RESUMEN
RESUMEN Fundamento: El cultivo celular permite el análisis directo de las células vivas mediante un microscopio. El estudio de las células contenidas en el líquido amniótico, mediante técnicas de cultivo, detecta anomalías en número y morfología de los cromosomas, que pueden relacionarse con enfermedades genéticas. Objetivo: Caracterizar las variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías. Metodología: Se realizó un estudio descriptivo transversal, en el Centro Provincial de Genética Médica de Camagüey, en el periodo de noviembre de 2016 a abril de 2018.La población de estudio estuvo constituida por 1571 muestras útiles de líquido amniótico obtenidas por amniocentesis, en gestantes en el segundo trimestre, evaluadas en consulta multidisciplinaria con criterios clínicos de estudios cromosómicos según lo establecido en el diagnóstico prenatal citogenético, previo consentimiento informado. Se utilizaron 20 mL de líquido amniótico para la siembra de células fetales, y se aplicaron tres variantes de cultivo abierto (directo, centrifugado y expandido). Se determinó el complemento cromosómico en cada variedad. Resultados: Predominó el complemento cromosómico normal. Las tetraploidías prevalecieron en el cultivo expandido. El índice mitótico fue similar en las tres variedades de cultivo y el cultivo directo tuvo el más bajo índice de poliploidías. Conclusiones: El cariotipo normal fue predominante. Las tetrapolidías fueron las alteraciones más frecuentes y prevalecieron en el cultivo expandido. En el cultivo directo se presentó el más bajo índice de errores inducidos in vitro.
ABSTRACT Background: Cell culture allows direct analysis of live cells under a microscope. The cell study contained in amniotic fluid, by culture techniques, detects abnormalities in chromosome number and morphology, which can be related to genetic diseases. Objective: To describe amniotic fluid culture strains for the in vitro diagnosis of polyploidy. Methodology: A cross-sectional descriptive study was conducted at the Camagüey Provincial Center of Medical Genetics, from November 2016 to April 2018.The study population consisted of 1571 useful amniotic fluid samples obtained by amniocentesis, in pregnant women in the second trimester, evaluated by multidisciplinary discussion with clinical criteria for chromosomal studies as established in the cytogenetic prenatal diagnosis, prior informed consent. 20 mL of amniotic fluid were used for fetal cell seeding, and three open culture strains (direct, centrifuged and expanded) were applied. Chromosomal complement was determined in each variety. Results: Normal chromosome complement was predominant. Tetraploidy prevailed in the expanded culture. The mitotic index was similar in the three culture strains and the direct culture had the lowest polyploidy index. Conclusions: Normal karyotype was predominant. Tetraploidy were the most frequent modifications and prevailed in the expanded culture. Direct culture had the lowest rate of the in vitro induced errors.
Asunto(s)
Poliploidía , Tetraploidía , Cultivo de Sangre , Líquido Amniótico/citologíaRESUMEN
Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.
Asunto(s)
Células Madre Adultas/citología , Cartílago Articular/efectos de los fármacos , Condrogénesis/genética , Osteoartritis/genética , Andamios del Tejido/química , Células Madre Adultas/trasplante , Líquido Amniótico/citología , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/ultraestructura , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Quitosano/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Microscopía Electrónica de Rastreo , Osteoartritis/patología , Osteoartritis/terapia , Polisacáridos Bacterianos/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Ingeniería de Tejidos/métodosRESUMEN
The aim of this study was to evaluate the lung maturity of premature and full-term lambs by analyzing amniotic fluid using the following methods: Clements test, Nile blue cytology test, hematoxylin-Shorr stain, lamellar body count, and radiographic tests. The use of these methods is intended to identify high-risk newborns and provide immediate clinical intervention after birth. Altogether, 56 animals (24 ewes and 32 lambs) were included in the study and divided into 3 groups. Group I consisted of 8 ewes that were at approximately 145 days of gestation; this group delivered 10 lambs naturally. Group II consisted of 8 ewes that were at 138 days' gestation; this group delivered 11 lambs by cesarean section. Group III consisted of 8 ewes at 138 days' gestation; this group was administered intramuscular dexamethasone (16mg/animal) 36 