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1.
Stroke ; 55(4): 1062-1074, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38436063

RESUMO

BACKGROUND: In preterm birth germinal matrix hemorrhages (GMHs) and the consequent posthemorrhagic hydrocephalus (PHH), the neuroepithelium/ependyma development is disrupted. This work is aimed to explore the possibilities of ependymal repair in GMH/PHH using a combination of neural stem cells, ependymal progenitors (EpPs), and mesenchymal stem cells. METHODS: GMH/PHH was induced in 4-day-old mice using collagenase, blood, or blood serum injections. PHH severity was characterized 2 weeks later using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Ependymal restoration and wall regeneration after stem cell treatments were tested in vivo and in an ex vivo experimental approach using ventricular walls from mice developing moderate and severe GMH/PHH. The effect of the GMH environment on EpP differentiation was tested in vitro. Two-tailed Student t or Wilcoxon-Mann-Whitney U test was used to find differences between the treated and nontreated groups. ANOVA and Kruskal-Wallis tests were used to compare >2 groups with post hoc Tukey and Dunn multiple comparison tests, respectively. RESULTS: PHH severity was correlated with the extension of GMH and ependymal disruption (means, 88.22% severe versus 19.4% moderate). GMH/PHH hindered the survival rates of the transplanted neural stem cells/EpPs. New multiciliated ependymal cells could be generated from transplanted neural stem cells and more efficiently from EpPs (15% mean increase). Blood and TNFα (tumor necrosis factor alpha) negatively affected ciliogenesis in cells committed to ependyma differentiation (expressing Foxj1 [forkhead box J1] transcription factor). Pretreatment with mesenchymal stem cells improved the survival rates of EpPs and ependymal differentiation while reducing the edematous (means, 18% to 0.5% decrease in severe edema) and inflammatory conditions in the explants. The effectiveness of this therapeutical strategy was corroborated in vivo (means, 29% to 0% in severe edema). CONCLUSIONS: In GMH/PHH, the ependyma can be restored and edema decreased from either neural stem cell or EpP transplantation in vitro and in vivo. Mesenchymal stem cell pretreatment improved the success of the ependymal restoration.


Assuntos
Doenças Fetais , Hidrocefalia , Células-Tronco Neurais , Nascimento Prematuro , Humanos , Feminino , Animais , Camundongos , Epêndima/patologia , Hidrocefalia/cirurgia , Hidrocefalia/metabolismo , Hemorragia Cerebral/terapia , Hemorragia Cerebral/metabolismo , Edema
2.
J Quant Spectrosc Radiat Transf ; 302: 108567, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36945203

RESUMO

Objective: To conduct a proof-of-concept study of the detection of two synthetic models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using polarimetric imaging. Approach: Two SARS-CoV-2 models were prepared as engineered lentiviruses pseudotyped with the G protein of the vesicular stomatitis virus, and with the characteristic Spike protein of SARS-CoV-2. Samples were prepared in two biofluids (saline solution and artificial saliva), in four concentrations, and deposited as 5-µL droplets on a supporting plate. The angles of maximal degree of linear polarization (DLP) of light diffusely scattered from dry residues were determined using Mueller polarimetry from87 samples at 405 nm and 514 nm. A polarimetric camera was used for imaging several samples under 380-420 nm illumination at angles similar to those of maximal DLP. Per-pixel image analysis included quantification and combination of polarization feature descriptors in 475 samples. Main results: The angles (from sample surface) of maximal DLP were 3° for 405 nm and 6° for 514 nm. Similar viral particles that differed only in the characteristic spike protein of the SARS-CoV-2, their corresponding negative controls, fluids, and the sample holder were discerned at 10-degree and 15-degree configurations. Significance: Polarimetric imaging in the visible spectrum may help improve fast, non-contact detection and identification of viral particles, and/or other microbes such as tuberculosis, in multiple dry fluid samples simultaneously, particularly when combined with other imaging modalities. Further analysis including realistic concentrations of real SARS-CoV-2 viral particles in relevant human fluids is required. Polarimetric imaging under visible light may contribute to a fast, cost-effective screening of SARS-CoV-2 and other pathogens when combined with other imaging modalities.

