RESUMO
FHL1 gene locates in the Xq26 region and encodes for four and half LIM domain protein 1. It plays a crucial role in muscle cells and mutations in FHL1 are related to muscular dystrophy (MD). Peripheral blood mononuclear cells (PBMCs) were obtained from 2 family patients with MD that carry a pathogenic missense mutation in FHL1 (c.377G > A, p.C126Y). Induced pluripotent stem cells (iPSCs) were generated by PBMCs reprogramming using the lentiviral-hSTEMCCA-loxP vector, obtaining FHL1-T and FHL1-V iPSCs lines from patients. FHL1 genotype was maintained, and stemness and pluripotency were confirmed in both iPSCs lines.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofias Musculares , Humanos , Mutação de Sentido Incorreto , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Musculares/genética , Mutação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genéticaRESUMO
The arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease characterized by the progressive replacement of contractile myocardium by fibro-fatty adipose tissue, that generates ventricular arrhythmias and sudden death in patients. The ACM has a genetic origin with alterations in desmosomal genes with the most commonly mutated being the PKP2 gene. We generated two CRISPR/Cas9 edited iPSCs lines, one iPSC line with a point mutation in PKP2 reported in patients with ACM and another iPSC line with a premature stop codon to knock-out the same gene.
Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Mutação Puntual , Células-Tronco Pluripotentes Induzidas/metabolismo , Displasia Arritmogênica Ventricular Direita/genética , Sistemas CRISPR-Cas/genética , Cardiomiopatias/genética , Mutação/genética , Placofilinas/genética , Placofilinas/metabolismoRESUMO
ABSTRACT: Background : Mesenchymal stem cells (MSCs) can be activated by different bacterial toxins. Lipopolysaccharides and Shiga Toxin (Stx) are the main toxins necessary for hemolytic uremic syndrome development. The main etiological event in this disease is endothelial damage that causes glomerular destruction. Considering the repairing properties of MSC, we aimed to study the response of MSC derived from induced pluripotent stem cells (iPSC-MSC) to LPS and/or Stx and its effect on the restoration of injured endothelial cells. Methods : iPSC-MSC were treated with LPS and or/Stx for 24 h and secretion of cytokines, adhesion, and migration were measured in response to these toxins. In addition, conditioned media from treated iPSC-MSC were collected and used for proteomics analysis and evaluation of endothelial cell healing and tubulogenesis using human microvascular endothelial cells 1 as a source of endothelial cells. Results : The results obtained showed that LPS induced a proinflammatory profile on iPSC-MSC, whereas Stx effects were less evident, even though cells expressed the Gb 3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to a gelatin substrate. Addition of conditioned media of iPSC-MSC treated with LPS + Stx, decreased the capacity of human microvascular endothelial cells 1 to close a wound, and did not favor tubulogenesis. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support the functional results described. Conclusions : iPSC-MSC activated by LPS acquired a proinflammatory profile that induces migration and adhesion to extracellular matrix proteins but the addition of Stx did not activate any repair program to ameliorate endothelial damage, indicating that the use of iPSC-MSC to regenerate endothelial injury caused by LPS and/or Stx in hemolytic uremic syndrome could not be the best option to consider to regenerate a tissue injury.
Assuntos
Síndrome Hemolítico-Urêmica , Células-Tronco Pluripotentes Induzidas , Humanos , Toxina Shiga , Lipopolissacarídeos/farmacologia , Células Endoteliais/metabolismo , Meios de Cultivo Condicionados , ProteômicaRESUMO
ABSTRACT: Vascularized composite allotransplantation of the face is an exceedingly complex procedure, requiring extensive planning and surgical precision in order to successfully manage patients with facial disfigurements. This review aims to present an overview of the salient anatomic considerations in facial transplantation, as well as give attention to unique patient populations and special considerations.A literature review was performed in search of articles pertaining to considerations in facial transplantation using the databases PubMed, Web of Science, and Cochrane. Articles selected for further review included full-text articles with an emphasis on specific anatomic defects and how they were addressed in the transplant process, as well as management of special patient populations undergoing facial transplantation. In total, 19 articles were deemed appropriate for inclusion.The use of computer-assisted technologies for the planning portion of the procedure, as well as intraoperative efficiency, has yielded favorable results and can be considered as part of the operative plan. The ultimate outcome is dependent upon the synchronization of subunits of the allograft and the desired functional outcomes, including osseous, ocular, oral, and otologic considerations. Management of specific pathology and subgroups of patients are critical aspects. Although pediatric face transplantation has not yet been performed, it is a likely a future step in the evolution of this procedure.When performing a face transplantation, many components must be considered pre-, intra-, and post-operatively. This systematic review presents specific anatomic considerations, as well as information about special patient populations within this crosssection of multidisciplinary microsurgery, psychiatry, and transplant immunology.
