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1.
Life Sci ; 284: 119923, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34481865

RESUMO

AIMS: Levetiracetam (LEV) is a broad-spectrum antiepileptic drug with neuroprotective properties and novel mechanisms of action. Some evidence suggests that LEV may impact adult neurogenesis, but the results are controversial. The present study was aimed to evaluate the effects of LEV on the proliferation and differentiation of rat embryonic neural stem cells (NSCs) and to explore the role of GABAB or NMDA receptors. MAIN METHODS: NSCs were isolated from rat fetal ganglionic eminence at embryonic day 14.5. The effects of LEV on viability, proliferation, neurosphere formation, and neuronal or astroglial differentiation of NSCs were assessed using resazurin, BrdU incorporation, immunocytochemistry, quantitative real-time PCR, and western blotting. Additionally, we addressed the relationship between treatment with NMDA and GABAB receptor antagonists (MK801 and saclofen, respectively) in combination with LEV on these parameters. KEY FINDINGS: The data showed that LEV (50 µM) significantly increased the number (p < 0.01) and diameter of neurospheres (p < 0.05), enhanced proliferation (p < 0.01), and promoted neuronal differentiation, as revealed by significantly increased expressions of DCX and NeuN. The expressions of astroglial markers, GFAP and Olig2, were markedly reduced. The addition of MK801 (10 µM) significantly diminished neurospheres growth (p < 0.001), decreased the number of proliferating cells (p < 0.01), and reduced the number of new neurons (p < 0.001) but increased the astroglial cells (p < 0.001) induced by LEV. Co-treatment with saclofen (25 µM) did not significantly affect LEV-induced NSCs proliferation and differentiation. SIGNIFICANCE: Our findings suggest that LEV may enhance rat embryonic neurogenesis mainly through an NMDA receptor-mediated mechanism.


Assuntos
Embrião de Mamíferos/fisiologia , Levetiracetam/farmacologia , Neurogênese/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Feminino , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos
2.
Anticancer Res ; 41(9): 4215-4228, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475041

RESUMO

BACKGROUND/AIM: Tyrosine kinase inhibitors (TKIs) are used for the treatment of both wild type and mutant non-small cell lung cancer (NSCLC); however, acquired resistance is a major clinical challenge. Herein, we aimed to investigate the effects of telmisartan (Tel), CFM 4.16 and sorafenib combination in rociletinib resistant NSCLC tumors. MATERIALS AND METHODS: 3D spheroid cultures and western blotting were used for evaluating cytotoxic effects and protein expression. An in vivo rociletinib resistant H1975 xenograft model of NSCLC was developed by subcutaneous injection of rociletinib resistant H1975 cells into nude mice. RESULTS: Tel, CFM 4.16 and sorafenib combination displayed superior anti-cancer effects in 3D spheroid cultures and a rociletinib resistant H1975 xenograft model of NSCLC by decreasing the protein expression of oncogenic and cancer stem cell markers (Nanog, Sox2 and Oct4). CONCLUSION: Tel facilitates effective penetration of CFM 4.16 and sorafenib in rociletinib resistant H1975 models of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Esferoides Celulares/citologia , Compostos de Espiro/administração & dosagem , Telmisartan/administração & dosagem , Tiadiazóis/administração & dosagem , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Esferoides Celulares/efeitos dos fármacos , Compostos de Espiro/farmacologia , Telmisartan/farmacologia , Tiadiazóis/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445168

RESUMO

Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that investigate the effects of OXT on a cellular/molecular level. In this study, we established a hypothalamic neuronal spheroid model to investigate the cellular response in a more realistic 3D setting. Our data indicate that the formation of spheroids itself does not alter the basic characteristics of the cell line and that markers of cellular morphology and connectivity are stably expressed. We found that both OXT and arginine vasopressin (AVP) treatment increase spheroid size (surface area and volume), as well as individual nucleus size, which serves as an indicator for cellular proliferation. The cellular response to both OXT and AVP seems mainly to be mediated by the AVP receptor 1a (V1aR); however, the OXT receptor (OXTR) contributes significantly to the observed proliferative effect. When we blocked the OXTR pharmacologically or knocked down the OXTR by siRNA, the OXT- or AVP-induced cellular proliferation decreased. In summary, we established a 3D cell culture model of the neuronal response to OXT and AVP and found that spheroids react to the treatment via their respective receptors but also via cross-talk between the two receptor types.