3.
Crit Rev Biotechnol ; 40(3): 283-291, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32054356

RESUMO

Recent advances in stem cell biology and molecular engineering have improved and simplified the methodology employed to create experimental chimeras, highlighting their value in basic research and broadening the spectrum of potential applications. Experimental chimeras have been used for decades during the generation of murine genetic models, this being especially relevant in developmental and regeneration studies. Indeed, their value for the research and modeling of human diseases was recognized by the 2007 Nobel Prize to Mario Capecchi, Martin Evans, and Oliver Smithies. More recently, their potential application in regenerative medicine has generated a lot of interest, particularly the enticing possibility to generate human organs for transplantation in livestock animals. In this review, we provide an update on interspecific chimeric organogenesis, its possibilities, current limitations, alternatives, and ethical issues.


Assuntos
Quimera/metabolismo , Animais , Temas Bioéticos , Quimera/classificação , Células-Tronco Embrionárias , Humanos , Camundongos , Modelos Genéticos , Organogênese , Medicina Regenerativa , Quimeras de Transplante
4.
J Neuroinflammation ; 13(1): 295, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27863501

RESUMO

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2. METHODS: Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNFα-induced changes in proteins of the NF-κB pathway and optimized western blot analysis to capture α-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls. RESULTS: LRRK2 silencing decreased α-synuclein protein levels in mutated neurons and modified NF-κB transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-κB and α-synuclein pathways converged and found that TNFα modulated α-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-κB dysregulation in mutated neurons, as shown by a protracted recovery of IκBα and a clear impairment in p65 nuclear translocation in the LRRK2 mutants. CONCLUSIONS: Altogether, our results show that LRRK2 mutations affect α-synuclein regulation and impair NF-κB canonical signaling in iPSC-derived neurons. TNFα modulated α-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PD.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , NF-kappa B/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Citocinas/metabolismo , Análise Mutacional de DNA , Dopamina/metabolismo , Fibroblastos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Antígenos Embrionários Estágio-Específicos/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo
5.
Cell Mol Life Sci ; 70(1): 121-36, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22773119

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson's disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.


Assuntos
Autofagia/genética , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/genética , Idoso , Substituição de Aminoácidos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macrolídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , ATPases Translocadoras de Prótons/antagonistas & inibidores
6.
Expert Opin Biol Ther ; 24(9): 933-954, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39162129

RESUMO

INTRODUCTION: Cell therapy development represents a critical challenge in amyotrophic lateral sclerosis (ALS) research. Despite more than 20 years of basic and clinical research, no definitive safety and efficacy results of cell-based therapies for ALS have been published. AREAS COVERED: This review summarizes advances using stem cells (SCs) in pre-clinical studies to promote clinical translation and in clinical trials to treat ALS. New technologies have been developed and new experimental in vitro and animal models are now available to facilitate pre-clinical research in this field and to determine the most promising approaches to pursue in patients. New clinical trial designs aimed at developing personalized SC-based treatment with biological endpoints are being defined. EXPERT OPINION: Knowledge of the basic biology of ALS and on the use of SCs to study and potentially treat ALS continues to grow. However, a consensus has yet to emerge on how best to translate these results into therapeutic applications. The selection and follow-up of patients should be based on clinical, biological, and molecular criteria. Planning of SC-based clinical trials should be coordinated with patient profiling genetically and molecularly to achieve personalized treatment. Much work within basic and clinical research is still needed to successfully transition SC therapy in ALS.


Assuntos
Esclerose Lateral Amiotrófica , Transplante de Células-Tronco , Esclerose Lateral Amiotrófica/terapia , Esclerose Lateral Amiotrófica/genética , Humanos , Animais , Modelos Animais de Doenças , Ensaios Clínicos como Assunto
7.
Neuron ; 112(17): 2886-2909.e16, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39079530

RESUMO

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.


Assuntos
Corpos de Inclusão , Células-Tronco Pluripotentes Induzidas , alfa-Sinucleína , Células-Tronco Pluripotentes Induzidas/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , Sinucleinopatias/genética , Neurônios/metabolismo , Neurônios/patologia , Encéfalo/metabolismo , Encéfalo/patologia
8.
Prog Neurobiol ; 217: 102330, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35872220

