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1.
Surv Ophthalmol ; 69(2): 179-189, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37778668

RESUMO

Diseases leading to retinal cell loss can cause severe visual impairment and blindness. The lack of effective therapies to address retinal cell loss and the absence of intrinsic regeneration in the human retina leads to an irreversible pathological condition. Progress in recent years in the generation of human three-dimensional retinal organoids from pluripotent stem cells makes it possible to recreate the cytoarchitecture and associated cell-cell interactions of the human retina in remarkable detail. These human three-dimensional retinal organoid systems made of distinct retinal cell types and possessing contextual physiological responses allow the study of human retina development and retinal disease pathology in a way animal model and two-dimensional cell cultures were unable to achieve. We describe the derivation of retinal organoids from human pluripotent stem cells and their application for modeling retinal disease pathologies, while outlining the opportunities and challenges for its application in academia and industry.


Assuntos
Células-Tronco Pluripotentes , Doenças Retinianas , Animais , Humanos , Retina , Células-Tronco Pluripotentes/metabolismo , Organoides/metabolismo , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Descoberta de Drogas , Diferenciação Celular
2.
Stem Cell Res ; 62: 102789, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35472829

RESUMO

Macular corneal dystrophy (MCD) an autosomal recessive disorder leading to severe visual impairment is caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) gene. Here, we describe generation of a iPSC line from peripheral blood mononuclear cells (PBMCs) from a 38 year old MCD patient harboring a homozygous mutation, p.V172L in the CHST6 gene. The iPSC line GLNNFi001-A was generated using an integration-free Sendai virus containing (KLF4, c-MYC, OCT4 and SOX2).


Assuntos
Distrofias Hereditárias da Córnea , Células-Tronco Pluripotentes Induzidas , Adulto , Distrofias Hereditárias da Córnea/genética , Homozigoto , Humanos , Leucócitos Mononucleares , Mutação/genética
3.
Cytotherapy ; 24(3): 235-248, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34656419

RESUMO

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses a never before seen challenge to human health and the economy. Considering its clinical impact, with no streamlined therapeutic strategies in sight, it is crucial to understand the infection process of SARS-CoV-2. Our limited knowledge of the mechanisms underlying SARS-CoV-2 infection impedes the development of alternative therapeutics to address the pandemic. This aspect can be addressed by modeling SARS-CoV-2 infection in the human context to facilitate drug screening and discovery. Human induced pluripotent stem cell (iPSC)-derived lung epithelial cells and organoids recapitulating the features and functionality of the alveolar cell types can serve as an in vitro human model and screening platform for SARS-CoV-2. Recent studies suggest an immune system asynchrony leading to compromised function and a decreased proportion of specific immune cell types in coronavirus disease 2019 (COVID-19) patients. Replenishing these specific immune cells may serve as useful treatment modality against SARS-CoV-2 infection. Here the authors review protocols for deriving lung epithelial cells, alveolar organoids and specific immune cell types, such as T lymphocytes and natural killer cells, from iPSCs with the aim to aid investigators in making relevant in vitro models of SARS-CoV-2 along with the possibility derive immune cell types to treat COVID-19.


Assuntos
COVID-19 , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Estudos Prospectivos , SARS-CoV-2
4.
Invest Ophthalmol Vis Sci ; 62(3): 2, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33646290

RESUMO

Purpose: To determine the status of proangiogenic factors in the tear fluid of preterm infants with and without retinopathy of prematurity (ROP). Methods: Preterm infants (n = 36) undergoing routine ROP screening included in the prospective study were categorized as No-ROP (n = 13, no ROP at any visits), ROP (if ROP was present at first visit; n = 18), or No-ROP to ROP (no disease at first visit, but developed ROP subsequently; n = 5). Infants with ROP were also grouped as progressing (n = 7) and regressing (n = 16) based on ROP evolution between the first and subsequent visits. Schirmer's strips were used to collect tear fluid and proangiogenic factors (VEGF, angiogenin, soluble vascular cell adhesion molecule, and fractalkine) levels (in picograms per milliliter) in tear fluid were measured by multiplex ELISA. Results: Lower levels of VEGF (135 ± 69; mean ± standard deviation) and higher levels of angiogenin (6568 ± 4975) were observed in infants with ROP compared with infants without ROP (172.5 ± 54.0; 4139 ± 3909) at the first visit. Significantly lower levels of VEGF were observed in the No-ROP to ROP group compared with the No-ROP and ROP groups. The VEGF and angiogenin levels at the first visit were significantly lower in infants with ROP with progressing disease. Angiogenin levels negatively correlated with birth weight and gestational age in ROP. The area under the curve (AUC) and odds ratio (OR) analysis demonstrated that angiogenin/birth weight (AUC = 0.776; OR, 8.6); angiogenin/gestational age (AUC = 0.706; OR, 7.3) and Angiogenin/VEGF (AUC = 0.806; OR, 14.3) ratios were able to differentiated preterm infants with and without ROP. Conclusions: The association between angiogenin and ROP suggests its possible role in ROP. The ratio of angiogenin level with birth weight, gestational age, and/or VEGF could serve as a potential noninvasive screening biomarker for ROP.


