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1.
Cytotherapy ; 26(7): 769-777, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38556961

RESUMO

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Mesenquimais , Humanos , Células HeLa , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Mesenquimais/citologia , Transformação Celular Neoplásica
2.
Biochem Biophys Res Commun ; 415(1): 141-6, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22020101

RESUMO

Nifekalant and azimilide, Class III antiarrhythmic agents, block the human ether-à-go-go-related gene K(+) (hERG) channel. However, when a depolarizing membrane potential is applied, they also increase the current at low potentials by shifting its activation curve towards hyperpolarizing voltages. This phenomenon is called 'facilitation'. In this study, we tried to address the mechanism underlying the facilitation by analyzing the effects of various compounds on hERG expressed in Xenopus oocytes. Like nifekalant, amiodarone, quinidine and carvedilol, but not by dofetilide, caused the current facilitation of hERG, suggesting that the facilitation is a common effect to a subset of hERG blockers. As the concentration of each compound was increased, the total hERG current was suppressed progressively, while the current at low potentials was augmented. Activation curves of the remaining hERG current in the facilitation condition could be described as the sum of two Boltzmann functions reflecting two populations of hERG currents having different activation curves. The voltage shift in the activation curve from control was constant for each compound even at different concentrations; -31 mV in amiodarone, -27 mV in nifekalant, -17 mV in quinidine and -12 mV in carvedilol. Therefore, the facilitation is based on the appearance of hERG whose voltage-dependence for the activation is shifted towards hyperpolarizing voltages.


Assuntos
Antiarrítmicos/farmacologia , Canais de Potássio Éter-A-Go-Go/agonistas , Amiodarona/farmacologia , Animais , Canal de Potássio ERG1 , Humanos , Hidantoínas , Imidazolidinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinonas/farmacologia , Xenopus laevis
4.
Growth Factors ; 27(4): 228-36, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19521893

RESUMO

We found that eosinophil cationic protein (ECP) stimulated the growth of mouse Balb/c 3T3 fibroblasts. ECP-treated 3T3 cells were more flattened and exhibited enhanced stress fiber formation. The enhancement of cytoskeleton after addition of recombinant ECP appeared stable and was able to inhibit disassembly of actin filaments that was induced by fibroblast growth factor-2. The ROCK inhibitor, Y-27632, abrogated this enhancement on stress fiber formation that was induced by ECP indicating the involvement of Rho/ROCK signaling pathway. The effect of ECP was assessed on the differentiation of primary cardiomyocytes derived from rat neonatal heart since the development of actin filaments is significantly related with organization of stress fibers. As the result, both beating rate and the expression of cardiac muscle specific markers such as atrial natriuretic factor were enhanced in the presence of ECP. Thus ECP may also function as a cardiomyocyte differentiation factor.


Assuntos
Proteína Catiônica de Eosinófilo/fisiologia , Miócitos Cardíacos/citologia , Células 3T3 , Actinas/metabolismo , Amidas/farmacologia , Animais , Diferenciação Celular , Citoesqueleto/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Piridinas/farmacologia , Ratos , Transdução de Sinais , Estresse Mecânico
5.
Cardiovasc Res ; 78(3): 458-65, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18296710

RESUMO

AIMS: In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. METHODS AND RESULTS: PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. CONCLUSION: We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Miócitos Cardíacos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Animais Recém-Nascidos , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Vetores Genéticos , Células HeLa , Humanos , Imunoprecipitação , Proteínas com Domínio LIM , Potenciais da Membrana , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenilefrina/farmacologia , Ligação Proteica , Proteína Quinase C , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Coelhos , Ratos , Técnicas do Sistema de Duplo-Híbrido
6.
Sci Rep ; 9(1): 18936, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831759

RESUMO

A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes , Retina , Transdução de Sinais , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Retina/citologia , Retina/metabolismo
7.
J Vet Med Sci ; 78(4): 691-5, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-26638898

RESUMO

A 10-year-old female spayed mixed breed cat with a subcutaneous mass on the right hind limb was revealed with bimodal monoclonal gammopathy composed of IgA by immunoelectrophoresis and immunofixation. Approximately 1 month after referral, the cat died due to renal failure. Postmortem immunohistopathologic evaluation of the subcutaneous mass revealed neoplastic cell proliferation of plasma cells and giant myeloma cells. Neoplastic cells were also present in the liver and spleen. These results led to the diagnosis of a rare case of feline myeloma-related disorders with extramedullary plasmacytoma infiltrating in multiple locations. This report emphasizes the necessity to accumulate cases with similar clinicopathologic findings in the future.