RESUMO

Animal models play a pivotal role in translational neuroscience but recurrent problems in data collection, analyses, and interpretation, lack of biomarkers, and a tendency to over-reliance on mice have marred neuroscience progress, leading to one of the highest attrition rates in drug translation. Global initiatives to improve reproducibility and model selection are being implemented. Notwithstanding, mice are still the preferred animal species to model human brain disorders even when the translation has been shown to be limited. Non-human primates are better positioned to provide relevant translational information because of their higher brain complexity and homology to humans. Among others, lack of resources and formal training, strict legislation, and ethical issues may impede broad access to large animals. We propose that instead of increasingly restrictive legislation, more resources for training, education, husbandry, and data sharing are urgently needed. The creation of multidisciplinary teams, in which veterinarians need to play a key role, would be critical to improve translational efficiency. Furthermore, it is not usually acknowledged by researchers and regulators the value of comparative studies in lower species, that are instrumental in toxicology, target identification, and mechanistic studies. Overall, we highlight here the need for a conceptual shift in neuroscience research and policies to reach the patients.


Assuntos
Neurociências , Animais , Humanos , Camundongos , Primatas , Reprodutibilidade dos Testes
9.
Stem Cells Transl Med ; 11(8): 805-813, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35830540

RESUMO

The development of cell-, gene- and tissue engineering (CGT)-based therapies must adhere to strict pharmaceutical quality management standards, as for any other biological or small-molecule drug. However, early developments often failed to fully comply with good laboratory practices (GLP) in non-clinical safety studies. Despite an upward trend of positive opinions in marketing authorization applications, evidence of adherence to the principles of GLP is not openly reported; therefore, their relative impact on the overall quality of the product development program is unknown. Herein we investigated the actual degree of GLP implementation and the underlying factors impeding full compliance in non-clinical developments of CGT-based marketed medicines in the EU and USA, including (i) the co-existence of diverse quality management systems of more strategic value for small organizations, particularly current Good Manufacturing Practices n(GMP); (ii) lack of regulatory pressure to pursue GLP certification; and (iii) the involvement of public institutions lacking a pharmaceutical mindset and resources. As a final reflection, we propose conformity to good research practice criteria not as a doctrinaire impediment to scientific work, but as a facilitator of efficient clinical translation of more effective and safer innovative therapies.


Assuntos
Terapias em Estudo , Preparações Farmacêuticas
10.
Animals (Basel) ; 12(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36428378

RESUMO

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

11.
Parkinsonism Relat Disord ; 96: 29-35, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35149357

RESUMO

INTRODUCTION: Blood and cerebrospinal fluid represent emerging candidate fluids for biomarker identification in Parkinson's disease (PD). METHODS: We studied 8 individuals carrying the E46K-SNCA mutation (3 PD dementia (PDD), 1 tremor-dominant PD, 2 young rigid-akinetic PD and 2 asymptomatic) and 8 age- and sex-matched healthy controls. We quantified the levels of total alpha-synuclein (a-syn), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), Tau and ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with SiMoA (Quanterix) in cerebrospinal fluid (CSF) of mutation carriers and in serum of all participants. The correlation between the concentration of biofluid markers and clinical outcomes was evaluated. RESULTS: Although based on a small number of cases, CSF a-syn was decreased in symptomatic E46K-SNCA carriers compared to the asymptomatic ones. Asymptomatic carriers exhibited similar serum biomarker levels as compared to matched controls, except for serum a-syn, which was higher in asymptomatic individuals. Carriers with PDD diagnosis displayed increased levels of serum NfL and GFAP compared to matched controls. These findings highly correlated with cognitive and motor status of E46K-SNCA carriers, but not with disease duration. CONCLUSIONS: Patients with familial forms of neurodegenerative disease exhibit variable penetrance of the phenotype and are exceptionally valuable for delineating biomarkers. Serum and CSF molecular biomarkers in E46K-SNCA mutation carriers show that a-syn might be suitable to track the conversion from asymptomatic to PD, whereas NfL and GFAP might serve to foresee the progression to PD dementia. These findings should be interpreted with caution and need to be replicated in other genetic synucleinopathy cohorts.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Parkinson , alfa-Sinucleína , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Humanos , Mutação , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/líquido cefalorraquidiano , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Doença de Parkinson/sangue , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , alfa-Sinucleína/sangue , alfa-Sinucleína/líquido cefalorraquidiano
12.
Stem Cells Transl Med ; 11(4): 343-355, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35348788

