Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.741
Filtrar
1.
FASEB J ; 35(11): e21896, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34634154

RESUMO

Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuritos/metabolismo , Seda/farmacologia , Aranhas/metabolismo , Vitronectina/farmacologia , Animais , Autoenxertos , Materiais Biocompatíveis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Humanos , Laminina/farmacologia , Camundongos , Neuritos/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/cirurgia , Engenharia de Proteínas/métodos , Ratos , Proteínas Recombinantes/farmacologia , Seda/genética , Vitronectina/genética
2.
PLoS One ; 16(8): e0255075, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34375370

RESUMO

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.


Assuntos
Células Endoteliais/patologia , Fibroblastos/patologia , Neovascularização Fisiológica , Doença Arterial Periférica/patologia , Acetilação/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Fibroblastos/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Isquemia/patologia , Isquemia/terapia , Laminina/farmacologia , Lipoproteínas LDL/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Fisiológica/efeitos dos fármacos , Perfusão , Lectinas de Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteoglicanas/farmacologia
3.
Exp Cell Res ; 405(2): 112710, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34174319

RESUMO

Immune cells not only constitute tumour microenvironment but they may even affect disease prognosis as a result of dual functional roles that they may play in tumour tissues. Two frequently used established immune cell lines (lymphocytic Jurkat and monocytic THP-1) were used to test whether microenvironmental factors, especially molecular components of extracellular matrix, can shape the phenotype of immune cells. Proliferation, morphological and phenotypical analyses were applied to compare behaviour of the immune cells, typically cultured as suspensions in culture medium, with their behaviour in collagen type I-based and Matrigel-based 3D cultures. Density of both immune cell types in routine suspension cultures affected their subsequent proliferation in extracellular matrices. THP-1 cells appeared to be more sensitive to their surrounding microenvironment as judged from extracellular matrix type-dependent changes in their cell doubling times and from slight increase in their diameters in both extracellular matrix-containing cell cultures. Moreover, even chemically uninduced monocytic THP-1 cells were present in a minor fraction as CD68 positive cell population in collagen type I matrix indicating their partial differentiation to macrophages. Observed modifications of immune cells by microenvironmental factors may have profound implications for their roles in healthy and pathological tissues.


Assuntos
Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Fenótipo , Microambiente Tumoral/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno/farmacologia , Colágeno Tipo I/metabolismo , Combinação de Medicamentos , Humanos , Laminina/metabolismo , Laminina/farmacologia , Proteoglicanas/metabolismo , Proteoglicanas/farmacologia
4.
Exp Cell Res ; 403(2): 112599, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848551

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.


Assuntos
Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Adulto , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Colágeno/farmacologia , Colagenases/farmacologia , Meios de Cultura/química , Combinação de Medicamentos , Feminino , Fibronectinas/farmacologia , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/análogos & derivados , Insulina/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Laminina/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Proteoglicanas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
5.
J Vis Exp ; (168)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33720119

RESUMO

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.


Assuntos
Neoplasias Encefálicas/patologia , Comunicação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Rastreamento de Células , Glioma/patologia , Humanos , Processamento de Imagem Assistida por Computador , Laminina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
6.
Cells ; 10(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668931

RESUMO

Human adipose-derived stromal cells (ADSCs) are receiving unprecedented attention as a potential cellular source for regenerative medicine-based therapies against various diseases and conditions. However, there still have significant issues concerning the translational development of ADSC-based therapies, such as its heterogeneity and being prone to aging. We developed a new simple and economical 3D semi-suspended expansion method in which 3D spheroids reside on an ADSC-derived self-feeder cell layer, producing cells with increased population homogeneity and strong stemness and ensuring that the proliferation and differentiation potency of the cells does not become notably reduced after at least ten passages in culture. To check the potential application of the 3D ADSC spheroids, we discovered that the combination of siEID3, which is a small interfering RNA of EP300 inhibitor of differentiation 3 (EID3), and laminin/poly-d-lysine matrix can rapidly result in trans-differentiation of the 3D spheroid cells to neural progenitor-like cells (NPLCs) in approximately 9 days in vitro. This approach provides a multidisciplinary tool for stem cell research and production in mesenchymal stem cell-related fields.