Assuntos
Hipotálamo/citologia , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Arginina Vasopressina/metabolismo , Linhagem Celular , Proliferação de Células , Hipotálamo/metabolismo , Ocitocina/metabolismo , Ratos , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
4.
Biomed Res Int ; 2021: 5540877, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337022

RESUMO

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/citologia , Esferoides Celulares/citologia , Adipogenia , Apoptose , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Ciclo Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Consumo de Oxigênio , Células-Tronco Pluripotentes/metabolismo , Esferoides Celulares/metabolismo
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360970

RESUMO

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.


Assuntos
Ligamento Cruzado Anterior/fisiologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Ligamento Cruzado Anterior/citologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Decorina/genética , Decorina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Masculino , Poliésteres/química , Coelhos , Regeneração , Esferoides Celulares/metabolismo , Estresse Mecânico
6.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202056

RESUMO

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Esferoides Celulares , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Isquemia/diagnóstico , Isquemia/etiologia , Isquemia/metabolismo , Isquemia/terapia , Imageamento por Ressonância Magnética , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/etiologia , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/terapia , Esferoides Celulares/citologia , Esferoides Celulares/ultraestrutura , Coloração e Rotulagem
7.
Int J Biol Macromol ; 185: 87-97, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34144066

RESUMO

The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N-octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology.


Assuntos
1-Octanol/química , Neoplasias Encefálicas/tratamento farmacológico , Quitosana/química , Glioblastoma/tratamento farmacológico , Esferoides Celulares/citologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrogéis , Esferoides Celulares/química , Esferoides Celulares/efeitos dos fármacos
8.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065977

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30-40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14+ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. METHODS: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. RESULTS: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14+CD206+ and CD64+CD206+ populations in CD11b+ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14+ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b+CD14+ population and induce alterations in the sphericity of the cell cultures. CONCLUSION: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.


Assuntos
Neoplasias Encefálicas/metabolismo , Técnicas de Cocultura/métodos , Glioblastoma/metabolismo , Macrófagos/citologia , Monócitos/citologia , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Mitocondriais/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Cultura Primária de Células , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células THP-1
9.
Chem Biol Interact ; 345: 109565, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34161784

RESUMO

In previous study, we examined the anticancer effects of novel Biginelli-hybrids against HeLa cell line on 2D monolayer culture. The five most effective compounds were chosen for further analysis of their anticancer activity against HeLa spheroids. Using the 3D models implies the possible differences in anticancer effects and mechanisms of activity of tested compounds. The compounds 4c and 4d exerted the strongest activity against 3D HeLa spheroids and induced to some extent loosened cell-cell contacts in spheroids, leading to the largest reduction in the diameter of the spheroids. Additionally, the highest accumulation of the cells in the subG1 phase of the cell cycle was observed after the treatment with compounds 4d and 4c, while the compound 4f led to the G2/M arrest. The invasion potential of treated HeLa cells in spheroids was monitored by imaging of spheroids embedded in a matrix made of matrigel and collagen and by determination of MMP2, MMP9, and VEGF gene expression levels. The compound 4l did not show invasion-suppressive activity, while the compounds 4c and 4d exerted the strongest anti-invasive activity.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Esferoides Celulares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Esferoides Celulares/citologia , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Int J Biol Macromol ; 184: 768-775, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174305