RESUMO

Graft versus host disease (GVHD) is a severe complication after allogenic hematopoietic cell transplantation (HSCT). Several clinical trials have reported the use of mesenchymal stromal cells (MSCs) for the treatment of GVHD. In March 2008, the Andalusian Health Care System launched a compassionate use program to treat steroid-resistant GVHD with MSC. Clinical-grade MSC were obtained under GMP conditions. MSC therapy was administered intravenously in four separate doses of 1 × 106 cells/kg. Sixty-two patients, 45 males (7 children) and 17 females (2 children), received the treatment. Patients had a median age of 39 years (range: 7-66) at the time of the allogenic HSCT. The overall response was achieved in 58.7% of patients with acute (a)GVHD. Two years' survival for aGVHD responders was 51.85%. The overall response for patients with chronic (c)GVHD was 65.50% and the 2-year survival rate for responders was 70%. Age at the time of HSCT was the only predictor found to be inversely correlated with survival in aGVHD. Regarding safety, four adverse events were reported, all recovered without sequelae. Thus, analysis of this compassionate use experience shows MSC to be an effective and safe therapeutic option for treating refractory GVHD, resulting in a significant proportion of patients responding to the therapy.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Doença Aguda , Adolescente , Adulto , Idoso , Criança , Ensaios de Uso Compassivo/efeitos adversos , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Pessoa de Meia-Idade , Esteroides/uso terapêutico , Adulto Jovem
13.
Sci Rep ; 12(1): 2356, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181702

RESUMO

Effective testing is essential to control the coronavirus disease 2019 (COVID-19) transmission. Here we report a-proof-of-concept study on hyperspectral image analysis in the visible and near-infrared range for primary screening at the point-of-care of SARS-CoV-2. We apply spectral feature descriptors, partial least square-discriminant analysis, and artificial intelligence to extract information from optical diffuse reflectance measurements from 5 µL fluid samples at pixel, droplet, and patient levels. We discern preparations of engineered lentiviral particles pseudotyped with the spike protein of the SARS-CoV-2 from those with the G protein of the vesicular stomatitis virus in saline solution and artificial saliva. We report a quantitative analysis of 72 samples of nasopharyngeal exudate in a range of SARS-CoV-2 viral loads, and a descriptive study of another 32 fresh human saliva samples. Sensitivity for classification of exudates was 100% with peak specificity of 87.5% for discernment from PCR-negative but symptomatic cases. Proposed technology is reagent-free, fast, and scalable, and could substantially reduce the number of molecular tests currently required for COVID-19 mass screening strategies even in resource-limited settings.


Assuntos
Exsudatos e Transudatos/virologia , Programas de Rastreamento/métodos , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Espectroscopia de Luz Próxima ao Infravermelho , Humanos , Testes Imediatos , Estudo de Prova de Conceito
14.
NPJ Regen Med ; 6(1): 20, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795700

RESUMO

Transplantation of several types of stem cells (SC) for the treatment of amyotrophic lateral sclerosis (ALS) has been evaluated in numerous Phase I/II clinical trials with inconclusive results. Here, we conducted a meta-analysis to systematically assess the outcome of SC therapy trials which report the evolution of each patient before and after cell administration. In this way, we aimed to determine the effect of the SC intervention despite individual heterogeneity in disease progression. We identified 670 references by electronic search and 90 full-text studies were evaluated according to the eligibility criteria. Eleven studies were included comprising 220 cell-treated patients who received mesenchymal (M) SC (n = 152), neural (N) SC (n = 57), or mononuclear cells (MNC: CD34, CD117, and CD133 positive cells) (n = 11). Our analyses indicate that whereas intrathecal injection of mesenchymal stromal cells appears to have a transient positive effect on clinical progression, as measured by the ALS functional rating score, there was a worsening of respiratory function measured by forced vital capacity after all interventions. Based on current evidence, we conclude that optimal cell product and route of administration need to be determined in properly controlled preclinical models before further advancing into ALS patients. In addition, in-depth understanding of disease mechanisms in subsets of patients will help tailoring SC therapy to specific targets and increase the likelihood of improving outcomes.

15.
Cells ; 10(9)2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34572024

RESUMO

Neural stem cells represent an attractive tool for the development of regenerative therapies and are being tested in clinical trials for several neurological disorders. Human neural stem cells can be isolated from the central nervous system or can be derived in vitro from pluripotent stem cells. Embryonic sources are ethically controversial and other sources are less well characterized and/or inefficient. Recently, isolation of NSC from the cerebrospinal fluid of patients with spina bifida and with intracerebroventricular hemorrhage has been reported. Direct reprogramming may become another alternative if genetic and phenotypic stability of the reprogrammed cells is ensured. Here, we discuss the advantages and disadvantages of available sources of neural stem cells for the production of cell-based therapies for clinical applications. We review available safety and efficacy clinical data and discuss scalability and quality control considerations for manufacturing clinical grade cell products for successful clinical application.