Assuntos
Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Transdiferenciação Celular , Células Alimentadoras/citologia , Células-Tronco Neurais/citologia , Polilisina/farmacologia , Esferoides Celulares/citologia , Células-Tronco/citologia , Tecido Adiposo/citologia , Proliferação de Células , Senescência Celular , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Laminina/farmacologia , Modelos Biológicos , RNA Interferente Pequeno/metabolismo
7.
Mol Cell Probes ; 56: 101694, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33429040

RESUMO

The ability to preserve and transport human cells in a stable medium over long distances is critical to collaborative efforts and the advancement of knowledge in the study of human disease. This is particularly important in the study of rare diseases. Recently, advancements in the understanding of renal ciliopathies has been achieved via the use of patient urine-derived cells (UDCs). However, the traditional method of cryopreservation, although considered as the gold standard, can result in decreased sample viability of many cell types, including UDCs. Delays in transportation can have devastating effects upon the viability of samples, and may even result in complete destruction of cells following evaporation of dry ice or liquid nitrogen, leaving samples in cryoprotective agents, which are cytotoxic at room temperature. The loss of any patient sample in this manner is detrimental to research, however it is even more so when samples are from patients with a rare disease. In order to overcome the associated limitations of traditional practices, new methods of preservation and shipment, including cell encapsulation within hydrogels, and transport in specialised devices are continually being investigated. Here we summarise and compare traditional methods with emerging novel alternatives for the preservation and shipment of cells, and consider the effectiveness of such methods for use with UDCs to further enable the study and understanding of kidney diseases.


Assuntos
Encapsulamento de Células/métodos , Ciliopatias/terapia , Criopreservação/métodos , Células Epiteliais/citologia , Doenças Raras/terapia , Alginatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Quitosana/farmacologia , Ciliopatias/patologia , Colágeno/farmacologia , Crioprotetores/farmacologia , Combinação de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/transplante , Gelatina/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Rim/patologia , Laminina/farmacologia , Proteoglicanas/farmacologia , Doenças Raras/patologia , Transportes/métodos , Urotélio/citologia
8.
Laryngoscope ; 131(8): 1732-1740, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33135799

RESUMO

OBJECTIVES: One of the greatest hurdles in tracheal tissue engineering is insufficient vascularization, which leads to delayed mucosal regeneration, inflammation, and restenosis. This study investigated whether a prevascularized segmental tracheal substitute using platysma can enhance tracheal mucosal regeneration. METHODS: Three-dimensional (3D) printed scaffolds with (group M) or without (group S) Matrigel coating were implanted under the feeding vessels of the platysma in New Zealand White rabbits (n = 3) to induce vascularization. After 1 or 2 weeks, tracheal defects were created and vascularized scaffolds with feeders of the platysma were transplanted as rotational flaps. As controls, scaffolds with or without Matrigel coating was transplanted into a tracheal defect without prevascularization. Airway patency and epithelization were examined using a rigid bronchoscope every 2 weeks. Surviving animals were euthanized at 24 weeks, and microcomputed tomography and histological evaluation were performed. RESULTS: Animals with 2 weeks of prevascularization showed longer survival than animals with 0 or 1 weeks of prevascularization regardless of the Matrigel coating. Wider airway patency was observed in group M than group S. Group M showed migration of epithelium over the scaffold from 4 weeks after transplantation and complete coverage with epithelium at 12 weeks, whereas group S showed migration of the epithelium from 14 weeks and incomplete coverage with epithelium even at 24 weeks. CONCLUSION: This two-step method, utilizing the platysma as an in vivo bioreactor, may be a promising approach to achieve long-term survival and enhanced luminal patency. Matrigel coating on the scaffold had a synergistic effect on epithelial regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1732-1740, 2021.


Assuntos
Regeneração/efeitos dos fármacos , Ritidoplastia/métodos , Retalhos Cirúrgicos/transplante , Traqueia/cirurgia , Remodelação das Vias Aéreas/fisiologia , Animais , Materiais Biocompatíveis/farmacologia , Colágeno/farmacologia , Combinação de Medicamentos , Laminina/farmacologia , Masculino , Modelos Animais , Impressão Tridimensional/normas , Proteoglicanas/farmacologia , Coelhos , Regeneração/fisiologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/transplante , Retalhos Cirúrgicos/irrigação sanguínea , Engenharia Tecidual/métodos , Engenharia Tecidual/estatística & dados numéricos , Tecidos Suporte , Microtomografia por Raio-X/métodos
9.
Transplant Proc ; 53(1): 450-456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32928554

