Cell Therapy For Neurodegenerative Diseases Research Articles

Role of Neural Stem Cells in Parkinsons Disease

Human fetal mesencephalic tissue can be successfully used in dopamine (DA) cell replacement therapy in Parkinsons disease (PD), nevertheless, further advances have been impeded due to limited tissue availability. Stem cell-based approaches have received much hype as potential treatments for neurodegenerative disorders, but have shown poor cell survival. The brains of three patients with Parkinsons disease who had undergone transplants of fetal tissue were examined postmortem and it was observed that some transplanted cells contained clumps of protein containing alpha-synuclein. Inducible pluripotent stem (iPS) cells derived from somatic cells and human embryonic stem cells, represent a novel renewable source of tissue precursors and therapeutic safety necessitate much more in-depth research before the initiation of human clinical trials. Keywords: Parkinson's disease, neural stem cells, replacement therapy, induction of pluripotent stem cells, Stem Cells, therapy in Parkinson's disease, neurodegenerative disorders, Inducible pluripotent stem (iPS) cells, neural transplantation, somatic cells and human embryonic stem cells, embryonic stem cells, Stem Cell, –, Based Regeneration, Human ES cell-derived dopaminergic neurons, cell therapy in neurodegenerative diseases, neural stem cells in Parkinson's disease, Gene therapy, Regeneration-based therapies, embryonic germ cells, epigenetic reprogramming, Stem Cell Research, stem cell-specific expertise, human embryonic pluripotent stem cells, adult stem cells, stem cell-based therapies, progenitor cell transplants, Transplanted dopaminergic

Protection of murine neural progenitor cells by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin in the low nanomolar concentration range

Stem cell-based approaches provide hope as a potential therapy for neurodegenerative diseases and stroke. One of the major scientific hurdles for stem cell therapy is the poor survival rate of the newly formed or transplanted neural stem cells. In this study, we found that low-dose treatment with the Heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heavily investigated anti-cancer drug, prevented neural progenitor cells from either naturally-occurring or stress-induced apoptosis, although it induced apoptosis at higher doses. This stress adaptation effect mediated by low-dose 17-AAG is accompanied by activation of multiple cell survival pathways, including the stress response pathway (induction of Hsp70), the MAPK pathway, and the PI3K/Akt pathway. When administered in vivo, 17-AAG led to Akt and glycogen synthase kinase 3β phosphorylation, and more 5-bromo-2'-deoxyuridine positive cells in the mouse brain. These findings could have profound implications in stem cell therapy for neurodegenerative diseases and stroke.

Neuron-Like Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

PurposeMesenchymal stem cells (MSCs) are multipotent and give rise to distinctly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechanically made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).Materials and MethodsBMSCs grown confluent were mechanically separated with cell scrapers and masses of separated cells were cultured to form cluster BMSCs. As described here cluster of BMSCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differentiated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immunocytochemistry to identify the expression of neural specific markers.ResultsFor the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Particularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neural cell phenotype, expressing several neuro-glial specific markers.Conclusionbone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could differentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neuronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.

Bone-marrow-derived mesenchymal stem cell therapy for neurodegenerative diseases

Background: Stem-cell-based therapy is a promising new approach to handling neurodegenerative diseases. One of the most promising cellular sources is bone-marrow-derived mesenchymal stem cells (MSCs) also termed multipotent stromal cells. MSCs represent an autologous source and are abundant and non-tumorigenic. Additionally, MSCs possess the useful characteristics of homing and chemokine secretion.Objective/methods: Since neurodegenerative diseases have many pathological processes in common, a specific therapeutic agent could potentially ameliorate the symptoms of several distinct neurodegenerative diseases. In this review we demonstrate the wide variety of mechanisms by which MSCs can influence neurodegenerative processes.Results/conclusions: The mechanisms by which transplanted MSCs influence neurodegenerative diseases can be broadly classified as cellular replacement or paracrine secretion, with the latter subdivided into trophic factor secretion or immunomodulation by cytokines. Emerging research suggests that genetic manipulations before transplantation could enhance the therapeutic potential of MSCs. Such manipulation could turn the cells into a ‘Trojan horse’, to deliver specific proteins, or promote reprogramming of the MSCs into the neural lineage. Clinical trials testing MSC-based therapies for familial amyotrophic lateral sclerosis and multiple sclerosis are in progress.

