RESUMEN
Adipose-tissue-derived stem cells (ASCs) have received considerable attention due to their easy access, expansion potential, and differentiation capacity. ASCs are believed to have the potential to differentiate into neurons. However, the mechanisms by which this may occur remain largely unknown. Here, we show that culturing ASCs under active proliferation conditions greatly improves their propensity to differentiate toward osteogenic, adipogenic, and neurogenic lineages. Neurogenic-induced ASCs express early neurogenic genes as well as markers of mature neurons, including voltage-gated ion channels. Nestin, highly expressed in neural progenitors, is upregulated by mitogenic stimulation of ASCs, and as in neural progenitors, then repressed during neurogenic differentiation. Nestin gene (NES) expression under these conditions appears to be regulated by epigenetic mechanisms. The neural-specific, but not muscle-specific, enhancer regions of NES are DNA demethylated by mitogenic stimulation, and remethylated upon neurogenic differentiation. We observe dynamic changes in histone H3K4, H3K9, and H3K27 methylation on the NES locus before and during neurogenic differentiation that are consistent with epigenetic processes involved in the regulation of NES expression. We suggest that ASCs are epigenetically prepatterned to differentiate toward a neural lineage and that this prepatterning is enhanced by demethylation of critical NES enhancer elements upon mitogenic stimulation preceding neurogenic differentiation. Our findings provide molecular evidence that the differentiation repertoire of ASCs may extend beyond mesodermal lineages.
Asunto(s)
Tejido Adiposo/citología , Epigénesis Genética , Proteínas de Filamentos Intermediarios/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/genética , Neurogénesis/fisiología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Metilación de ADN , Histonas/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/metabolismo , Canales Iónicos/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/metabolismo , Regiones Promotoras GenéticasRESUMEN
The stromal compartment of adipose tissue harbors mesenchymal stem cells (MSCs) (also called stromal stem cells) that display extensive proliferative capacity and multilineage differentiation potential. Such cells offer a practical avenue of generating patient-matched tissue for use in regenerative medicine. It is relatively easy to isolate these cells from adipose tissue in large enough quantities (tens of millions) to allow for their clinical use in a native, uncultured form. Alternatively, MSCs from adipose tissue can be expanded and differentiated into the desired tissue type in vitro using straightforward cell culture techniques. In this chapter, we outline procedures for isolating large numbers of highly purified MSCs from human adipose tissue in their native, uncultured form and methods for their subsequent expansion and differentiation in vitro.
Asunto(s)
Tejido Adiposo/citología , Separación Celular , Células Madre Mesenquimatosas/citología , Técnicas de Cultivo de Célula , Células Cultivadas , HumanosRESUMEN
Mesenchymal stem cells (MSCs) can differentiate into multiple mesodermal cell types in vitro; however, their differentiation capacity is influenced by their tissue of origin. To what extent epigenetic information on promoters of lineage-specification genes in human progenitors influences transcriptional activation and differentiation potential remains unclear. We produced bisulfite sequencing maps of DNA methylation in adipogenic, myogenic, and endothelial promoters in relation to gene expression and differentiation capacity, and unravel a similarity in DNA methylation profiles between MSCs isolated from human adipose tissue, bone marrow (BM), and muscle. This similarity is irrespective of promoter CpG content. Methylation patterns of MSCs are distinct from those of hematopoietic progenitor cells (HPCs), pluripotent human embryonic stem cells (hESCs), and multipotent hESC-derived mesenchymal cells (MCs). Moreover, in vitro MSC differentiation does not affect lineage-specific promoter methylation states, arguing that these methylation patterns in differentiated cells are already established at the progenitor stage. Further, we find a correlation between lineage-specific promoter hypermethylation and lack of differentiation capacity toward that lineage, but no relationship between weak promoter methylation and capacity of transcriptional activation or differentiation. Thus, only part of the restriction in differentiation capacity of tissue-specific stem cells is programmed by promoter DNA methylation: hypermethylation seems to constitute a barrier to differentiation, however, no or weak methylation has no predictive value for differentiation potential.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Linaje de la Célula/fisiología , Metilación de ADN , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Regiones Promotoras Genéticas/genética , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo/citología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Islas de CpG/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Expresión Génica/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Musculares/citología , Células Musculares/metabolismo , Músculo Esquelético/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genéticaRESUMEN
The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle-related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage-induced apoptosis. 8-CPT-cAMP sensitizes ASCs to gamma-irradiation-induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated up-regulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress-induced apoptosis in mesenchymal stem cells.