RESUMO

BACKGROUND: Islets transplanted under the ear skin would allow easy observation of the graft response and survival in vivo. This research was designed to establish an efficient mouse islet transplant model to probe the dynamic cellular interplay in vivo. METHODS: Green fluorescent protein transgenic mice and BALB/c mice were used as donors and recipients. All recipients were divided into 6 groups of 6 mice each. First, we treated the transplant recipients, including diabetes induction, autologous epididymal fat pad, and MATRIGEL transplant to the ears. Then, 1. we transplanted isolated islets to the ear/ear with fat/ear with MATRIGEL; and 2. transplanted islets with collagen + basic fibroblast growth factor or islets with collagen + vascular endothelial growth factor. Mice in the control group received a sham transplantation with phosphate buffer saline. All recipients were then observed for 30 days with blood glucose (BG) monitoring. Finally, ears were removed with graft on day 28 for histologic examination. RESULTS: It was suggested that transplant of islets alone could not correct hyperglycemia. Fat, MATRIGEL, collagen, and growth factors have the similar function to form a microenvironment conducive to islet survival. The effect of islet transplantation for correcting hyperglycemia of the fat modification group was better than other groups (P < .05). BG could be normalized, and living islets were detected by anti-insulin immunohistochemistry. CONCLUSIONS: Transplant islets into the ear with transplanted autologous fat is the optimal way which can be used to analyze the allograft response in vivo and track cell population and migration using labels by confocal microscopy.


Assuntos
Tecido Adiposo/transplante , Colágeno/farmacologia , Modelos Animais de Doenças , Orelha , Transplante das Ilhotas Pancreáticas , Laminina/farmacologia , Proteoglicanas/farmacologia , Animais , Glicemia/metabolismo , Colágeno/metabolismo , Diabetes Mellitus Experimental/sangue , Combinação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Sobrevivência de Enxerto , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante Homólogo/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Bioorg Chem ; 107: 104516, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33293057

RESUMO

An analog of γ1 laminin (RDIAEIIKDI) decapeptide has been used to augment neuronal survival and regeneration after injuries, during aging and other CNS disorder. As a prime synthetic peptide, KDI, is responsible for the neurite outgrowth of human embryonic neurons. In this study, we have designed, modified a KDI derivative and synthesized by replacing isoleucine (I) with Pro (P) amino acid at C-terminal to enhance its potency towards neurite growth. -Cys-Gly-Cys (-CGC) N2S2 motif was also incorporated in the present design for peptide radiolabeling. The modified peptide showed a better binding with the desired 3T1M receptor for neurite growth. The peptide was synthesized using solid phase peptide synthesis and Fmoc-strategy with more than 80% yield. The receptor binding studies of 99mTc-N2S2-KDP in Neuro2A cell lines showed Kd value in 31 nM range and the complex showed appreciable brain uptake in mice. The results on human SH-SY5Y indicate that the unlabeled N2S2-KDP may perhaps be useful for neurite growth in neurodegenerative disorder.


Assuntos
Laminina/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Compostos Radiofarmacêuticos/farmacologia , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/diagnóstico por imagem , Linhagem Celular Tumoral , Galectinas/metabolismo , Humanos , Laminina/síntese química , Laminina/metabolismo , Laminina/farmacocinética , Camundongos Nus , Simulação de Acoplamento Molecular , Imagem Molecular , Ligação Proteica , Coelhos , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética
11.
J Gerontol A Biol Sci Med Sci ; 76(4): 586-590, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33284954

RESUMO

Anabolic resistance to a mechanical stimulus may contribute to the loss of skeletal muscle mass observed with age. In this study, young and aged mice were injected with saline or human LM-111 (1 mg/kg). One week later, the myotendinous junction of the gastrocnemius muscle was removed via myotenectomy (MTE), thus placing a chronic mechanical stimulus on the remaining plantaris muscle for 2 weeks. LM-111 increased α7B integrin protein expression and clustering of the α7B integrin near DAPI+ nuclei in aged muscle in response to MTE. LM-111 reduced CD11b+ immune cells, enhanced repair, and improved the growth response to loading in aged plantaris muscle. These results suggest that LM-111 may represent a novel therapeutic approach to prevent and/or treat sarcopenia.