Assuntos
Células-Tronco Neurais/fisiologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Reprogramação Celular/fisiologia , Humanos , Neurônios/fisiologia , Células-Tronco Pluripotentes/fisiologia , Medicina Regenerativa/métodos
16.
J Clin Neurosci ; 94: 76-85, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34863466

RESUMO

With an expanding elderly population, an increasing number of older adults will experience spinal cord injury (SCI) and might be candidates for cell-based therapies, yet there is a paucity of research in this age group. The objective of the present study was to analyze how aged rats tolerate behavioral testing, surgical procedures, post-operative complications, intra-spinal cell transplantation and immunosuppression, and to examine the effectiveness of human iPSC-derived Neural Progenitor Cells (IMR90-hiPSC-NPCs) in a model of SCI. We performed behavioral tests in rats before and after inducing cervical hemi-contusions at C4 level with a fourth-generation Ohio State University Injury Device. Four weeks later, we injected IMR90-hiPSC-NPCs in animals that were immunosuppressed by daily cyclosporine injection. Four weeks after injection we analyzed locomotor behavior and mortality, and histologically assessed the survival of transplanted human NPCs. As rats aged, their success at completing behavioral tests decreased. In addition, we observed high mortality rates during behavioral training (41.2%), after cervical injury (63.2%) and after cell injection (50%). Histological analysis revealed that injected cells survived and remained at and around the grafted site and did not cause tumors. No locomotor improvement was observed in animals four weeks after IMR90-hiPSC-NPC transplantation. Our results show that elderly rats are highly vulnerable to interventions, and thus large groups of animals must be initially established to study the potential efficacy of cell-based therapies in age-related chronic myelopathies.


Assuntos
Medula Cervical , Traumatismos da Medula Espinal , Idoso , Animais , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Ratos , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/terapia , Transplante de Células-Tronco
17.
Stem Cell Res Ther ; 11(1): 64, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070420

RESUMO

BACKGROUND: Graft-versus-host disease (GvHD) is the main life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Thirty to 80% of GvHD patients do not respond to first-line treatment and a second-line treatment is not universally established. Based on their immunomodulatory properties, mesenchymal stromal cells (MSC) have been proposed for the prevention and the treatment of GvHD in patients undergoing HSCT. Unfortunately, previous studies reported conflicting results regarding the prophylactic and therapeutic effects of MSC for GvHD. Consequently, we carried out a meta-analysis to clarify whether MSC administration can improve the dismal outcome of these patients. METHODS: We carried out a systematic review and selected studies (2004-2019) reporting data about the administration of allogeneic MSC for the prevention (n = 654 patients) or treatment of acute (n = 943 patients) or chronic (n = 76 patients) GvHD after HSCT. Our primary outcome was overall survival at the last follow-up. The secondary outcomes were the response and development of GvHD. Subgroup analyses included age, MSC dose, first infusion day after HSCT, number of organs and organ-specific involvement, acute GvHD grade (I-IV), and chronic GvHD grade (limited or extensive). RESULTS: Patients infused with MSC for GvHD prophylaxis showed a 17% increased overall survival (95% CI, 1.02-1.33) and a reduced incidence of acute GvHD grade IV (RR = 0.22; 95% CI, 0.06-0.81) and chronic GvHD (RR = 0.64; 95% CI, 0.47-0.88) compared with controls. Overall survival of acute GvHD patients (0.50; 95% CI, 0.41-0.59) was positively correlated with MSC dose (P = 0.0214). The overall response was achieved in 67% (95% CI, 0.61-0.74) and was complete in 39% (95% CI, 0.31-0.48) of acute patients. Organ-specific response was higher for the skin. Twenty-two percent (95% CI, 0.16-0.29) of acute patients infused with MSC developed chronic GvHD. Sixty-four percent (95% CI, 0.47-0.80) of chronic patients infused with MSC survived; the overall response was 66% (95% CI, 0.55-0.76) and was complete in 23% (95% CI 0.12-0.34) of patients. CONCLUSIONS: Our meta-analysis indicates that allogeneic MSC could be instrumental for the prophylaxis and treatment of GvHD. Future trials should investigate the effect of the administration of MSC as an adjuvant therapy for the treatment of patients with GvHD from the onset of the disease.