Assuntos
Indutores da Angiogênese/metabolismo , Biomarcadores/metabolismo , Recém-Nascido Prematuro/metabolismo , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/metabolismo , Lágrimas/metabolismo , Área Sob a Curva , Peso ao Nascer , Quimiocina CX3CL1/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/metabolismo , Feminino , Seguimentos , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Razão de Chances , Projetos Piloto , Estudos Prospectivos , Ribonuclease Pancreático/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Cytotherapy ; 23(6): 471-482, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33257213

RESUMO

The end of 2019 saw the beginning of the coronavirus disease 2019 (COVID-19) pandemic that soared in 2020, affecting 215 countries worldwide, with no signs of abating. In an effort to contain the spread of the disease and treat the infected, researchers are racing against several odds to find an effective solution. The unavailability of timely and affordable or definitive treatment has caused significant morbidity and mortality. Acute respiratory distress syndrome (ARDS) caused by an unregulated host inflammatory response toward the viral infection, followed by multi-organ dysfunction or failure, is one of the primary causes of death in severe cases of COVID-19 infection. Currently, empirical management of respiratory and hematological manifestations along with anti-viral agents is being used to treat the infection. The quest is on for both a vaccine and a more definitive management protocol to curtail the spread. Researchers and clinicians are also exploring the possibility of using cell therapy for severe cases of COVID-19 with ARDS. Mesenchymal stromal cells are known to have immunomodulatory properties and have previously been used to treat viral infections. This review explores the potential of mesenchymal stromal cells as cell therapy for ARDS.


Assuntos
COVID-19/epidemiologia , COVID-19/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Pandemias , Síndrome do Desconforto Respiratório/epidemiologia , Síndrome do Desconforto Respiratório/cirurgia , SARS-CoV-2 , Animais , COVID-19/virologia , Ensaios Clínicos como Assunto , Comorbidade , Humanos , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Síndrome do Desconforto Respiratório/virologia , Resultado do Tratamento
6.
Ocul Surf ; 18(2): 237-248, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31756391

RESUMO

PURPOSE: Corneal dystrophies (CD) are classified as rare eye diseases that results in visual impairment and requires corneal transplant in advanced stages. Ocular surface inflammatory status in different types of CD remains underexplored. Hence, we studied the levels of tear soluble factors in the tears of patients with various types of corneal dystrophies. METHODS: 17 healthy subjects and 30 CD subjects (including epithelial, stromal and endothelial CD) were included in the study. Schirmer's strips were used to collect the tear fluid in all subjects. 27 soluble factors including cytokines, chemokines, soluble cell adhesion molecules and growth factors were measured in the eluted tears by multiplex ELISA or single analyte sandwich ELISA. RESULTS: Percentages of subjects with detectable levels of tear soluble factors were significantly higher in CD compared to controls. Significant higher level of IL-2 was observed in both epithelial and stromal CD. IL-4, TGFß1 and IgE were significantly higher in stromal CD. VCAM, IL-13 and Fractalkine were significantly elevated in epithelial and macular CD. IL-1α, IL-8, IL-12, ANG, Eotaxin, MCP1, RANTES, ICAM1, L-selectin and P-selectin were significantly higher in epithelial CD. TGFBIp was significantly elevated in lattice CD and endothelial CD. CONCLUSION: Distinct set of the tear soluble factors were dysregulated in various types of CD. Increase in tear inflammatory factors was observed in majority of the CD subjects depending on their sub-types. This suggests a plausible role of aberrant inflammation in CD pathobiology. Hence, modulating inflammation could be a potential strategy in improving the prognosis of CD.


