This study could provide evidence for future transplantation route selection, possibly leading to stem cell transplantation through lumbar puncture. Grant: National natural science grant (30270491), Outstanding Science-technology program of Guangdong Province (2000)25.     

The Preventive and Therapeutic Effects of Intravenous Human Adipose-Derived Stem Cells in Alzheimer’s Disease Mice

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid plaques and neurofibrillary tangles accompanied by cognitive dysfunction. The aim of the present study was to elucidate preventive and therapeutic potential of stem cells for AD. Among stem cells, autologous human adipose-derived stem cells (hASCs) elicit no immune rejection responses, tumorigenesis, or ethical problems. We found that intravenously transplanted hASCs passed through the BBB and migrated into the brain. The learning, memory and pathology in an AD mouse model (Tg2576) mice greatly improved for at least 4 months after intravenous injection of hASC. The number of amyloid plaques and Aβ levels decreased significantly in the brains of hASC-injected Tg mice compared to those of Tg-sham mice. Here, we first report that intravenously or intracerebrally transplanted hASCs significantly rescues memory deficit and neuropathology, in the brains of Tg mice by up-regulating IL-10 and VEGF and be a possible use for the prevention and treatment of AD.     

Modelling Sporadic Alzheimer’s Disease Using Induced Pluripotent Stem Cells

Developing cellular models of sporadic Alzheimer’s disease (sAD) is challenging due to the unknown initiator of disease onset and the slow disease progression that takes many years to develop in vivo. The use of human induced pluripotent stem cells (iPSCs) has revolutionised the opportunities to model AD pathology, investigate disease mechanisms and screen potential drugs. The majority of this work has, however, used cells derived from patients with familial AD (fAD) where specific genetic mutations drive disease onset. While these provide excellent models to investigate the downstream pathways involved in neuronal toxicity and ultimately neuronal death that leads to AD, they provide little insight into the causes and mechanisms driving the development of sAD. In this review we compare the data obtained from fAD and sAD iPSC-derived cell lines, identify the inconsistencies that exist in sAD models and highlight the potential role of Aβ clearance mechanisms, a relatively under-investigated area in iPSC-derived models, in the study of AD. We discuss the development of more physiologically relevant models using co-culture and three-dimensional culture of iPSC-derived neurons with glial cells. Finally, we evaluate whether we can develop better, more consistent models for sAD research using genetic stratification of iPSCs and identification of genetic and environmental risk factors that could be used to initiate disease onset for modelling sAD. These considerations provide exciting opportunities to develop more relevant iPSC models of sAD which can help drive our understanding of disease mechanisms and identify new therapeutic targets.     

Deferoxamine Preconditioning of Neural-Like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells as a Strategy to Promote Their Tolerance and Therapeutic Potential: An In Vitro Study

Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton’s jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H₂O₂). HWJ-MSCs were differentiated toward neural-like cells for 14 days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100 µM DFO, as a known hypoxia mimetic agent for 48 h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H₂O₂ to DFO-treated cells resulted in higher resistance to H₂O₂-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.     

Targeting Glial Cells to Elucidate the Pathogenesis of Huntington’s Disease

Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by expended CAG repeats in the Huntingtin (Htt) gene. The resultant mutant Htt (mHtt) forms aggregates in neurons and causes neuronal dysfunctions. The major characteristic of HD is the selective loss of neurons in the striatum and cortex, which leads to movement disorders, dementia, and eventual death. Expression of mHtt was also found in non-neuronal cells in the brain, suggesting non-cell-autonomous neurotoxicity in HD. As was documented in many different neurodegenerative disorders, elevated inflammatory responses are also reported in HD. To date, effective treatments for this devastating disease remain to be developed. This review focuses on the importance of glial cells and inflammation in HD pathogenesis. Potential anti-inflammatory interventions for HD are also discussed.     

CCR7 Expressing Mesenchymal Stem Cells Potently Inhibit Graft-versus-Host Disease by Spoiling the Fourth Supplemental Billingham’s Tenet

The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham’s tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.     

Neuroprotective strategies for NMDAR-mediated excitotoxicity in Huntington’s Disease

BACKGROUND

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease causing severe neurodegeneration of the striatum as well as marked cognitive and motor disabilities. Excitotoxicity, caused by overstimulation of NMDA receptors (NMDARs) has been shown to have a key role in the neuropathogenesis of HD, suggesting that targeting NMDAR-dependent signaling may be an effective clinical approach for HD. However, broad NMDAR antagonists are generally poor therapeutics in clinical practice. It has been suggested that GluN2A-containing, synaptically located NMDARs activate cell survival signaling pathways, while GluN2B-containing, primarily extrasynaptic NMDARs trigger cell death signaling. A better approach to development of effective therapeutics for HD may be to target, specifically, the cell-death specific pathways associated with extrasynaptic GluN2B NMDAR activation, while maintaining or potentiating the cellsurvival activity of GluN2A-NMDARs.

OBJECTIVE

This review outlines the role of NMDAR-mediated excitotoxicity in HD and overviews current efforts to develop better therapeutics for HD where NMDAR excitotoxicity is the target.

METHODS

A systematic review process was conducted using the PubMed search engine focusing on research conducted in the past 5-10 years. 235 articles were consulted for the review, with key search terms including “Huntington’s Disease,” “excitotoxicity,” “NMDAR” and “therapeutics.”

RESULTS

A wide range of NMDAR excitotoxicity-based targets for HD were identified and reviewed, including targeting NMDARs directly by blocking GluN2B, extrasynaptic NMDARs and/or potentiating GluN2A, synaptic NMDARs, targeting glutamate release or uptake, or targeting specific downstream cell-death signaling of NMDARs.

CONCLUSION

The current review identifies NMDAR-mediated excitotoxicity as a key player in HD pathogenesis and points to various excitotoxicity-focused targets as potential future preventative therapeutics for HD.

     

The Effect of Lentivirus-Mediated PSPN Genetic Engineering Bone Marrow Mesenchymal Stem Cells on Parkinson’s Disease Rat Model

Persephin (PSPN) is one of the neurotrophic factors of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) which have been found to promote the survival of specific populations of neurons. The aim of this study was to assess the potential therapeutic function of gene-modified mesenchymal stem cells (MSCs)-Lv-PSPN-MSCs in 6-OHDA-induced Parkinson’s disease (PD) rats models. Here, we worked on the isolation, purification, identification and amplification of MSCs in vitro. The expression analysis revealed that several of the neural marker proteins like nestin, GFAP and S100 were expressed by rat MSCs. MES23.5 cells co-cultured with Lv-PSPN-MSCs showed less 6-OHDA induced cell death than control cells in vitro. When Lv-PSPN-MSCs were injected into the striatum of PD rats, we observed the survival rate, migration, differentiation and the behavior change of PD rats. We found that Lv-PSPN-MSCs showed higher survival rate in rat brain compared with Lv-null-MSCs. Rotational behavior showed that rats receiving Lv-PSPN-MSCs showed the most significant improvement compared with those in other groups. HPLC results showed the content of DA in striatum of rats which received Lv-PSPN-MSCs was highest compared with those in other groups. In conclusion, our results suggest that transplantation of Lv-PSPN-MSCs can lead to remarkable therapeutic effects in PD rats.     

Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.