hours prior to a cesarean section. Group III delivered11 lambs. Cytological tests were performed using a microscope with a maximum magnification of 1000x, while the Clements test was visually observed by one of the researchers. Amnioticfluid lamellar body counts were measured using transmission electron microscopy. Among the staining methods, hematoxylin-Shorr was reliable, and Group III had a greater number of orangeophilic cells when compared to Group II, probably due to corticoid administration. The Clements test showed pulmonary maturity in approximately 20% of Group I lambs and Group II showed 9.1% of bubbles; however, Group III had the highest pulmonary maturity percentage (36.4%). The lamellar bodies were measured, and all groups had sizes between 0.019 and 0.590μm. Radiographic evaluation revealed that the majority of lambs presented some level of pulmonary radiodensity, indicating an acinar pattern at birth. These results are in line with the expectations of each group. We found that the normal group showed greater pulmonary maturity, whereas Group II presented pulmonary immaturity, which is expected because this group comprised lambs born prematurely and Group III showed pulmonary maturity almost comparable to the normal delivery group (Group I). This is due to the fact that although these animals are premature, the use of dexamethasone helped in pulmonary maturation. Therefore, these pulmonary maturity tests are considered effective when more than one technique is used and can be used routinely in the care of a pregnant ewe in labor, where a simple collection of amniotic fluid can predict a high-risk pregnancy and alert the veterinarian if the newborn needs intensive supportive treatment.(AU)
O objetivo deste estudo foi avaliar a maturidade pulmonar de cordeiros prematuros e a termo por meio da análise do líquido amniótico utilizando os seguintes métodos: teste de Clements, teste de citologia do azul do Nilo, coloração de hematoxilina-Shorr, contagem de corpos lamelares e testes radiográficos. Um desses métodos tem por objetivo identificar recém-nascidos de alto risco e fornecer intervenção clínica imediata após o nascimento. Ao todo, 56 animais (24 ovelhas e 32 cordeiros) foram incluídos no estudo e divididos em 3 grupos. O grupo I foi composto por 8 ovelhas com aproximadamente 145 dias de gestação; este grupo deu à luz 10 cordeiros naturalmente. O Grupo II foi composto por 8 ovelhas com 138 dias de gestação; este grupo deu à luz 11 cordeiros por cesariana. Grupo III consistiu de 8 ovelhas com 138 dias de gestação; este grupo recebeu dexametasona intramuscular (16 mg / animal) 36 horas antes de uma cesariana. O Grupo III entregou 11 cordeiros. Os testes citológicos foram realizados em microscópio com aumento máximo de 1000x, enquanto o teste de Clements foi observado visualmente por um dos pesquisadores. A contagem de corpos lamelares de líquido amniótico foi medida usando microscopia eletrônica de transmissão. Dentre os métodos de coloração, o hematoxilina-Shorr foi confiável, sendo que o Grupo III apresentou maior número de células orangeofílicas quando comparado ao grupo II, provavelmente devido à administração de corticóide. O teste de Clements mostrou maturidade pulmonar em aproximadamente 20% dos cordeiros do Grupo I e o Grupo II apresentou 9,1% de bolhas; entretanto, o Grupo III apresentou o maior percentual de maturidade pulmonar (36,4%). Os corpos lamelares foram medidos e todos os grupos apresentaram tamanhos entre 0,019 e 0,590μm. A avaliação radiográfica revelou que a maioria dos cordeiros apresentava algum grau de radiodensidade pulmonar, indicando padrão acinar ao nascimento. Esses resultados estão alinhados com as expectativas de cada grupo. Verificamos que o grupo normal apresentou maior maturidade pulmonar, enquanto o Grupo II apresentou imaturidade pulmonar, o que é esperado por se tratar de cordeiros nascidos prematuramente e o Grupo III apresentou maturidade pulmonar quase comparável ao grupo de parto normal (Grupo I). Isso se deve ao fato de que, embora esses animais sejam prematuros, o uso da dexametasona auxiliou na maturação pulmonar. Portanto, esses testes de maturidade pulmonar são considerados eficazes quando mais de uma técnica são utilizadas e podem ser usadas rotineiramente no cuidado de uma ovelha gestante em trabalho de parto, onde uma simples coleta de líquido amniótico pode prever uma gravidez de alto risco e alertar o veterinário se o recém-nascido precisa de tratamento de suporte intensivo.(AU)
Asunto(s)
Animales , Femenino , Embarazo , Recien Nacido Prematuro , Trabajo de Parto/fisiología , Ovinos , Líquido Amniótico/citología , Pulmón/anomalíasRESUMEN