Assuntos
Distrofias Hereditárias da Córnea , Citocinas , Ensaio de Imunoadsorção Enzimática , Olho , Humanos , Lágrimas
7.
Stem Cell Res Ther ; 9(1): 287, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30359313

RESUMO

Human-induced pluripotent stem cells (hiPSCs) provide a personalized approach to study conditions and diseases including those of the eye that lack appropriate animal models to facilitate the development of novel therapeutics. Corneal disease is one of the most common causes of blindness. Hence, significant efforts are made to develop novel therapeutic approaches including stem cell-derived strategies to replace the diseased or damaged corneal tissues, thus restoring the vision. The use of adult limbal stem cells in the management of corneal conditions has been clinically successful. However, its limited availability and phenotypic plasticity necessitate the need for alternative stem cell sources to manage corneal conditions. Mesenchymal and embryonic stem cell-based approaches are being explored; nevertheless, their limited differentiation potential and ethical concerns have posed a significant hurdle in its clinical use. hiPSCs have emerged to fill these technical and ethical gaps to render clinical utility. In this review, we discuss and summarize protocols that have been devised so far to direct differentiation of human pluripotent stem cells (hPSCs) to different corneal cell phenotypes. With the summarization, our review intends to facilitate an understanding which would allow developing efficient and robust protocols to obtain specific corneal cell phenotype from hPSCs for corneal disease modeling and for the clinics to treat corneal diseases and injury.


Assuntos
Transplante de Córnea , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Doenças da Córnea/terapia , Epitélio Corneano/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fenótipo , Pesquisa Translacional Biomédica
8.
Stem Cell Reports ; 2(4): 520-33, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24749075

RESUMO

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.


Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Animais , Biomarcadores , Metilação de DNA , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética
9.
J Neurochem ; 130(4): 472-89, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24773031

RESUMO

Dopamine replacement therapy in Parkinson's disease is associated with several unwanted effects, of which dyskinesia is the most disabling. The development of new therapeutic interventions to reduce the impact of dyskinesia in Parkinson's disease is therefore a priority need. This review summarizes the key molecular mechanisms that underlie dyskinesia. The role of dopamine receptors and their associated signaling mechanisms including dopamine-cAMP-regulated neuronal phosphoprotein, extracellular signal-regulated kinase, mammalian target of rapamycin, mitogen and stress-activated kinase-1 and Histone H3 are summarized, along with an evaluation of the role of cannabinoid and nicotinic acetylcholine receptors. The role of synaptic plasticity and animal behavioral results on dyskinesia are also evaluated. The most recent therapeutic advances to treat Parkinson's disease are discussed, with emphasis on the possibilities and limitations of non-pharmacological interventions such as physical activity, deep brain stimulation, transcranial magnetic field stimulation and cell replacement therapy. The review suggests new prospects for the management of Parkinson's disease-associated motor symptoms, especially the development of dyskinesia. This review aims at summarizing the key molecular mechanisms underlying dyskinesia and the most recent therapeutic advances to treat Parkinson's disease with emphasis on non-pharmacological interventions such as physical activity, deep brain stimulation (DBS), transcranial magnetic field stimulation (TMS) and cell replacement therapy. These new interventions are discussed from both the experimental and clinical point of view, describing their current strength and limitations.


Assuntos
Discinesias/etiologia , Discinesias/terapia , Doença de Parkinson/complicações , Doença de Parkinson/terapia , Animais , Canabinoides/farmacologia , Transplante de Células , Cromatina/efeitos dos fármacos , Estimulação Encefálica Profunda , Dopaminérgicos/uso terapêutico , Fosfoproteína 32 Regulada por cAMP e Dopamina/fisiologia , Discinesias/fisiopatologia , Histonas/metabolismo , Humanos , Levodopa/farmacologia , Levodopa/uso terapêutico , Atividade Motora/fisiologia , Doença de Parkinson/fisiopatologia , Fosforilação , Desempenho Psicomotor/fisiologia , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estimulação Magnética Transcraniana
10.
Biol Open ; 1(8): 693-704, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213462