RESUMO

Polysaccharide hydrogels are promising candidate matrices for recapitulating the characteristics of extracellular matrix (ECM) in breast tumors in terms of their structure and composition. Herein, to obtain an ECM-mimetic matrix, hydroxyethyl chitosan (HECS) hydrogels were prepared through Schiff-base crosslinking reaction using dialdehyde hyaluronic acid as crosslinker. The obtained HECS hydrogels displayed a highly porous structure, a stiffness comparable to that of breast tissue, and a fast water-absorption speed. The amount of crosslinker had great effects on the swelling and rheological behaviors of the HECS hydrogels. Preliminary results from in vitro biological assessments confirmed that MCF-7 cells incubated within HECS hydrogels preferred to grow into three-dimensional spheroids. Importantly, the cells displayed enhanced migrative capability and upregulated expression levels of MMP-2, TGF-ß and VEGF in comparison to two-dimension cultured cells. Hence, the HECS hydrogels show great promise as a biomimetic ECM in constructing breast tumor models.


Assuntos
Neoplasias da Mama/metabolismo , Quitosana/química , Matriz Extracelular/metabolismo , Ácido Hialurônico/química , Hidrogéis/síntese química , Esferoides Celulares/citologia , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Porosidade , Bases de Schiff , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
J Adv Res ; 30: 103-112, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34026290

RESUMO

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- ß1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.


Assuntos
Folículo Piloso/citologia , Folículo Piloso/metabolismo , Cabelo/fisiologia , Queratinócitos/metabolismo , Regeneração , Animais , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/metabolismo , Derme/metabolismo , Epitélio/metabolismo , Humanos , Camundongos , Camundongos Nus , Fenótipo , Pele/metabolismo , Esferoides Celulares/citologia
12.
Sci Rep ; 11(1): 9356, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931678

RESUMO

The endocannabinoid system (ECS) plays a complex role in the development of neural circuitry during fetal brain development. The cannabinoid receptor type 1 (CB1) controls synaptic strength at both excitatory and inhibitory synapses and thus contributes to the balance of excitatory and inhibitory signaling. Imbalances in the ratio of excitatory to inhibitory synapses have been implicated in various neuropsychiatric disorders associated with dysregulated central nervous system development including autism spectrum disorder, epilepsy, and schizophrenia. The role of CB1 in human brain development has been difficult to study but advances in induced pluripotent stem cell technology have allowed us to model the fetal brain environment. Cortical spheroids resemble the cortex of the dorsal telencephalon during mid-fetal gestation and possess functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization. We first characterized the ECS using STORM microscopy and observed synaptic localization of components similar to that which is observed in the fetal brain. Next, using the CB1-selective antagonist SR141716A, we observed an increase in excitatory, and to a lesser extent, inhibitory synaptogenesis as measured by confocal image analysis. Further, CB1 antagonism increased the variability of spontaneous activity within developing neural networks, as measured by microelectrode array. Overall, we have established that cortical spheroids express ECS components and are thus a useful model for exploring endocannabinoid mediation of childhood neuropsychiatric disease.


Assuntos
Encéfalo/fisiologia , Córtex Cerebral/fisiologia , Feto/fisiologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto/farmacologia , Esferoides Celulares/fisiologia , Sinapses/fisiologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Antagonistas de Receptores de Canabinoides/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feto/citologia , Feto/efeitos dos fármacos , Humanos , Transdução de Sinais , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Sinapses/efeitos dos fármacos
13.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946876

RESUMO

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.