Generation and properties of a new human ventral mesencephalic neural stem cell line

Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH +) after differentiation, which also produce dopamine. In addition to proneural genes ( NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

Immune physiology and oogenesis in fetal and adult humans, ovarian infertility, and totipotency of adult ovarian stem cells

It is still widely believed that while oocytes in invertebrates and lower vertebrates are periodically renewed throughout life, oocytes in humans and higher vertebrates are formed only during the fetal/perinatal period. However, this dogma is questioned, and clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal from ovarian stem cells (OSCs) in adult human ovaries, and of the role of third-party bone marrow-derived cells (monocyte-derived tissue macrophages and T lymphocytes) could help provide a better understanding of the causes of ovarian infertility, its prevention, and potential treatment. We have reported differentiation of distinct cell types from OSC and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. OSCs are also capable of producing neural/neuronal cells in vitro after sequential stimulation with sex steroid combinations. Hence, OSC represent a unique type of totipotent adult stem cells, which could be utilized for autologous treatment of premature ovarian failure and also for autologous stem cell therapy of neurodegenerative diseases without use of allogeneic embryonic stem cells or somatic cell nuclear transfer. The in vivo application of sex steroid combinations may augment the proliferation of existing neural stem cells and their differentiation into mature neuronal cells (systemic regenerative therapy). Such treatment may also stimulate the transdifferentiation of autologous neural stem cell precursors into neural stem cells useful for topical or systemic regenerative treatment.

Potentials of ES Cell Therapy in Neurodegenerative Diseases

Neurodegenerative diseases comprise a heterogeneous spectrum of neural disorders that cause severe and progressive cognitive and motor deficits. A histological hallmark of these disorders is the occurrence of disease-specific cell death in specific regional subpopulations of neurons, such as the loss of dopaminergic neurons in the substantia nigra in Parkinson's disease. Neurodegenerative disease can also possibly occur from the loss or dysfunction of selected glial cell subsets, such as the dysfunction of supportive glial cells around somatic motor neurons in amyotrophic lateral sclerosis. The central nervous system (CNS), unlike many other tissues, has a very limited capacity for self-repair. Mature nerve cells lack the ability to regenerate, although endogenous neural stem cells exist in the adult brain that do have very limited ability to generate new functional neurons in response to injury. Rapid advances in stem cell biology have opened an alternative, fascinating perspective of neurogenesis by activation of endogenous neural stem cells and/or transplantation of in vitro-expanded stem cells and/or their neuronal- or glial-differentiated progeny. Embryonic stem (ES) cells, because of their ability to provide seemingly unlimited supply of specific cell types, their amenability to genetic engineering manipulations, and their broad developmental potential, are expected to become a cell source and biological delivery system for use in a variety of neurodegenerative diseases, and are likely to play a role in the development of novel cell-based therapies for these indications. However, before the full potential of ES cells can be realized for regenerative medicine, we need to understand mechanisms regulating their proliferation, differentiation into therapeutically relevant cells, and most importantly in the case of neuronal and glial lineages, to characterize their functional properties. In the present review we will be focusing on the factors and methodologies responsible for differentiation of ES cell into different neural precursors and neural cell lineages with particular emphasis on the potential research and clinical applications of ES cells in the field of neurodegenerative disease.