The aim of this study was to evaluate the lung maturity of premature and full-term lambs by analyzing amniotic fluid using the following methods: Clements test, Nile blue cytology test, hematoxylin-Shorr stain, lamellar body count, and radiographic tests. The use of these methods is intended to identify high-risk newborns and provide immediate clinical intervention after birth. Altogether, 56 animals (24 ewes and 32 lambs) were included in the study and divided into 3 groups. Group I consisted of 8 ewes that were at approximately 145 days of gestation; this group delivered 10 lambs naturally. Group II consisted of 8 ewes that were at 138 days' gestation; this group delivered 11 lambs by cesarean section. Group III consisted of 8 ewes at 138 days' gestation; this group was administered intramuscular dexamethasone (16mg/animal) 36 hours prior to a cesarean section. Group III delivered11 lambs. Cytological tests were performed using a microscope with a maximum magnification of 1000x, while the Clements test was visually observed by one of the researchers. Amnioticfluid lamellar body counts were measured using transmission electron microscopy. Among the staining methods, hematoxylin-Shorr was reliable, and Group III had a greater number of orangeophilic cells when compared to Group II, probably due to corticoid administration. The Clements test showed pulmonary maturity in approximately 20% of Group I lambs and Group II showed 9.1% of bubbles; however, Group III had the highest pulmonary maturity percentage (36.4%). The lamellar bodies were measured, and all groups had sizes between 0.019 and 0.590μm. Radiographic evaluation revealed that the majority of lambs presented some level of pulmonary radiodensity, indicating an acinar pattern at birth. These results are in line with the expectations of each group. We found that the normal group showed greater pulmonary maturity, whereas Group II presented pulmonary immaturity, which is expected because this group comprised lambs born prematurely and Group III showed pulmonary maturity almost comparable to the normal delivery group (Group I). This is due to the fact that although these animals are premature, the use of dexamethasone helped in pulmonary maturation. Therefore, these pulmonary maturity tests are considered effective when more than one technique is used and can be used routinely in the care of a pregnant ewe in labor, where a simple collection of amniotic fluid can predict a high-risk pregnancy and alert the veterinarian if the newborn needs intensive supportive treatment.(AU)
O objetivo deste estudo foi avaliar a maturidade pulmonar de cordeiros prematuros e a termo por meio da análise do líquido amniótico utilizando os seguintes métodos: teste de Clements, teste de citologia do azul do Nilo, coloração de hematoxilina-Shorr, contagem de corpos lamelares e testes radiográficos. Um desses métodos tem por objetivo identificar recém-nascidos de alto risco e fornecer intervenção clínica imediata após o nascimento. Ao todo, 56 animais (24 ovelhas e 32 cordeiros) foram incluídos no estudo e divididos em 3 grupos. O grupo I foi composto por 8 ovelhas com aproximadamente 145 dias de gestação; este grupo deu à luz 10 cordeiros naturalmente. O Grupo II foi composto por 8 ovelhas com 138 dias de gestação; este grupo deu à luz 11 cordeiros por cesariana. Grupo III consistiu de 8 ovelhas com 138 dias de gestação; este grupo recebeu dexametasona intramuscular (16 mg / animal) 36 horas antes de uma cesariana. O Grupo III entregou 11 cordeiros. Os testes citológicos foram realizados em microscópio com aumento máximo de 1000x, enquanto o teste de Clements foi observado visualmente por um dos pesquisadores. A contagem de corpos lamelares de líquido amniótico foi medida usando microscopia eletrônica de transmissão. Dentre os métodos de coloração, o hematoxilina-Shorr foi confiável, sendo que o Grupo III apresentou maior número de células orangeofílicas quando comparado ao grupo II, provavelmente devido à administração de corticóide. O teste de Clements mostrou maturidade pulmonar em aproximadamente 20% dos cordeiros do Grupo I e o Grupo II apresentou 9,1% de bolhas; entretanto, o Grupo III apresentou o maior percentual de maturidade pulmonar (36,4%). Os corpos lamelares foram medidos e todos os grupos apresentaram tamanhos entre 0,019 e 0,590μm. A avaliação radiográfica revelou que a maioria dos cordeiros apresentava algum grau de radiodensidade pulmonar, indicando padrão acinar ao nascimento. Esses resultados estão alinhados com as expectativas de cada grupo. Verificamos que o grupo normal apresentou maior