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1.
Clin Transl Sci ; 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34719115

RESUMO

On October 2020, the US Food and Drug Administration (FDA) approved remdesivir as the first drug for the treatment of coronavirus disease 2019 (COVID-19), increasing remdesivir prescriptions worldwide. However, potential cardiovascular (CV) toxicities associated with remdesivir remain unknown. We aimed to characterize the CV adverse drug reactions (ADRs) associated with remdesivir using VigiBase, an individual case safety report database of the World Health Organization (WHO). Disproportionality analyses of CV-ADRs associated with remdesivir were performed using reported odds ratios and information components. We conducted in vitro experiments using cardiomyocytes derived from human pluripotent stem cell cardiomyocytes (hPSC-CMs) to confirm cardiotoxicity of remdesivir. To distinguish drug-induced CV-ADRs from COVID-19 effects, we restricted analyses to patients with COVID-19 and found that, after adjusting for multiple confounders, cardiac arrest (adjusted odds ratio [aOR]: 1.88, 95% confidence interval [CI]: 1.08-3.29), bradycardia (aOR: 2.09, 95% CI: 1.24-3.53), and hypotension (aOR: 1.67, 95% CI: 1.03-2.73) were associated with remdesivir. In vitro data demonstrated that remdesivir reduced the cell viability of hPSC-CMs in time- and dose-dependent manners. Physicians should be aware of potential CV consequences following remdesivir use and implement adequate CV monitoring to maintain a tolerable safety margin.

2.
Materials (Basel) ; 14(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34576409

RESUMO

The skin protects the body from external barriers. Certain limitations exist in the development of technologies to rapidly prepare skin substitutes that are therapeutically effective in surgeries involving extensive burns and skin transplantation. Herein, we fabricated a structure similar to the skin layer by using skin-derived decellularized extracellular matrix (dECM) with bioink, keratinocytes, and fibroblasts using 3D-printing technology. The therapeutic effects of the produced skin were analyzed using a chimney model that mimicked the human wound-healing process. The 3D-printed skin substitutes exhibited rapid re-epithelialization and superior tissue regeneration effects compared to the control group. These results are expected to aid the development of technologies that can provide customized skin-replacement tissues produced easily and quickly via 3D-printing technology to patients.

3.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063742

RESUMO

Three-dimensional (3D) printing is perceived as an innovative tool for change in tissue engineering and regenerative medicine based on research outcomes on the development of artificial organs and tissues. With advances in such technology, research is underway into 3D-printed artificial scaffolds for tissue recovery and regeneration. In this study, we fabricated artificial scaffolds by coating bone demineralized and decellularized extracellular matrix (bdECM) onto existing 3D-printed polycaprolactone/tricalcium phosphate (PCL/TCP) to enhance osteoconductivity and osteoinductivity. After injecting adipose-derived stem cells (ADSCs) in an aggregate form found to be effective in previous studies, we examined the effects of the scaffold on ossification during mandibular reconstruction in beagle dogs. Ten beagles were divided into two groups: group A (PCL/TCP/bdECM + ADSC injection; n = 5) and group B (PCL/TCP/bdECM; n = 5). The results were analyzed four and eight weeks after intervention. Computed tomography (CT) findings showed that group A had more diffuse osteoblast tissue than group B. Evidence of infection or immune rejection was not detected following histological examination. Goldner trichrome (G/T) staining revealed rich ossification in scaffold pores. ColI, Osteocalcin, and Runx2 gene expressions were determined using real-time polymerase chain reaction. Group A showed greater expression of these genes. Through Western blotting, group A showed a greater expression of genes that encode ColI, Osteocalcin, and Runx2 proteins. In conclusion, intervention group A, in which the beagles received the additional ADSC injection together with the 3D-printed PCL/TCP coated with bdECM, showed improved mandibular ossification in and around the pores of the scaffold.