Assuntos
Cervos/anatomia & histologia , Folículo Piloso/citologia , Antígeno AC133/biossíntese , Antígeno AC133/genética , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Biomarcadores , Agregação Celular , Técnicas de Cultura de Células , Divisão Celular , Células Cultivadas , Cervos/genética , Regulação da Expressão Gênica , Ontologia Genética , Cabelo , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Mesoderma/citologia , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Especificidade da Espécie , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Transcriptoma , Versicanas/biossíntese , Versicanas/genética
14.
Life Sci ; 278: 119554, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932444

RESUMO

AIMS: Chemoresistance remains a persistent challenge in advanced prostate cancer therapy. Probenecid reportedly inhibits multiple drug-efflux transporters; hence, it can be employed as a potential sensitizer for chemotherapy. In the present study, we evaluated the effects of probenecid on three-dimensional (3D)-cultures of prostate cancer cells. MAIN METHODS: Prostate cancer cell lines, 22Rv1 and PC-3 were cultured as multicellular tumor spheroids. The effects of probenecid were evaluated using the MTT assay for viability, microscopy for spheroid size, and soft agar colony formation assay for anchorage-independent growth. KEY FINDINGS: The 3D-cultured 22Rv1 cells were less sensitive to cisplatin and doxorubicin than two-dimensional (2D) cell culture. Co-administration of probenecid at a low (100 or 300 µM), but not high (500 µM), concentration increased the sensitivity to cisplatin or doxorubicin in 22Rv1 spheroids. Probenecid increased the expression of ABCG2, a multidrug resistance transporter, in a dose-dependent manner. Furthermore, treatment with probenecid alone reduced the growth of 22Rv1 spheroids. Conversely, probenecid inhibited spheroid compaction rather than growth inhibition in 3D-cultured PC-3 cells. Moreover, probenecid inhibited colony formation of 22Rv1 and PC-3 cells in soft agar, as well as downregulated focal adhesion kinase (FAK), a crucial factor in anchorage-independent growth. SIGNIFICANCE: In 3D-cultured prostate cancer cells, probenecid demonstrated pleiotropic effects such as chemosensitization, growth suppression, inhibition of spheroid compaction, and suppression of anchorage-independent growth. Elucidating the detailed mechanism underlying these probenecid actions could result in the identification of novel therapeutic targets toward the advanced prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Probenecid/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
15.
Parasit Vectors ; 14(1): 213, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879231

RESUMO

BACKGROUND: Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation. METHODS: A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection. RESULTS: Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq. CONCLUSIONS: These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.


Assuntos
Ductos Biliares/parasitologia , Clonorquíase/genética , Clonorchis sinensis/metabolismo , Proteínas de Helminto/metabolismo , Esferoides Celulares/parasitologia , Animais , Ductos Biliares/citologia , Clonorquíase/metabolismo , Clonorquíase/parasitologia , Clonorchis sinensis/genética , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Perfilação da Expressão Gênica , Proteínas de Helminto/genética , Humanos , Masculino , Coelhos , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
16.
Cells ; 10(4)2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33916870

RESUMO

Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell-cell and cell-matrix interplay within the tumor-stroma microenvironment.


Assuntos
Tecido Adiposo/citologia , Bioimpressão , Neoplasias da Mama/patologia , Diferenciação Celular , Modelos Biológicos , Esferoides Celulares/citologia , Adipogenia , Sobrevivência Celular , Matriz Extracelular/metabolismo , Feminino , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Impressão Tridimensional , Células Estromais/citologia
17.
Cells ; 10(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804895

RESUMO

The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Esferoides Celulares/citologia , Geleia de Wharton/citologia , Biomarcadores/metabolismo , Forma Celular , Sobrevivência Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo
18.
Sci Rep ; 11(1): 9149, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33911091