maturidade pulmonar, enquanto o Grupo II apresentou imaturidade pulmonar, o que é esperado por se tratar de cordeiros nascidos prematuramente e o Grupo III apresentou maturidade pulmonar quase comparável ao grupo de parto normal (Grupo I). Isso se deve ao fato de que, embora esses animais sejam prematuros, o uso da dexametasona auxiliou na maturação pulmonar. Portanto, esses testes de maturidade pulmonar são considerados eficazes quando mais de uma técnica são utilizadas e podem ser usadas rotineiramente no cuidado de uma ovelha gestante em trabalho de parto, onde uma simples coleta de líquido amniótico pode prever uma gravidez de alto risco e alertar o veterinário se o recém-nascido precisa de tratamento de suporte intensivo.(AU)
Asunto(s)
Animales , Femenino , Embarazo , Recien Nacido Prematuro , Trabajo de Parto/fisiología , Ovinos , Líquido Amniótico/citología , Pulmón/anomalíasRESUMEN
First discovered by Friedenstein in 1976, mesenchymal stem cells (MSCs) are adult stem cells found throughout the body that share a fixed set of characteristics. Discovered initially in the bone marrow, this cell source is considered the gold standard for clinical research, although various other sources-including adipose tissue, dental pulp, mobilised peripheral blood and birth-derived tissues-have since been identified. Although similar, MSCs derived from different sources possess distinct characteristics, advantages and disadvantages, including their differentiation potential and proliferation capacity, which influence their applicability. Hence, they may be used for specific clinical applications in the fields of regenerative medicine and tissue engineering. This review article summarises current knowledge regarding the various sources, characteristics and therapeutic applications of MSCs.
Asunto(s)
Células de la Médula Ósea/citología , Médula Ósea/microbiología , Trasplante de Células Madre Mesenquimatosas , Líquido Amniótico/citología , Médula Ósea/fisiología , Diferenciación Celular , Pulpa Dental/metabolismo , Pulpa Dental/trasplante , Sangre Fetal/citología , Sangre Fetal/trasplante , Humanos , Trasplante de Células Madre Mesenquimatosas/métodos , Trasplante de Células Madre Mesenquimatosas/tendencias , Células Madre Mesenquimatosas/metabolismo , Literatura de Revisión como Asunto , Ingeniería de Tejidos/métodos , Ingeniería de Tejidos/tendenciasRESUMEN
BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs. METHODS: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors. RESULTS: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression. CONCLUSION: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro.
Asunto(s)
Adipocitos/metabolismo , Silenciador del Gen , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Antígenos Thy-1/genética , Adipocitos/citología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Líquido Amniótico/citología , Líquido Amniótico/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Diferenciación Celular , Proliferación Celular , Pulpa Dental/citología , Pulpa Dental/metabolismo , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunofenotipificación , Lentivirus/genética , Lentivirus/metabolismo , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Antígenos Thy-1/metabolismoRESUMEN
We report that a 30-year-old woman with mental retardation was referred for prenatal diagnoses during pregnancy. An ultrasound scan showed that the heart structure and function of the fetus were normal. Cytogenetic analysis showed that the female karyotype was 47,XX, t(17; 22) (q21; q11), +21. The woman's husband had a normal male karyotype and was phenotypically normal. During this first pregnancy, an amniocentesis, which was done at 19 weeks, revealed that the fetal karyotype was 46,XX, t(17; 22) (q21; q11). Fluorescence in situ hybridization testing of amniotic fluid gave a normal result for chromosome 21. The child was a phenotypically normal female baby.
Asunto(s)
Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 22/genética , Síndrome de Down/genética , Patrón de Herencia/genética , Madres , Núcleo Familiar , Translocación Genética , Adulto , Líquido Amniótico/citología , Bandeo Cromosómico , Femenino , Humanos , Cariotipificación , FenotipoRESUMEN
OBJECTIVES: Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis. METHODS: A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey's test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05. RESULTS: hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48 h. The treated group had less fibrosis and proteinuria at 2 months after injury. CONCLUSION: hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.