Assuntos
Envelhecimento/fisiologia , Laminina/farmacologia , Músculo Esquelético , Sarcopenia , Envelhecimento/efeitos dos fármacos , Anabolizantes/farmacologia , Animais , Matriz Extracelular/fisiologia , Integrinas/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Condicionamento Físico Animal/fisiologia , Regeneração/efeitos dos fármacos , Sarcopenia/metabolismo , Sarcopenia/prevenção & controle , Sarcopenia/terapia
12.
Cells ; 9(12)2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333852

RESUMO

In highly metastatic tumors, vasculogenic mimicry (VM) involves the acquisition by tumor cells of endothelial-like traits. Poly-(ADP-ribose) polymerase (PARP) inhibitors are currently used against tumors displaying BRCA1/2-dependent deficient homologous recombination, and they may have antimetastatic activity. Long non-coding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To evaluate the impact of olaparib treatment in the context of non-coding RNA, we have analyzed the expression of lncRNA after performing unbiased whole-transcriptome profiling of human uveal melanoma cells cultured to form VM. RNAseq revealed that the non-coding transcriptomic landscape differed between olaparib-treated and non-treated cells: olaparib significantly modulated the expression of 20 lncRNAs, 11 lncRNAs being upregulated, and 9 downregulated. We subjected the data to different bioinformatics tools and analysis in public databases. We found that copy-number variation alterations in some olaparib-modulated lncRNAs had a statistically significant correlation with alterations in some key tumor suppressor genes. Furthermore, the lncRNAs that were modulated by olaparib appeared to be regulated by common transcription factors: ETS1 had high-score binding sites in the promoters of all olaparib upregulated lncRNAs, while MZF1, RHOXF1 and NR2C2 had high-score binding sites in the promoters of all olaparib downregulated lncRNAs. Finally, we predicted that olaparib-modulated lncRNAs could further regulate several transcription factors and their subsequent target genes in melanoma, suggesting that olaparib may trigger a major shift in gene expression mediated by the regulation lncRNA. Globally, olaparib changed the lncRNA expression landscape during VM affecting angiogenesis-related genes.


Assuntos
Redes Reguladoras de Genes/genética , Neovascularização Fisiológica/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Longo não Codificante/genética , Sequência de Bases , Linhagem Celular Tumoral , Colágeno/farmacologia , Combinação de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Laminina/farmacologia , Neoplasias/genética , Neovascularização Fisiológica/efeitos dos fármacos , Proteoglicanas/farmacologia , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
13.
Sci Rep ; 10(1): 19071, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149250

RESUMO

The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers' response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.


Assuntos
Líquido Amniótico/citologia , Técnicas de Reprogramação Celular/métodos , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Líquido Amniótico/metabolismo , Diferenciação Celular , Forma Celular , Células Cultivadas , Colágeno/farmacologia , Combinação de Medicamentos , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/farmacologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Fenótipo , Proteoglicanas/farmacologia , Testes de Toxicidade/métodos , Troponina I/genética , Troponina I/metabolismo
14.
Bull Exp Biol Med ; 170(1): 158-163, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33231802

RESUMO

In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of ßIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Meios de Cultura/farmacologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Mucosa Olfatória/citologia , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Fibronectinas/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Laminina/farmacologia , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Cultura Primária de Células , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/terapia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
15.
Biotechniques ; 69(5): 339-346, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32867513

RESUMO

Invasion is a hallmark of cancer and therefore in vitro invasion assays are important tools in cancer research. We aimed to describe in vitro 2D transwell assays and 3D spheroid assays to quantitatively determine the invasive behavior of glioblastoma stem cells in response to the chemoattractant SDF-1α. Matrigel was used as a matrix in both assays. We demonstrated quantitatively that SDF-1α increased invasive behavior of glioblastoma stem cells in both assays. We conclude that the 2D transwell invasion assay is easy to perform, fast and less complex whereas the more time-consuming 3D spheroid invasion assay is physiologically closer to the in vivo situation.


Assuntos
Bioensaio/métodos , Neoplasias Encefálicas/patologia , Quimiocina CXCL12/farmacologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteoglicanas/farmacologia , Receptores CXCR4/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
16.
Blood ; 136(14): 1579-1589, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777816

RESUMO

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.


Assuntos
Eritrócitos/metabolismo , Hemólise , Baço/metabolismo , Baço/fisiopatologia , Animais , Biomarcadores , Envelhecimento Eritrocítico/efeitos dos fármacos , Deformação Eritrocítica , Membrana Eritrocítica , Transfusão de Eritrócitos , Eritrócitos/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Histocitoquímica , Humanos , Imunofenotipagem , Laminina/farmacologia , Macrófagos/metabolismo , Camundongos , Fagocitose
17.
J Am Heart Assoc ; 9(16): e015841, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32783519