RESUMO

Ovarian cancer is associated with a high mortality rate due to diagnosis at advanced stages. Dissemination often occurs intraperitoneally within the ascites fluid. The microenvironment can support dissemination through several mechanisms. One potential ascites factor which may mediate dissemination are EVs or extracellular vesicles that can carry information in the form of miRNAs, proteins, lipids, and act as mediators of cellular communication. We present our observations on EVs isolated from ascitic supernatants from patients diagnosed with high grade serous ovarian carcinoma in augmenting motility, growth, and migration towards omental fat. MicroRNA profiling of EVs from malignant ascitic supernatant demonstrates high expression of miR 200c-3p, miR18a-5p, miR1246, and miR1290 and low expression of miR 100- 5p as compared to EVs isolated from benign ascitic supernatant. The migration of ovarian cancer spheroids towards omental fat is enhanced in the presence of malignant ascitic EVs. Gene expression of these cells showed increased expression of ZBED2, ZBTB20, ABCC3, UHMK1, and low expression of Transgelin and MARCKS. We present evidence that ovarian ascitic EVs increase the growth of ovarian cancer spheroids through miRNAs.


Assuntos
Líquido Ascítico/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias Ovarianas/patologia , Idoso , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neoplasias Ovarianas/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Microambiente Tumoral , Regulação para Cima/efeitos dos fármacos
19.
Dev Cell ; 56(9): 1326-1345.e6, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33887203

RESUMO

The interplay between hypothalamic neurons and microglia as they integrate stressors to regulate homeostasis is of growing interest. We asked if microglia in the embryonic hypothalamus were likewise stress responsive and, if so, whether their precocious activation perturbs nearby neural stem cell (NSC) programs. We performed single-cell transcriptomics to define embryonic hypothalamic microglia heterogeneity and identified four microglial subsets, including a subpopulation adjacent to NSCs that was responsive to gestational cold stress. Stress exposure elevated CCL3 and CCL4 secretion, but only in male brains, and ex vivo CCL4 treatment of hypothalamic NSCs altered proliferation and differentiation. Concomitantly, gestational stress decreased PVN oxytocin neurons only in male embryos, which was reversed by microglia depletion. Adult offspring exposed to gestational stress displayed altered social behaviors, which was likewise microglia dependent, but only in males. Collectively, immature hypothalamic microglia play an unappreciated role in translating maternal stressors to sexually dimorphic perturbation of neurodevelopmental programs.


Assuntos
Embrião de Mamíferos/citologia , Microglia/citologia , Células-Tronco Neurais/citologia , Estresse Fisiológico , Animais , Comportamento Animal , Contagem de Células , Diferenciação Celular/genética , Proliferação de Células/genética , Temperatura Baixa , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Masculino , Camundongos , Microglia/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Oligodendroglia/citologia , Núcleo Hipotalâmico Paraventricular/citologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Análise de Célula Única , Comportamento Social , Esferoides Celulares/citologia
20.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923671

RESUMO

Stem cell therapy is one of the most promising candidate treatments for spinal cord injury. Research has shown optimistic results for this therapy, but clinical limitations remain, including poor viability, engraftment, and differentiation. Here, we isolated novel peripheral nerve-derived stem cells (PNSCs) from adult peripheral nerves with similar characteristics to neural-crest stem cells. These PNSCs expressed neural-crest specific markers and showed multilineage differentiation potential into Schwann cells, neuroglia, neurons, and mesodermal cells. In addition, PNSCs showed therapeutic potential by releasing the neurotrophic factors, including glial cell-line-derived neurotrophic factor, insulin-like growth factor, nerve growth factor, and neurotrophin-3. PNSC abilities were also enhanced by their development into spheroids which secreted neurotrophic factors several times more than non-spheroid PNSCs and expressed several types of extra cellular matrix. These features suggest that the potential for these PNSC spheroids can overcome their limitations. In an animal spinal cord injury (SCI) model, these PNSC spheroids induced functional recovery and neuronal regeneration. These PNSC spheroids also reduced the neuropathic pain which accompanies SCI after remyelination. These PNSC spheroids may represent a new therapeutic approach for patients suffering from SCI.


Assuntos
Esferoides Celulares/transplante , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal , Transplante de Células-Tronco/métodos , Animais , Células Cultivadas , Células-Tronco Neurais/citologia , Neurogênese , Nervos Periféricos/citologia , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Esferoides Celulares/citologia
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