Assuntos
Tecido Adiposo/citologia , Fosfatos de Cálcio/química , Matriz Extracelular/fisiologia , Mandíbula/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Poliésteres/química , Células-Tronco/citologia , Tecidos Suporte/química , Adipócitos/citologia , Animais , Regeneração Óssea/efeitos dos fármacos , Cães , Osteoblastos/efeitos dos fármacos , Impressão Tridimensional , Engenharia Tecidual/métodos
4.
Biomaterials ; 266: 120472, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33120201

RESUMO

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as 'condensed round shape' and 'spread shape' in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.


Assuntos
Células Progenitoras Endoteliais , Animais , Química Click , Modelos Animais de Doenças , Membro Posterior , Humanos , Isquemia/diagnóstico por imagem , Isquemia/terapia , Neovascularização Fisiológica , Células-Tronco , Tomografia
5.
Biomedicines ; 8(11)2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33121085

RESUMO

Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.

6.
Antiviral Res ; 184: 104955, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33091434

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , COVID-19/virologia , Miócitos Cardíacos/virologia , Células-Tronco Pluripotentes/citologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Amidas/farmacologia , Animais , Antimaláricos/farmacologia , COVID-19/complicações , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Cloroquina/farmacologia , Eletrocardiografia , Citometria de Fluxo , Cardiopatias/complicações , Humanos , Hidroxicloroquina/farmacologia , Microscopia de Fluorescência , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/virologia , Pirazinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Ensaio de Placa Viral
7.
Free Radic Biol Med ; 161: 92-101, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33011273

RESUMO

NADPH oxidases (NOXs) are comprised of different isoforms, NOX1 to 5 and Duox1 and 2, and they trigger diabetic nephropathy (DN) in the patients with diabetes mellitus. Recently, it was shown that, compared to the other isoforms, the expression of NOX5 was increased in the patients with DN and, NOX5 has been suggested to be important in the development of therapeutic agents. The effect of pan-NOX inhibition by APX-115 has also been investigated in type 2 diabetic mice. However, since NOX5 is absent in mice, we evaluated the effect of pan-NOX inhibition by APX-115 in Nox5 transgenic mouse. Wild type and renal podocyte specific NOX5 transgenic mice (NOX5 pod+) were fed with high-fat diet (60% kcal fat) and treated with APX-115 (60 mg/kg) by oral gavage for 14 weeks. APX-115 significantly improved pancreatic beta cell function by decreased fasting blood glucose levels and increased insulin levels. Further, the total serum cholesterol, triglycerides, and urinary albumin/creatinine levels were also significantly decreased by APX-115 treatment. Increased NOX5 mRNA expressions, increased desmin levels, and reduced podocin protein expressions in the kidney of NOX5 pod + mice were also significantly restored to normal levels by APX-115 treatment. Moreover, APX-115 inhibited the expression of inflammation-related proteins such as TRAF6. Collectively, these data suggest that APX-115 might be a promising therapeutic agent for the treatment of DN because of its pan-NOX inhibitory activity, including its NOX5 inhibitory activity, and also owing to its anti-inflammatory effect.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/prevenção & controle , Humanos , Camundongos , Camundongos Transgênicos , NADPH Oxidase 5 , NADPH Oxidases/genética , Pirazóis , Piridinas , Espécies Reativas de Oxigênio
8.
Biomater Res ; 24: 10, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32514370

RESUMO

Background: We investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology. Methods: Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested. Results: Defined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs. Conclusions: These results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.

9.
PLoS One ; 15(5): e0232899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392240

RESUMO

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.


Assuntos
Técnicas de Cocultura/instrumentação , Células Epiteliais , Células-Tronco Mesenquimais , Nanoestruturas , Animais , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Propriedades de Superfície
10.
Genes Dev ; 34(7-8): 526-543, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32079652

RESUMO

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ferroptose/genética , Metabolismo dos Lipídeos/genética , PPAR alfa/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Proteínas de Ciclo Celular/genética , Glioblastoma/fisiopatologia , Células HCT116 , Humanos , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Ratos , Proteína Supressora de Tumor p53/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
11.
Curr Eye Res ; 45(9): 1136-1143, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31951764