RESUMO

Background Extracellular matrix, especially laminin-221, may play crucial roles in viability and survival of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) after in vivo transplant. Then, we hypothesized laminin-221 may have an adjuvant effect on therapeutic efficacy by enhancing cell viability and survival after transplantation of 3-dimensional engineered cardiac tissue (ECT) to a rat model of myocardial infarction. Methods and Results In vitro study indicates the impacts of laminin-221 on hiPS-CMs were analyzed on the basis of mechanical function, mitochondrial function, and tolerance to hypoxia. We constructed 3-dimensional ECT containing hiPS-CMs and fibrin gel conjugated with laminin-221. Heart function and in vivo behavior were assessed after engraftment of 3-dimensional ECT (laminin-conjugated ECT, n=10; ECT, n=10; control, n=10) in a rat model of myocardial infarction. In vitro assessment indicated that laminin-221 improves systolic velocity, diastolic velocity, and maximum capacity of oxidative metabolism of hiPS-CMs. Cell viability and lactate dehydrogenase production revealed that laminin-221 improved tolerance to hypoxia. Furthermore, analysis of mRNA expression revealed that antiapoptotic genes were upregulated in the laminin group under hypoxic conditions. Left ventricular ejection fraction of the laminin-conjugated ECT group was significantly better than that of other groups 4 weeks after transplantation. Laminin-conjugated ECT transplantation was associated with significant improvements in expression levels of rat vascular endothelial growth factor. In early assessments, cell survival was also improved in laminin-conjugated ECTs compared with ECT transplantation without laminin-221. Conclusions In vitro laminin-221 enhanced mechanical and metabolic function of hiPS-CMs and improved the therapeutic impact of 3-dimensional ECT in a rat ischemic cardiomyopathy model. These findings suggest that adjuvant laminin-221 may provide a clinical benefit to hiPS-CM constructs.


Assuntos
Sobrevivência Celular , Células-Tronco Pluripotentes Induzidas/citologia , Laminina/farmacologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/efeitos dos fármacos , Engenharia Tecidual , Animais , Apoptose/genética , Hipóxia Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Transplante de Coração , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , L-Lactato Desidrogenase/biossíntese , Masculino , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Neovascularização Fisiológica , RNA Mensageiro/metabolismo , Ratos , Ratos Nus , Proteínas Recombinantes/farmacologia , Volume Sistólico , Engenharia Tecidual/métodos , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Remodelação Ventricular
18.
J Assist Reprod Genet ; 37(9): 2137-2150, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32671735

RESUMO

PURPOSE: Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. METHODS: Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. RESULTS: Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, ß-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. CONCLUSIONS: LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.


Assuntos
Matriz Extracelular/química , Laminina/farmacologia , Ovário/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos , Adulto , Cromatografia Líquida , Colágeno/química , Colágeno/farmacologia , Meios de Cultura/farmacologia , Combinação de Medicamentos , Matriz Extracelular/genética , Feminino , Células da Granulosa , Humanos , Laminina/química , Pessoa de Meia-Idade , Folículo Ovariano , Ovário/efeitos dos fármacos , Proteoglicanas/química , Proteoglicanas/farmacologia , RNA-Seq , Análise de Célula Única , Espectrometria de Massas em Tandem
19.
Macromol Biosci ; 20(9): e2000197, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32691517

RESUMO

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride-triflouroethylene (PVDF-TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF-TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF-TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.


Assuntos
Eletricidade , Hidrocarbonetos Fluorados/química , Polivinil/química , Tecidos Suporte/química , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalização , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Laminina/farmacologia , Camundongos , Células NIH 3T3 , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Resistência à Tração
20.
Adv Biosyst ; 4(8): e2000008, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32700474

RESUMO

Human mesenchymal stromal cells (hMSCs) have enormous potential for the treatment of various inflammatory and degenerative diseases. Their manufacturing for cell-based therapies requires extensive ex vivo expansion and optimal growth conditions. To support cell adhesion, spreading, and growth in serum-free culture conditions, the applied plasticware needs to be functionalized with essential biochemical cues. By employing a recently developed screening tool, a chemically defined functional matrix composed of dextran sulfate and a bone-related extracellular matrix peptide is identified, which supports long-term culture of bone marrow-derived hMSCs in serum-free culture conditions. Cells grown under these conditions display rapid proliferation and high viability while maintaining their differentiation and immunomodulatory capacity, characteristic cell morphology, expression of hMSC-specific surface antigens as well as important markers of stemness and differentiation potential. The chemically defined, serum-free culture environment enables reliable and reproducible expansion of hMSCs important for cell based-therapies, drug screening, and disease modeling.


Assuntos
Materiais Biomiméticos/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Sulfato de Dextrana/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Peptídeos/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno/farmacologia , Meios de Cultura Livres de Soro/química , Matriz Extracelular/química , Fibronectinas/farmacologia , Expressão Gênica , Humanos , Laminina/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Vitronectina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...