Abstract: To develop cell therapies for damaged nervous tissue with human neural stem/progenitor cells (hNPCs), the risk of an immune response and graft rejection must be considered. There are conflicting results and lack of knowledge concerning the immunocompetence of hNPCs of different origin. Here, we studied the immunogenicity and immunomodulatory potentials of hNPCs cultured under equivalent conditions after derivation from human embryonic stem cells (hESC-NPCs) or human fetal spinal cord tissue (hfNPCs). The expression patterns of human leukocyte antigen, co-stimulatory and adhesion molecules in hESC-NPCs and hfNPCs were relatively similar and mostly not affected by inflammatory cytokines. Unstimulated hfNPCs secreted more transforming growth factor-β1 (TGF-β1) and β2 but similar level of interleukin (IL)-10 compared to hESC-NPCs. In contrast to hfNPCs, hESC-NPCs displayed 4-6 fold increases in TGF-β1, TGF-β2 and IL-10 under inflammatory conditions. Both hNPCs reduced the alloreaction between allogeneic peripheral blood mononuclear cells (PBMCs) and up-regulated CD4(+)CD25(+)forkhead box P3 (FOXP3)(+) T cells. However, hESC-NPCs but not hfNPCs dose-dependently triggered PBMC proliferation, which at least partly may be due to TGF-β signaling. To conclude, hESC-NPCs and hfNPCs displayed similarities but also significant differences in their immunocompetence and interaction with allogeneic PBMCs, differences may be crucial for the outcome of cell therapy.
Abstract: The capability to integrate into degenerative environment, release neurotrophic cytokines, contrast oxidative stress and an inherent differentiation potential towards siteappropriate phenotypes are considered crucial for the use of stem cells in tissue repair and regeneration. Naïve human chorial villi- (hCVCs) and amniotic fluid- (hAFCs) derived cells, whose properties and potentiality have not been extensively investigated, may represent two novel foetal cell sources for stem cell therapy. We previously described that long-term transplantation of hAFCs in the lateral ventricles of wobbler and healthy mice was feasible and safe. In the present study we examine the in vitro intrinsic stem potential of hCVCs and hAFCs for future therapeutic applications in neurodegenerative disorders. Presence of stem lineages was evaluated assessing the expression pattern of relevant candidate markers by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Release of cytokines that may potentialy sustain endogenous neurogenesis and/or activate neuroprotective pathways was quantified by enzyme-linked immunosorbent assays (ELISAs). We also performed an in vitro neurorescue assay, wherein a neuroblastoma cell line damaged by 6-hydroxydopamine (6-OHDA) was treated with hCVC/hAFC-derived conditioned medium (CM). Naïve hCVCs/hAFCs show a neurogenic/angiogenic predisposition. Both cell types express several specific neural stem/progenitor markers, such as nestin and connexin 43, and release significant amounts of brain-derived neurotrophic factor, as well as vascular endothelial growth factor. hCVC and hAFC populations comprise several interesting cell lineages, including mesenchymal stem cells (MSCs) and cells with neural-like phenotypes. Moreover, although CMs obtained from both cell cultures actively sustained metabolic activity in a 6-OHDA-induced Parkinson's disease (PD) cell model, only hCVC-derived CMs significantly reduced neurotoxin-induced apoptosis. In conclusion, this study demonstrates that naïve hAFCs and hCVCs may enhance cell-recovery following neuronal damage through multiple rescue mechanisms, and may provide a suitable means of stem cell therapy for neurodegenerative disorders including PD.
Abstract: Although amniotic fluid cells can differentiate into several mesenchymal lineages and have been proposed as a valuable therapeutic cell source, their ability to undergo terminal neuronal differentiation remains a cause of controversy. The aim of this study was to investigate the neuronal differentiation ability of the c-Kit-positive population from GFP-transgenic rat amniotic fluid, amniotic fluid stem (AFS) cells, and to assess how they affected injury response in avian embryos. AFS cells were found to express several neural stem/progenitor cell markers. However, no overt neuronal differentiation was apparent after either treatment with small molecules known to stimulate neuronal differentiation, attempts to differentiate AFS cell-derived embryoid body-like structures, or grafting AFS cells into environments known to support neuronal differentiation (organotypic rat hippocampal cultures, embryonic chick nervous system). Nonetheless, AFS cells significantly reduced hemorrhage and increased survival when grafted at the site of an extensive thoracic crush injury in E2.5 chick embryos. Increased embryo survival was induced neither by desmopressin treatment, which also reduced hemorrhage, nor by grafting other mesenchymal or neural cells, indicating a specific effect of AFS cells. This was found to be mediated by soluble factors in a transwell coculture model. Altogether, this study shows that AFS cells reduce tissue damage and increase survival in injured embryos, providing a potentially valuable tool as therapeutic agents for tissue repair, particularly prenatal/perinatal repair of defects diagnosed during gestation, but this effect is mediated via paracrine mechanisms rather than the ability of AFS cells to fully differentiate into neuronal cells.