Highly Efficient Neural Differentiation of Human Somatic Stem Cells, Isolated by Minimally Invasive Periodontal Surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Pharmacologically active microcarriers releasing glial cell line – derived neurotrophic factor: Survival and differentiation of embryonic dopaminergic neurons after grafting in hemiparkinsonian rats

To improve the outcome of foetal dopaminergic cell transplantation for the treatment of Parkinson's disease, pharmacologically active microcarriers (PAM) were developed. PAM are able to convey cells on their surface and release a growth factor to improve cell survival, differentiation and integration after brain implantation. Lysozyme-releasing PAM were first produced and characterized. They served as a model system for the development of glial cell line-derived neurotrophic factor (GDNF)-releasing PAM conveying foetal ventral mesencephalic (FVM) cells. The effects of the intrastriatal implantation of this system were studied in hemiparkinsonian rats during a 6-week period. This study reports on the degradation of coated and non-coated PAM and the release of lysozyme and of biologically active GDNF for 42 days. Unloaded and GDNF-loaded PAM conveying FVM cells allowed a high improvement of the grafted cell survival and of fibre outgrowth, when compared to the cells transplanted alone. The animals receiving the PAM showed an earlier improvement in amphetamine-induced rotational behaviour compared to animals receiving FVM cells only; behaviour that appears to be more regular and stable with the GDNF-releasing PAM. The use of PAM to convey foetal cells is thus an efficient strategy for cell therapy in neurodegenerative diseases, as it allows improvement of cell survival and fibre outgrowth inducing a rapid recovery of behaviour using only low amounts of cells.

Induction of Neurotrophin Expression via Human Adult Mesenchymal Stem Cells: Implication for Cell Therapy in Neurodegenerative Diseases

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFR(p75)) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.

Highly Efficient Neural Differentiation of Human Peripheral Blood Haematopoietic Stem Cells.

Neural stem cells are a potential source of cells for cell therapy of neurodegenerative diseases or drug screening. Ethical and practical considerations limit the application of neural stem cells derived from human embryonic stem cells or adult brain tissue. Therefore, alternative sources of adult human neural stem cells are of high interest for basic research as well as potential clinical use. These sources have to satisfy the demands of easy accession, rapid expansion in serum-free media and reliable induction to a neural fate.CD133 positive hematopoietic stem cells (HSCs) isolated from mobilized leukapheresis were cultured and expanded in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2/bFGF) in serum-free medium. After two weeks of culture, neurosphere formation was observed. Subsequent culture in serum-free, EGF and FGF-2 containing medium resulted in large numbers of CD133−, Nestin+ and Sox-2+ neural stem cells. Neural stem cells derived from HSCs (hNSCs) are highly proliferative and are able to migrate in response to chemotactic stimuli described to induce migration of neural stem cells. We used immunocytochemical techniques and PCR to assess neural differentiation. Adherence to poly-D-lysine/laminin and growth factor deprivation resulted in cells of neuronal morphology and high expression of neuronal differentiation markers. We furthermore show that after retinoic acid treatment neuronal induction was greatly enhanced with more than 90% of cells expressing neuronal differentiation markers as beta-III-tubulin and neurofilaments. In addition, hNSCs were able to differentiate into cells of the glial lineage as shown by expression of glial fibrillary acidic protein (GFAP). Our novel method provides nearly limitless numbers of neural stem cells from an easily accessible autologous adult human source, which could be used as a starting point for further experimental studies and potential therapeutic use.

Neural transdifferentiation of mesenchymal stem cells – a critical review

The classic concept of stem cell differentiation can be illustrated as driving into a series of one-way streets, where a given stem cell through generations of daughter cells becomes correspondingly restricted and committed towards a definitive lineage with fully differentiated cells as end points. According to this concept, tissue-derived adult stem cells can only give rise to cells and cell lineages found in the natural, specified tissue of residence. During the last few years it has, however, been reported that under certain experimental conditions adult stem cells may lose their tissue or germ layer-specific phenotypes and become reprogrammed to transdifferentiate into cells of other germ layers and tissues. The transdifferentiation process is referred to as "stem cell plasticity". Mesenchymal stem cells, present in various tissues, including bone marrow, have--besides differentiation into bone, cartilage, smooth muscle and skeletal muscle--also been reported to transdifferentiate into skin, liver and brain cells (neurons and glia). Conversely, neural stem cells have been reported to give rise to blood cells. The actual occurrence of transdifferentiation is currently much debated, but would have immense clinical potential in cell replacement therapy and regenerative medicine. Controlled neural differentiation of human mesenchymal stem cells might thus become an important source of cells for cell therapy of neurodegenerative diseases, since autologous adult mesenchymal stem cells are more easily harvested and effectively expanded than corresponding neural stem cells. This article provides a critical review of the reports of neural transdifferentiation of mesenchymal stem cells, and proposes a set of criteria to be fulfilled for validation of transdifferentiation.