Assuntos
Doenças do Gato/imunologia , Mieloma Múltiplo/veterinária , Paraproteinemias/veterinária , Animais , Gatos , Feminino , Imunoglobulina A , Mieloma Múltiplo/complicações , Mieloma Múltiplo/imunologia , Paraproteinemias/etiologia , Paraproteinemias/imunologia
8.
Channels (Austin) ; 1(3): 198-208, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18690032

RESUMO

Chemicals and toxins are useful tools to elucidate the structure-function relationship of various proteins including ion channels. The HERG channel is blocked by many compounds and this may cause life-threatening cardiac arrhythmia. Besides block, some chemicals such as the class III anti-arrhythmic agent nifekalant stimulate HERG at low potentials by shifting its activation curve towards hyperpolarizing voltages. This is called "facilitation". Here, we report mutations and simulations analyzing the association between nifekalant and channel pore residues for block and facilitation. Alanine-scanning mutagenesis was performed in the pore region of HERG. The mutations at the base of the pore helix (T623A), the selectivity filter (V625A) and the S6 helix (G648A, Y652A and F656A) abolished and S624A attenuated both block and facilitation induced by the drug. On the other hand, the mutation of other residues caused either an increase or a decrease in nifekalant-induced facilitation without affecting block. An open-state homology model of the HERG pore suggested that T623, S624, Y652 and F656 faced the central cavity, and were positioned within geometrical range for the drug to be able to interact with all of them at the same time. Of these, S649 was the only polar residue located within possible interaction distance from the drug held in its blocking position. Further mutations and flexible-docking simulations suggest that the size, but not the polarity, of the side chain at S649 is critical for drug induced facilitation.


Assuntos
Antiarrítmicos/farmacologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Mutação , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Pirimidinonas/farmacologia , Animais , Simulação por Computador , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Técnicas de Transferência de Genes , Humanos , Potenciais da Membrana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos , Conformação Proteica , Fatores de Tempo , Xenopus laevis
9.
Biochem Biophys Res Commun ; 327(4): 1105-13, 2005 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-15652511

RESUMO

Cardiac hypertrophy is triggered in response to mechanical stress and various neurohumoral factors, such as G-protein coupling receptor (GPCR) and gp130 cytokine receptor agonists. Recent studies have suggested cardiac Z-disc plays a pivotal role to regulate these cellular responses. Here, we demonstrate stimulations with GPCR agonists (norepinephrine, angiotensin II, and endothelin 1) and phorbol ester activated and translocated protein kinase D1 (PKD1) to the Z-discs in neonatal rat cardiomyocytes in a protein kinase C (PKC)-dependent manner, whereas gp130 agonist did not. Especially, upon the alpha-adrenergic receptor agonist stimulations, following the PKCepsilon-PKD1 complex formation, PKCepsilon-dependent activation of PKD1 was essential to induce hypertrophic responses. Constitutively active mutant of either PKD1 or PKCepsilon also induced cardiac hypertrophy ex vivo. Taken together, the PKCepsilon-PKD1 complex at Z-discs could play a pivotal role in the cardiac hypertrophy induced by GPCR agonists, at least alpha-adrenergic receptor agonist.


Assuntos
Cardiomegalia/metabolismo , Coração/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais , Agonistas alfa-Adrenérgicos/farmacologia , Angiotensina II/farmacologia , Animais , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Células Cultivadas , Endotelina-1/farmacologia , Mutação/genética , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Norepinefrina/farmacologia , Ésteres de Forbol/farmacologia , Proteína Quinase C/genética , Proteína Quinase C-épsilon , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos
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