Assuntos
Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Condicionamento Pré-Transplante/efeitos adversos , Feminino , Humanos , Masculino
18.
Stem Cells Transl Med ; 9(9): 1085-1101, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32475061

RESUMO

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.


Assuntos
Hemorragia Cerebral/líquido cefalorraquidiano , Recém-Nascido Prematuro/líquido cefalorraquidiano , Células-Tronco Neurais/patologia , Antígeno AC133/metabolismo , Animais , Hemorragia Cerebral/genética , Endoscopia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Nus , Células-Tronco Neurais/transplante
19.
Brain ; 131(Pt 8): 2127-39, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18669499

RESUMO

The identity and functional potential of dopamine neurons derived in vitro from embryonic stem cells are critical for the development of a stem cell-based replacement therapy for Parkinson's disease. Using a parthenogenetic primate embryonic stem cell line, we have generated dopamine neurons that display persistent expression of midbrain regional and cell-specific transcription factors, which establish their proper identity and allow for their survival. We show here that transplantation of parthenogenetic dopamine neurons restores motor function in hemi-parkinsonian, 6-hydroxy-dopamine-lesioned rats. Exposure to Wnt5a and fibroblast growth factors (FGF) 20 and 2 at the final stage of in vitro differentiation enhanced the survival of dopamine neurons and, correspondingly, the extent of motor recovery of transplanted animals. Importantly for future development of clinical applications, dopamine neurons were post-mitotic at the time of transplantation and there was no tumour formation. These data provide proof for the concept that parthenogenetic stem cells are a suitable source of functional neurons for therapeutic applications.


Assuntos
Células-Tronco Embrionárias/transplante , Neurônios/transplante , Transtornos Parkinsonianos/cirurgia , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Dopamina/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Mesencéfalo , Atividade Motora , Neurônios/metabolismo , Oxidopamina , Transtornos Parkinsonianos/metabolismo , Partenogênese , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Proteínas Wnt/farmacologia , Proteína Wnt-5a
20.
Parkinsonism Relat Disord ; 65: 139-145, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31178336

RESUMO

BACKGROUND AND OBJECTIVE: In 2004 we described the E46K mutation in alpha-synuclein gene (E46K-SNCA), a rare point mutation causing an aggressive Lewy body disease with early prominent non-motor features and small fiber denervation of myocardium. Considering the potential interest of the skin as a target for the development of biomarkers in Parkinson's Disease (PD), in this work we aimed to evaluate structural and functional integrity of small autonomic nerve fibers and phosphorylated alpha-synuclein (p-synuclein) deposition in the skin of E46K-SNCA carriers as compared to those observed in parkin gene mutation (PARK2) carriers and healthy controls. PATIENTS AND METHODS: We studied 7 E46K-SNCA carriers (3 dementia with Lewy bodies, 2 pure autonomic failure, 1 PD and 1 asymptomatic), 2 PARK2 carriers and 2 healthy controls to quantify intraepidermal nerve fiber density and p-synuclein deposition with cervical skin punch biopsies (immunohistochemistry against anti PGP9.5/UCHL-1, TH and p-synuclein) and sudomotor function with electrochemical skin conductance (ESC) (SudoScan). RESULTS: All E46K-SNCA carriers had moderate to severe p-synuclein deposits and small fiber neurodegeneration in different epidermal and dermal structures including nerve fascicles and glands, especially in carriers with Pure Autonomic Failure, while p-synuclein aggregates where absent in healthy controls and in one of two PARK2 carriers. The severity of the latter skin abnormalities in E46K-SNCA were correlated with sudomotor dysfunction (lower ESC) in hands (p = 0.035). INTERPRETATION: These results together with our previous findings support the relevance of E46K-SNCA mutation as a suitable model to study small fiber neuropathy in Lewy body diseases.


Assuntos
Heterozigoto , Mutação Puntual/fisiologia , Pele/metabolismo , Neuropatia de Pequenas Fibras/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/fisiologia , Pele/patologia , Neuropatia de Pequenas Fibras/diagnóstico , Neuropatia de Pequenas Fibras/genética , alfa-Sinucleína/genética
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