RESUMO

PURPOSE: Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. METHODS: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. RESULTS: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. CONCLUSION: Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Herpesvirus Humano 4/fisiologia , NADPH Oxidase 4/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Western Blotting , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , NADPH Oxidases/antagonistas & inibidores , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/virologia , Transdução de Sinais/efeitos dos fármacos
12.
Biomaterials ; 225: 119534, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31590118

RESUMO

3D culture of stem cells can improve therapeutic effects. However, there is limited research on how to deliver cultured stem cell spheroids to the desired target. Here, we developed lotus seedpod-inspired hydrogel (LoSH) containing microwells for culture and delivery of stem cell spheroids. Human adipose-derived stem cells (hADSCs) inside the square microwells (200 or 400 µm in width with various depths) spontaneously formed spheroids with high viability (94.08 ±â€¯1.56%), and fibronectins conjugated to the hydrogel successfully gripped the spheroids, similar to the funiculus gripping seeds in the lotus seedpod. The spheroids slightly bound to the LoSH surface at 37 °C were detached by the expansion of LoSH at lower temperature of 4 °C. After spheroid formation, LoSH was placed on the target substrate upside-down, expanded at 4 °C for 10 min, and removed from the target. As a result, the spheroids within the microwell were successfully transferred to the target substrate with high transfer efficiency (93.78 ±â€¯2.30%). A delivery of spheroids from LoSH to full-thickness murine skin wound with chimney model showed significant enhancement of the number of SMA-positive vessels at day 21 compared to the group received the same number of spheroids by injection. Together, our findings demonstrate LoSH as a one-step platform that can culture and deliver spheroids to a large target area, which will be useful for various biomedical applications.


Assuntos
Materiais Biomiméticos/farmacologia , Técnicas de Cultura de Células/métodos , Hidrogéis/farmacologia , Lotus/química , Sementes/química , Esferoides Celulares/transplante , Células-Tronco/citologia , Animais , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Regeneração/efeitos dos fármacos , Pele/patologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos
13.
Microvasc Res ; 126: 103912, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31433972

RESUMO

Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.


Assuntos
Eritrosina , Isquemia/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Trombose/fisiopatologia , Animais , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Membro Posterior , Isquemia/induzido quimicamente , Luz , Masculino , Camundongos Endogâmicos ICR , Processos Fotoquímicos , Fluxo Sanguíneo Regional , Trombose/induzido quimicamente , Fatores de Tempo
14.
Mater Sci Eng C Mater Biol Appl ; 103: 109729, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349510

RESUMO

Graphene and its derivatives have seen a rapid rise in interest as promising biomaterials especially in the field of tissue engineering, regenerative medicine, and cell biology of late. Despite its proven potential in numerous biological applications, information regarding the relationship between the different forms of graphene and cell lineages is still lacking partly due to its topical emergence in cellular studies. Herein, we explore the biocompatibility of four types of graphene substrates (chemical vapor deposition grown graphene, mechanically exfoliated graphene, chemically exfoliated graphene oxide, and reduced graphene oxide) with three types of somatic cells (keratinocytes, hepatocytes, endothelial cells) derived from the three germ layers in relation to cell adhesion, proliferation, morphology, and gene expression. The results revealed exceptional cell adhesion for all tested groups but enhanced proliferation and cytoskeletal interconnectivity in graphene oxide and reduced graphene oxide substrates. We were unable to detect any adverse effects in gene expression and survivability during a week of culture. We further show topographic changes to graphene substrates under fetal bovine serum adsorption to better illustrate the actual microenvironment of inhabitant cells. This study highlights the extraordinary synergy between graphene and somatic cells, suggesting the discretionary use of extracellular matrix components for in vitro cultivation.


Assuntos
Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Grafite , Hepatócitos , Células Endoteliais da Veia Umbilical Humana , Queratinócitos , Grafite/química , Grafite/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo
15.
Nat Commun ; 10(1): 3123, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311935

RESUMO

Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.