RESUMO

Meso-diencephalic dopaminergic (mdDA) neurons are critical for motor control and cognitive functioning and their loss or dysfunction is associated with disorders such as Parkinson's disease (PD), schizophrenia and addiction. However, relatively little is known about the molecular mechanisms underlying mdDA neuron development and maintenance. Here, we determined the spatiotemporal map of genes involved in the development of mdDA neurons to gain further insight into their molecular programming. Genome-wide gene expression profiles of the developing ventral mesencephalon (VM) were compared at different developmental stages leading to the identification of novel regulatory roles of neuronal signaling through nicotinic acthylcholine receptors (Chrna6 and Chrnb3 subunits) and the identification of novel transcription factors (Oc2 and 3) involved in the generation of the mdDA neuronal field. We show here that Pitx3, in cooperation with Nurr1, is the critical component in the activation of the Chrna6 and Chrnb3 subunits in mdDA neurons. Furthermore, we provide evidence of two divergent regulatory pathways resulting in the expression of Chrna6 and Chrnb3 respectively.

11.
Neuropsychopharmacology ; 34(12): 2489-96, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19675532

RESUMO

Although serum autoantibodies directed against basal ganglia (BG) implicate autoimmunity in the pathogenesis of obsessive-compulsive disorder (OCD), it is unclear whether these antibodies can cross the blood-brain barrier to bind against BG or other components of the OCD circuit. It is also unclear how they might lead to hyperactivity in the OCD circuit. We examined this by investigating the presence of autoantibodies directed against the BG or thalamus in the serum as well as CSF of 23 OCD patients compared with 23 matched psychiatrically normal controls using western blot. We further investigated CSF amino acid (glutamate, GABA, taurine, and glycine) levels and also examined the extent to which these levels were related to the presence of autoantibodies. There was evidence of significantly more binding of CSF autoantibodies to homogenate of BG as well as to homogenate of thalamus among OCD patients compared with controls. There was no significant difference in binding between patient and control sera except for a trend toward more bands to BG and thalamic protein corresponding to 43 kD among OCD patients compared with controls. CSF glutamate and glycine levels were also significantly higher in OCD patients compared with controls, and further multivariate analysis of variance showed that CSF glycine levels were higher in those OCD patients who had autoantibodies compared with those without. The results of our study implicate autoimmune mechanisms in the pathogenesis of OCD and also provide preliminary evidence that autoantibodies against BG and thalamus may cause OCD by modulating excitatory neurotransmission.


Assuntos
Autoanticorpos/metabolismo , Gânglios da Base/imunologia , Neurotransmissores/metabolismo , Transtorno Obsessivo-Compulsivo/imunologia , Transtorno Obsessivo-Compulsivo/metabolismo , Tálamo/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Autoanticorpos/líquido cefalorraquidiano , Feminino , Ácido Glutâmico/líquido cefalorraquidiano , Glicina/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neurotransmissores/líquido cefalorraquidiano , Taurina/líquido cefalorraquidiano , Adulto Jovem , Ácido gama-Aminobutírico/líquido cefalorraquidiano
12.
Biol Chem ; 389(7): 791-8, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18627319

RESUMO

The Ras signaling pathway plays a predominant role during development and controls diverse biological process in all eukaryotic cells. It is a member of the large family of GTPases proteins that binds and hydrolyzes GTP. Ras is a lipid-anchored protein on the intracellular membrane compartments, and cycles between inactive GDP-bound and the signaling competent GTP-bound conformation. Studies have demonstrated Ras to be a central regulator in signal transduction pathways responding to diverse extracellular and intracellular stimuli. Much progress has been made towards delineating specific genes involved in the process of pluripotency and differentiation of stem cells. Here, we discuss recent aspects of Ras signaling pathways in mediating stem cell properties.


Assuntos
Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas ras/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
13.
Eur J Neurosci ; 25(7): 1971-81, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17439485

RESUMO

The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt/PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction.


Assuntos
Dopamina/metabolismo , Mesencéfalo/citologia , Neurônios/fisiologia , Fármacos Neuroprotetores/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Oxidopamina/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Técnicas de Patch-Clamp , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Proteínas ras/genética
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