Asunto(s)
Lesión Renal Aguda/terapia , Líquido Amniótico/citología , Riñón/citología , Daño por Reperfusión/terapia , Trasplante de Células Madre , Células Madre/citología , Lesión Renal Aguda/patología , Animales , Diferenciación Celular , Rastreo Celular , Células Cultivadas , Femenino , Humanos , Riñón/patología , Masculino , Embarazo , Ratas Wistar , Daño por Reperfusión/patologíaRESUMEN
The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long-term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.
Asunto(s)
Líquido Amniótico/citología , Bovinos/fisiología , Técnicas de Cultivo de Célula/veterinaria , Supervivencia Celular , Criopreservación/veterinaria , Fragmentación del ADN , Cordón Umbilical/citología , Animales , Técnicas de Cultivo de Célula/métodos , Citogenética , Factores de TiempoRESUMEN
Amniotic fluid is a complex mixture composed of water, salts and different cells types derived from embryo exfoliation. Some of these cells present similar characteristics to mesenchymal stem cells as adherent properties, typical surface antigens and differentiation capacity. These cells are called amniotic fluid-derived mesenchymal stem cells (AFMSCs) and are easily obtained by amniocentesis, propagated in culture and differentiated in several cell types with specific inductions. In this study, we observe the ability of simvastatin, a 3-HMG-CoA reductase inhibitor, to induce AFSMCs osteogenic differentiation. When AFSMCs were incubated with medium containing simvastatin, it was observed morphological changes, calcium deposits formation confirmed by Alizarin Red stain. Differentiated cells also expressed typical osteogenic genes, as osteopontin and osteocalcin. In conclusion, simvastatin could be used as an optional osteogenic induction agent for amniotic fluid-derived mesenchymal stem cells.
Asunto(s)
Líquido Amniótico/citología , Diferenciación Celular/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteocalcina/genética , Osteopontina/genética , Simvastatina/farmacología , Calcio/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Femenino , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Quantitative fluorescent polymerase chain reaction (QF-PCR) is an accurate and reliable method for rapid detection of aneuploidy; however, it is not routinely used in China. We aimed to validate QF-PCR as a means for prenatal common aneuploidy screening and to analyze the heterozygosities of short tandem repeat (STR) markers in the Chinese population. The sequences of 19 STR markers in chromosomes 21, 18, 13, X, and Y were designed; three kinds of fluoresceins were used to label the primers, and the QF-PCR detecting conditions were explored and optimized. The results of analysis of 210 prenatal samples by multiplex QF-PCR were compared with karyotyping analysis. All cases were successfully tested by QF-PCR and conventional cytogenetic analysis. QF-PCR results were consistent with the results of cytogenetic analyses, with the exception of two cases. The sensitivity and specificity of QF-PCR to diagnose common aneuploidies were 94.74 and 100%, respectively. The heterozygosities of most of the markers were lower than reported for Western populations, but relatively similar to those of other Asian populations. We conclude that QF-PCR is able to detect the common aneuploidies for prenatal diagnosis with high detection efficacy; therefore it is suitable for rapid prenatal diagnosis and for large-scale testing in laboratories. However, we need to add new STR markers or to find alternative STR markers with high heterozygosity in order to make this technique useful for routine diagnosis.