Assuntos
Coração/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Regeneração , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Injeções Intralesionais , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Ratos , Ratos Endogâmicos F344 , Resultado do Tratamento
16.
Colloids Surf B Biointerfaces ; 180: 384-392, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31082776

RESUMO

Precise detection of undifferentiated human pluripotent stem cells (hPSCs) and their entire subsequent elimination are incredibly important in preventing teratoma formations after transplantation. Recently, electrochemical sensing platforms have demonstrated immense potential as a new tool to detect remaining hPSCs in label-free and non-destructive manner. Nevertheless, one of the critical huddles of this electrochemical sensing approach is its low sensitivity since even low concentrations of remaining hPSCs were reported to form teratoma once transplanted. To address this issue, in this study, we report an engineering-based approach to improve the sensitivity of electrochemical sensing platform for hPSC detection. By optimizing the density of gold nanostructure and the matrigel concentration to improve both electro-catalytic property and biocompatibility, the sensitivity of the developed platform toward hESCs detection could reach 12,500 cells/chip, which is close to the known critical concentration of hPSCs (˜10,000 cells) that induce teratoma formation in vivo. Remarkably, the electrochemical signals were not detectable from other types of stem cell-derived endothelial cells (CB-EPCs) even at high concentrations of CB-EPCs (40,000 cells/chip), proving the high selectivity of the developed platform toward hPSC detection. Hence, the developed platform could be highly useful to solve the safety issues that are related with clinical application of hPSC-derived cells.


Assuntos
Eletroquímica/métodos , Ouro/química , Células-Tronco Embrionárias Humanas/citologia , Nanoestruturas/química , Eletrodos , Células Endoteliais/citologia , Sangue Fetal/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Nanoestruturas/ultraestrutura , Compostos de Estanho/química
17.
Wound Repair Regen ; 27(4): 345-359, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30835922

RESUMO

Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3ß, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.


Assuntos
Fibroblastos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/patologia , Animais , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Cicatrização/fisiologia
18.
Stem Cells ; 37(5): 623-630, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30721559

RESUMO

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.


Assuntos
Diferenciação Celular/genética , Células Endoteliais/transplante , Células-Tronco Embrionárias Humanas/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Membro Posterior/patologia , Membro Posterior/transplante , Humanos , Isquemia/terapia , Camundongos , Técnicas de Transferência Nuclear
19.
Cell ; 176(3): 564-580.e19, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30580964

RESUMO

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.


Assuntos
Ácido Mevalônico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Colesterol/metabolismo , Feminino , Genes Supressores de Tumor , Células HCT116 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Regiões Promotoras Genéticas , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Terpenos/metabolismo
20.
J Invest Dermatol ; 139(3): 692-701, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30393080

RESUMO

Much of our understanding of human biology and the function of mammalian cells in tissue regeneration have been derived from mechanistically and genetically manipulated rodent models. However, current models examining epidermal wound repair fail to address both the cross-species mechanistic and immunogenic differences simultaneously. Herein, we describe a multifaceted approach intended to better recapitulate human skin recovery in rodent models. First, immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice were intravenously inoculated with human hematopoietic stem cells to become, in essence, humanized, and capable of initiating an adaptive immune response. Next, a chimney-shaped mechanical device was implanted onto the excisional wound site to prevent healing by primary intention (contraction) and expedite cell transplantation. Subsequently, cell therapy was administered by transplanting cord blood-derived endothelial progenitor cells or human pluripotent stem cell-derived endothelial cells into the wound site to examine the regeneration process at a histological level. This study demonstrates human cutaneous repair in a murine model by addressing both the mechanistic and immunogenic differences in the epidermis. We further show human leukocyte recruitment in damaged tissue and improved healing by secondary intention in the transplanted groups, highlighting the need for useful preclinical animal models to better understand leukocyte function in human (tissue repair and) regeneration.


Assuntos
Imunidade Adaptativa/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Pele/lesões , Cicatrização/fisiologia , Ferimentos e Lesões/terapia , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Células Endoteliais/transplante , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Regeneração/fisiologia , Ferimentos e Lesões/imunologia
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