Assuntos
Transplante de Face , Alotransplante de Tecidos Compostos Vascularizados , Criança , Transplante de Face/métodos , Humanos , Microcirurgia , Transplante Homólogo , Alotransplante de Tecidos Compostos Vascularizados/métodosRESUMO
Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death.
Assuntos
Aprendizado Profundo , Interpretação de Imagem Assistida por Computador , Linguagens de Programação , Morte Celular , Humanos , Células MCF-7 , MicroscopiaRESUMO
PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted exclusively to germline cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in pluripotent, neural, cardiac and even cancer cells. However, controversy remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNA transcripts, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences of amplification loop (ping-pong) were found. Genes hosting piRNA transcripts in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that these piRNAs might have a role in fine-tuning the expression of genes involved in differentiation of pluripotent cells to cardiomyocytes.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/genética , Adulto , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismoRESUMO
Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.
Assuntos
Cavalos , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Animais , Diferenciação Celular/genética , Fibroblastos/citologia , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Técnicas de Transferência NuclearRESUMO
Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell-substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.
Assuntos
Diferenciação Celular , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteômica , Linhagem Celular , Vesículas Extracelulares/ultraestrutura , Humanos , Análise de Componente Principal , Células Estromais/metabolismo , Espectrometria de Massas em Tandem , Geleia de Wharton/citologiaRESUMO
Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3ß activity. ABBREVIATIONS: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III ß-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.
Assuntos
Ciclina E/genética , Regulação da Expressão Gênica , Neurônios/citologia , Neurônios/metabolismo , Proteínas Oncogênicas/genética , Células-Tronco Pluripotentes/citologia , Proliferação de Células , Células Cultivadas , Ciclina E/metabolismo , Fatores de Transcrição E2F/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Fase G2 , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mitose , Células-Tronco Neurais/metabolismo , Proteínas Oncogênicas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula/genética , Regulação da Expressão Gênica , Mesoderma/metabolismo , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Células Cultivadas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesoderma/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Leukemia inhibitory factor (LIF) is a growth factor with pleiotropic biological functions. It has been reported that LIF acts at different stages during mesoderm development. Also, it has been shown that LIF has a cytoprotective effect on neonatal murine cardiomyocytes (CMs) in culture, but little is known about the role of LIF during human cardiogenesis. Thus, we analyzed the effects of LIF on human pluripotent stem cells (PSC) undergoing cardiac differentiation. We first showed that LIF is expressed in the human heart during early development. We found that the addition of LIF within a precise time window during the in vitro differentiation process significantly increased CMs viability. This finding was associated to a decrease in the expression of pro-apoptotic protein Bax, which coincides with a reduction of the apoptotic rate. Therefore, the addition of LIF may represent a promising strategy for increasing CMs survival derived from PSCs.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Fator Inibidor de Leucemia/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
Please note that Carolina Blüguermann's surname was misspelled (as Blugüermann) in this article as originally published.
RESUMO
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3ß signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3ß inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3ß, and thus results relevant for PSC survival.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Oncogênica v-akt/metabolismo , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais , Sobrevivência Celular , Células Cultivadas , Regulação da Expressão Gênica , HumanosRESUMO
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.
Assuntos
Compostos de Anilina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Sulfonamidas/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/farmacologia , Linhagem Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismoRESUMO
Although BMP4-induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC-enriched cell lines (GSC-ECLs) from high-grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC-ECLs' behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC-ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC-ECL. Additionally, treatment of GSC-ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC-ECLs adopted an unexpected smooth muscle-like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Transporte/metabolismo , Glioma/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Idoso , Antígenos CD/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/fisiologiaRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes. METHODS: In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement. RESULTS: We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum. CONCLUSIONS: This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.