Asunto(s)
Amniocentesis/métodos , Aneuploidia , ADN/análisis , Líquido Amniótico/citología , China , Femenino , Genotipo , Humanos , Repeticiones de Microsatélite/genética , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Determinar el valor predictivo del índice de líquido amniótico en las complicaciones neonatales. Se seleccionaron 120 embarazadas en las que se evaluó el valor del índice de líquido amniótico, complicaciones neonatales y eficacia diagnóstica. Las pacientes fueron divididas según el punto de corte del índice de líquido amniótico (grupo A: índice de líquido amniótico menor de 60 mm y grupo B índice de líquido amniótico igual o mayor a 60 mm). Servicio de Obstetricia y Ginecología. Hospital Central Dr. Urquinaona. Maracaibo. Estado Zulia. Las pacientes del grupo A presentaron una duración mayor del trabajo de parto y recién nacidos con menos peso al nacer que las pacientes del grupo B (P < 0,05). Con respecto a las complicaciones perinatales, la frecuencia de recién nacidos con sufrimiento fetal y con puntuación de Apgar menor o igual de 6 puntos al minuto fue estadísticamente superior en las pacientes del grupo A comparado con aquellas del grupo B (P < 0,05). El valor de corte de 60 mm en la predicción de sufrimiento fetal tiene una sensibilidad del 22,2 por ciento, especificidad del 96,4 por ciento, valor predictivo positivo del 72,3 por ciento y valor predictivo negativo del 74,3 por ciento; en la predicción de puntuación de Apgar menor o igual de 6 puntos al minuto tiene una sensibilidad del 25,0 por ciento, especificidad del 96,4 por ciento, valor predictivo positivo del 69,2 por ciento y valor predictivo negativo del 74,7 por ciento. El índice de líquido amniótico tiene valor en la predicción de sufrimiento fetal y puntuación de Apgar
To determine the predictive value of amniotic fluid index in perinatal complications. One hundred and twenty patients were selected. Amniotic fluid index, perinatal complications and diagnostic accuracy was evaluated. Patients were divided according to cut-off point of amniotic fluid index (group A: amniotic fluid index less than 60 mm and group B amniotic fluid index same or higher than 60 mm). Servicio de Obstetricia y Ginecologia. Hospital Central Dr. Urquinaona. Maracaibo. Estado Zulia. Patients in group A presented a longer labor and there newborns had less weight than those of patients in group B (P < 0.05). In relation to perinatal complications, the frequency of fetal distress and Apgar Score less than 6 points at minute was statically superior in patients of group A compared with those in group B (P < 0.05). The cut-off value of 60 mm for prediction of fetal distress has a sensivity of 22.2 percent, specificity of 96.4 percent, positive predictive value of 72.3 percent and negative predictive value of 74.3 percent; in the prediction of Apgar score equal or less than 6 points at minute had a sensivity of 25.0 percent, specificity of 96.4 percent, positive predictive value of 69.2 percent and negative predictive value of 74.7 percent. Amniotic fluid index has a value for prediction of fetal distress and Apgar score
Asunto(s)
Embarazo , Complicaciones del Embarazo , Líquido Amniótico/citología , Predicción/métodos , Recién Nacido/líquido cefalorraquídeo , Neonatología , ObstetriciaRESUMEN
BACKGROUND: Chronic allograft vasculopathy (CAV) is an important cause of graft loss. Considering the immune inflammatory events involved in the development of CAV, therapeutic approaches to target this process are of relevance. Human amniotic fluid-derived stem cells (hAFSCs), a class of fetal, pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, display immunomodulatory properties. hAFSCs express mesenchymal and embryonic markers, show high proliferation rates; however, they do not induce tumor formation, and their use does not raise ethical issues. Thus, we sought to investigate the effect of hAFSC on CAV in a model of aorta transplantation. METHODS: Orthotopic aorta transplantation was performed using Fisher (F344) rats as donors and Lewis rats as recipients. Rats were divided into three groups: syngeneic (SYNG), untreated F344 receiving aorta from F344 (n = 8); allogeneic (ALLO), Lewis rats receiving allogeneic aorta from F344 (n = 8); and ALLO + hAFSC, ALLO rats treated with hAFSC (10(6) cells; n = 8). Histological analysis and immunohistochemistry were performed 30 days posttransplantation. RESULTS: The ALLO group developed a robust aortic neointimal formation (208.7 ± 25.4 µm) accompanied by a significant high number of ED1+ (4845 ± 841 cells/mm2) and CD43+ cells (4064 ± 563 cells/mm2), and enhanced expression of α-smooth muscle actin in the neointima (25 ± 6%). Treatment with hAFSC diminished neointimal thickness (180.7 ± 23.7 µm) and induced a significant decrease of ED1+ (1100 ± 276 cells/mm2), CD43+ cells (1080 ± 309 cells/µm2), and α-smooth muscle actin expression 8 ± 3% in the neointima. CONCLUSIONS: These preliminary results showed that hAFSC suppressed inflammation and myofibroblast migration to the intima, which may contribute to ameliorate vascular changes in CAV.