Transcription Factors Analysis in Neuronal-Like Cells Derived from Adult Human Bone Marrow.

Stem cells are immature cells with self-renewal capacity and ability to differentiate into multiple cell types including bone, cartilage, fat, tendon and muscle. Transplantation of stem cells or their derivatives to the adult brain have been proposed as future therapy for neurodegenerative diseases. Parkinson's disease (PD) is a progressive neurodegenerative disease involving the deterioration of dopaminergic neurons of the substantia nigra, a part of the midbrain. Most of the currently used drugs are based on dopamine replacement which, although it reduces symptoms is limited owing to severe side effects. Alternatively, strategies of cell therapy for the treatment of PD focused on replacing damaged neurons with cells that may improve the functioning of the damaged cell population. In this study, we demonstrate that bone marrow stromal cells isolated from human iliac crest can be induced to differentiate into neural-like cells. Human bone marrow stromal cells differentiation was induced with N2 supplements, retinoic acid, butylated hydroxyanisole, isobutylmethylxanthine and dibutyryl-cyclic-AMP. A few hours after differentiation the cells changed their phenotype to neural-like cells, spindle shaped, with cell bodies and long branches. Moreover, immunostaining demonstrated the expression of specific neuronal markers such as nestin, neurofilaments and neuron specific enolase. To define the transcription factors associated with the differentiation, we used the TranSignal Protein/DNA kit (Panomics Inc). This new array-based technology allows functional analysis of dozens of eukaryotic transcription factors. Nuclear proteins were purified and incubated with mix labeled DNA binding oligonucleotides corresponding to the consensus sequences of 96 transcription factors. The DNA probes were then dissociated from the complexes and used to hybridize the TranSignal Array. We found that the differentiated human bone marrow stromal cells induced the expression of several neuron-specific transcription factors such as GAG (glycosaminoglycan), FKHR (human forkhead in rhabdomyosarcoma) and DR-2. The activation of these transcription factors in a time course manner was evident also by electrophoretic mobility shift assay. In conclusion, we show that the induced differentiation of neuronal like cells from bone marrow is associated with activation of specific neuronal transcription factors. Our study advances the possible use of autologous transplantation as a new strategy for cell therapy in neurodegenerative diseases.

Stem Cell Therapy for Neurodegenerative Diseases: The Issue of Transdifferentiation

In the past few years research on stem cells has exploded as a tool to develop potential therapies to treat incurable neurodegenerative diseases. Stem cell transplantation has been effective in several animal models, but the underlying restorative mechanisms are still unknown. Several events such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation (adult cell acquisition of new unexpected identities) may explain therapeutic success, in addition to replacement of lost cells. This issue needs to be clarified further to maximize the potential for effective therapies. Preliminary stem transplantation trials have already been performed for some neurodegenerative diseases. There is no effective pharmacological treatment for amyotrophic lateral sclerosis, but recent preliminary data both in experimental and clinical settings have targeted it as an ideal candidate disease for the development of stem cell therapy in humans. This review summarizes recent advances gained in stem cell research applied to neurodegenerative diseases with a special emphasis to the criticisms put forward.

Growth factors regulate the survival and fate of cells derived from human neurospheres.

Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations. Neurospheres can also be generated from the embryonic human brain and in some cases have been expanded for extended periods of time in culture. However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.