Assuntos
Plaquetas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Pluripotentes/citologia , Antígenos de Superfície/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Metilação de DNA , Células-Tronco Embrionárias Humanas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras GenéticasRESUMO
Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.
Assuntos
Antibacterianos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Antibacterianos/toxicidade , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciprofloxacina/farmacologia , Ciprofloxacina/toxicidade , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/microbiologia , Humanos , Cariótipo , Macrolídeos/farmacologia , Macrolídeos/toxicidade , Mycoplasma/efeitos dos fármacosRESUMO
El presente trabajo aborda el debate que se ha dado en la última década a propósito del tema del mejoramiento en sus diferentes variantes. Sobresale el escaso consenso a la hora de definir el concepto y respecto de la necesidad de permitir o prohibir estas prácticas. El único punto de amplio acuerdo es la necesidad de no admitir mejoramientos que impliquen riesgos para la salud, aplazando el debate basado en argumentos que superen el mero peligro para la salud física. De esta manera, parecería que los criterios científicos son los que determinan el destino de los comportamientos sociales. En este sentido, la categoría de medicalización permitirá incorporar la crítica a la sociedad contemporánea y a la hegemonía de la visión médico-científica.
This work addresses the debate mounted in the last decade on the subject of improvement in its different variations. There is a striking lack of consensus when it comes to defining the concept and the need to permit or prohibit such practices. The only point on which there is widespread agreement is the need to block improvements that involve health hazards, thereby postponing the debate based on arguments that go beyond the mere risk to physical wellbeing. Thus, it seems as though scientific criteria are what determines the fate of social behavior. In this sense, the category of medicalization will allow for accommodating a critique of contemporary society and the hegemony of the medical-scientific vision.
O presente trabalho aborda o debate que vem sendo dado na última década a respeito do tema do melhoramento em suas diferentes variantes. Salienta o escasso consenso na hora de definir o conceito e respeito da necessidade de permitir ou proibir essas práticas. O único ponto de amplo acordo é a necessidade de não admitir melhoramentos que impliquem riscos para a saúde, adiando o debate baseado em argumentos que superem o mero perigo para a saúde física. Dessa maneira, pareceria que os critérios científicos são os que determinam o destino dos comportamentos sociais. Nesse sentido, a categoria de medicalização permitirá incorporar a crítica à sociedade contemporânea e à hegemonia da visão médico-científica.
Assuntos
Humanos , Bioética , Terapia Biológica , Melhoramento Biomédico , Melhoramento Genético , MedicalizaçãoRESUMO
Se expone el caso de un niño de catorce meses de edad, con tetralogía de Fallot asociada a síndrome de cimitarra, cuyo diagnóstico se realizó mediante ecocardiografía, cateterismo cardiaco y angio-TAC. Se hace referencia a las consideraciones clínicas y quirúrgicas de este caso que hasta la fecha es el quinto reportado en la literatura.
We describe the case of a fourteen-month-old boy with tetralogy of Fallot associated with scimitar syndrome, whose diagnosis was made by echocardiography, cardiac catheterization and angio-CT. We relate the clinical and surgical considerations in this case which is so far the fifth reported in the literature.
Assuntos
Humanos , Síndrome de Cimitarra , Tetralogia de FallotRESUMO
La obtención del acceso vascular es fundamental en la realización de diferentes procedimientos de cateterismo cardíaco. Los cateterismos a repetición y las cirugías cardíacas múltiples pueden resultar en una marcada incidencia de pérdida de estos sitios de acceso, razón por la cual, en algunas oportunidades, es necesario el uso de técnicas no convencionales. Se reporta el caso de un paciente de un año de edad en quien fue necesario el abordaje retroperitoneal de la arteria ilíaca común.
Obtaining vascular access is essential in the realization of different cardiac catheterization procedures. The recurrent catheterizations and multiple cardiac surgeries may result in marked incidence of loss of these access sites, which is why sometimes it is necessary to use unconventional techniques. We report the case of a one year old patient that necessitated the retroperitoneal approach of the common iliac artery.