Abstract: Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.
Abstract: The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.
Abstract: Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.
Abstract: Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.
Abstract: Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, nowadays considered as suitable candidate for autologous stem therapy with bone marrow (BM). A careful characterization of BM stem cell (SC) compartment is mandatory before its extensive application to clinic. Indeed, widespread systemic involvement has been recently advocated given that non-neuronal neighboring cells actively influence the pathological neuronal loss. We therefore investigated BM samples from 21 ALS patients and reported normal hematopoietic biological properties while an atypical behavior and impaired SC capabilities affected only the mesenchymal compartment. Moreover, by quantitative real-time approach, we observed altered Collagen IV and Metalloproteinase-9 levels in patients' derived mesenchymal stem cells (MSCs). Widespread metalloproteinase (MMPs) and their tissue inhibitor (TIMPs) alterations were established by multiplex ELISA analysis, demonstrating diffuse enzymatic variations in MSC compartment. Since MMPs act as fundamental effectors of extra-cellular matrix remodeling and stem cell mobilization, their modifications in ALS may influence reparative mechanisms effective in counteracting the pathology. In conclusion, ALS is further confirmed to be a systemic disease, not restricted to the nervous system, but affecting also the BM stromal compartment, even in sporadic cases. Therefore, therapeutic implantation of autologous BM derived SC in ALS patients needs to be carefully reevaluated.
Abstract: Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.
Abstract: Given the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), compelling preclinical data on stem cell research has targeted this disease as a candidate for stem cell treatment. Stem cell transplantation has been effective in several animal models, but the underlying biological pathways of restorative processes are still unresolved. Several mechanisms such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation may explain positive therapeutic results in preclinical animal models, in addition to replacement of lost motor neurons. The clinical target in ALS has shifted from being neuroncentered to focus on the interaction between motor neurons and non-neuronal cells (mainly astroglial or microglial). In fact, one of the fundamental unanswered questions in ALS is whether and how much motor neuron death depends on neighboring cells, and how wildtype non-neuronal cells may protect motor neurons expressing an ALS-causing mutation. Lately, motor neuron replacement has been successfully achieved in animal models with reinnervation of the muscle target. Even if many biological issues need to be solved in preclinical models, preliminary stem cell transplantation trials have been performed in ALS patients with conflicting results. The review discusses relevant topics regarding the application of stem cell research to ALS focusing on their therapeutic relevance and mechanisms of action.
Abstract: 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.
Abstract: Post-transcriptional regulation exerted by neural-specific RNA-binding proteins plays a pivotal role in the development and maintenance of the nervous system. Neural ELAV proteins are key inducers of neuronal differentiation through the stabilization and/or translational enhancement of target transcripts bearing the AU-rich elements (AREs), whereas Musashi-1 maintains the stem cell proliferation state by acting as a translational repressor. Since the gene encoding Musashi-1 (Msi1) contains a conserved ARE in its 3' untranslated region, we focused on the possibility of a mechanistic relationship between ELAV proteins and Musashi-1 in cell fate commitment. Colocalization of neural ELAV proteins with Musashi-1 clearly shows that ELAV proteins are expressed at early stages of neural commitment, whereas interaction studies demonstrate that neural ELAV proteins exert an ARE-dependent binding activity on the Msi1 mRNA. This binding activity has functional effects, since the ELAV protein family member HuD is able to stabilize the Msi1 ARE-containing mRNA in a sequence-dependent way in a deadenylation/degradation assay. Furthermore activation of the neural ELAV proteins by phorbol esters in human SH-SY5Y cells is associated with an increase of Musashi-1 protein content in the cytoskeleton. We propose that ELAV RNA-binding proteins exert an important post-transcriptional control on Musashi-1 expression in the transition from proliferation to neural differentiation of stem/progenitor cells.
Abstract: The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.
Abstract: Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.
Abstract: Among the array of microangiopathies that may occur during pregnancy, HELLP syndrome and thrombotic thrombocytopenic purpura (TTP) produce similar laboratory findings (hemolytic anemia and thrombocytopenia), although neurological symptoms prevail in TTP and abnormal liver function in HELLP syndrome. It is clinically important to distinguish the two entities given that their managements differ (prompt induction of delivery in HELLP syndrome, plasma exchange in TTP). The purpose of this study was to evaluate whether or not ADAMTS-13, the metalloprotease that disposes ultralarge, highly thrombogenic multimers of von Willebrand factor (VWF) and is severely deficient or undetectable in many patients with TTP, is deficient in HELLP syndrome.