Asunto(s)
Líquido Amniótico/citología , Aorta Abdominal/trasplante , Enfermedades de la Aorta/prevención & control , Células Madre Fetales/trasplante , Trasplante de Órganos/efectos adversos , Células Madre Pluripotentes/trasplante , Actinas/metabolismo , Animales , Aorta Abdominal/inmunología , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Biomarcadores/metabolismo , Movimiento Celular , Células Cultivadas , Células Madre Fetales/inmunología , Células Madre Fetales/metabolismo , Humanos , Inmunohistoquímica , Masculino , Miofibroblastos/metabolismo , Miofibroblastos/patología , Neointima , Células Madre Pluripotentes/inmunología , Células Madre Pluripotentes/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Factores de TiempoRESUMEN
Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me(2)SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me(2)SO and glycerol presented workable viability ratios.
Asunto(s)
Líquido Amniótico/citología , Criopreservación/métodos , Crioprotectores/farmacología , Células Madre Mesenquimatosas/citología , Análisis de Varianza , Antígenos CD/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Citometría de Flujo , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.
Asunto(s)
Líquido Amniótico/metabolismo , Criopreservación , Proteínas de Homeodominio/genética , Cariotipificación , Células Madre Mesenquimatosas/metabolismo , Factores de Transcripción SOXB1/genética , Líquido Amniótico/citología , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog , EmbarazoRESUMEN
Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells.
Asunto(s)
Alantoides/citología , Líquido Amniótico/citología , Investigación con Células Madre , Células Madre/citología , Adipogénesis , Alantoides/inmunología , Alantoides/metabolismo , Líquido Amniótico/inmunología , Líquido Amniótico/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Condrogénesis , Medios de Cultivo/metabolismo , Perros , Femenino , Edad Gestacional , Inmunofenotipificación , Proteínas de Filamentos Intermediarios/metabolismo , Queratina-18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Osteogénesis , Embarazo , Células Madre/inmunología , Células Madre/metabolismo , Vimentina/metabolismoRESUMEN
The cytology of extraembrionic fluids in cows in the first, second, and third trimester of pregnancy was evaluated. For each trimester, 10 pregnant uteri, collected in the slaughterhouse were used. A volume of 20mL of amniotic and allantoic fluids was collected, and centrifuged at 200g for 10 minutes. The sediment was taken and processed on a slide for cytology examination after staining. The results showed that the total cells number of the amniotic fluid increased during gestation, with the panoptic staining method being efficient to evaluate the amniotic fluid cytology in crossbreed Holstein x Zebu cows.
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Animales , Femenino , Líquido Amniótico/citología , Líquidos Corporales/citología , Reproducción/fisiología , Bovinos , Placentación/fisiologíaRESUMEN
The cytology of extraembrionic fluids in cows in the first, second, and third trimester of pregnancy was evaluated. For each trimester, 10 pregnant uteri, collected in the slaughterhouse were used. A volume of 20mL of amniotic and allantoic fluids was collected, and centrifuged at 200g for 10 minutes. The sediment was taken and processed on a slide for cytology examination after staining. The results showed that the total cells number of the amniotic fluid increased during gestation, with the panoptic staining method being efficient to evaluate the amniotic fluid cytology in crossbreed Holstein x Zebu cows. (AU)
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Animales , Femenino , Líquido Amniótico/citología , Líquidos Corporales/citología , Reproducción/fisiología , Placentación/fisiología , BovinosRESUMEN
O cão e um excelente modelo pre clinico para o estudo de doencas, testes farmacologicos e novas terapias para uma futura aplicacao em humanos. Desta forma, estudamos o modelo canino como fonte de celulas tronco de anexos fetais, o liquido amniotico, alantoide e o conteudo vitelino. ...
The dog is an excellent preclinical model for the study of diseases, pharmacological tests and new terapies for future aplicatiom in humans. Thus, we studied the canine model as source of stem cells from fetal membranes, amniotic fluid, alantois and fluid yolk. ...
Asunto(s)
Perros , Alantoides/citología , Alantoides/crecimiento & desarrollo , Células Madre Fetales/citología , Líquido Amniótico/citología , Líquido Amniótico , Saco Vitelino/anatomía & histología , Saco Vitelino/citología , Cuerpo Adiposo/anatomía & histologíaRESUMEN
Este artículo pretende destacar la importancia de las intervenciones de enfermería en pacientes que presentan Ruptura Prematura de Membranas. Se subraya la función que cumple el Líquido Amniótico en la prevención de infecciones y en la amenaza de parto prematuro. Estas acciones están dirigidas a favorecer la evolución del embarazo, conservando la salud de la paciente y la vida del feto.