Asunto(s)
Adipocitos/fisiología , Apoptosis/fisiología , AMP Cíclico/metabolismo , Ciclina E/metabolismo , Daño del ADN , Células Madre Mesenquimatosas/fisiología , Proteínas Oncogénicas/metabolismo , Adipocitos/citología , Adipocitos/efectos de la radiación , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Forma de la Célula , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de la radiación , Neuronas/citología , Neuronas/fisiología , Proteínas Oncogénicas/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
BACKGROUND: Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation. RESULTS: ASC clones cultured to senescence display reduced adipogenic differentiation capacity in vitro, on the basis of limited lipogenesis and reduced transcriptional upregulation of FABP4 and LPL, two adipogenic genes, while LEP and PPARG2 transcription remains unaffected. In undifferentiated senescent cells, PPARG2 and LPL expression is unaltered, whereas LEP and FABP4 transcript levels are increased but not in all clones. Bisulfite sequencing analysis of DNA methylation reveals overall relative stability of LEP, PPARG2, FABP4 and LPL promoter CpG methylation during senescence and upon differentiation. Mosaicism in methylation profiles is maintained between and within ASC clones, and any CpG-specific methylation change detected does not necessarily relate to differentiation potential. One exception to this contention is CpG No. 21 in the LEP promoter, whose senescence-related methylation may impair upregulation of the gene upon adipogenic stimulation. CONCLUSION: Senescent ASCs display reduced in vitro differentiation ability and transcriptional activation of adipogenic genes upon differentiation induction. These restrictions, however, cannot in general be attributed to specific changes in DNA methylation at adipogenic promoters. There also seems to be a correlation between CpGs that are hypomethylated and important transcription factor binding sites.
Asunto(s)
Adipogénesis/genética , Tejido Adiposo/citología , Senescencia Celular/genética , Metilación de ADN , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Células Madre/citología , Tejido Adiposo/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Células Clonales/citología , Islas de CpG/genética , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Leptina/genética , Lipoproteína Lipasa/genética , PPAR gamma/genéticaRESUMEN
In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported, and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes, CD31 and CD144, in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells, freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes, marginal upregulation of CD31, and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however, no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters, which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Fosfatos de Dinucleósidos/genética , Endotelio Vascular/citología , Regiones Promotoras Genéticas , Células Madre/citología , Células Madre/fisiología , Tejido Adiposo/fisiología , Antígenos CD , Humanos , Inmunofenotipificación , Metilación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Venas UmbilicalesRESUMEN
The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. Stromal stem cells offer a potentially large therapeutic potential in the field of regenerative medicine. Adipose tissue contains a large number of stromal stem cells, is relatively easy to obtain in large quantities, and thus constitutes a very convenient source of stromal stem cells. Importantly, the number of stem cells obtained is compatible with extensive analyses of the cells in an uncultured, freshly isolated, form. This chapter describes procedures for isolating millions of highly purified stromal stem cells from human adipose tissue and methods of establishing polyclonal and monoclonal cultures of adipose tissue-derived stem cells.
Asunto(s)
Tejido Adiposo/citología , Técnicas de Cultivo de Célula/métodos , Células Madre/citología , Células del Estroma/citología , Diferenciación Celular , Separación Celular , Células Cultivadas , Colagenasas/metabolismo , Eritrocitos/citología , Humanos , Antígenos Comunes de Leucocito/biosíntesis , Leucocitos/citología , Mesodermo/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesisRESUMEN
Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.
Asunto(s)
Adipogénesis/genética , Tejido Adiposo/citología , Islas de CpG/genética , Metilación de ADN , Células Madre Mesenquimatosas/citología , Regiones Promotoras Genéticas/genética , Linaje de la Célula , Separación Celular , Células Cultivadas , Células Clonales , Femenino , Regulación de la Expresión Génica , Humanos , Leptina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADNRESUMEN
The functional reprogramming of a differentiated cell to pluripotency may present beneficial applications in regenerative medicine. Somatic cell nuclear transfer may offer this possibility, but technical hurdles and ethical guidelines currently prevent application of this technology in several countries. As a result, alternative approaches are being developed for altering cell fate. Recent non-nuclear transfer-based approaches for reprogramming somatic cells are discussed as well as ways to enhance their differentiation potential. These approaches include the fusion of differentiated cells with embryonic stem cells and the use of extract from pluripotent cells to reprogramme differentiated cells into multipotent or pluripotent cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Extractos Celulares/farmacología , Animales , Carcinoma , Diferenciación Celular/genética , Fusión Celular , Núcleo Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Regulación hacia Abajo , Epigénesis Genética , Humanos , Óvulo , Células Madre Pluripotentes , Células Madre/efectos de los fármacosRESUMEN
Stromal stem cells identified in various adult mesenchymal tissues (commonly called mesenchymal stem cells [MSCs]) have in past years received more attention as a result of their potential interest as replacement cells in regenerative medicine. An abundant and easily accessible source of adult human MSCs are stem cells harvested from liposuction material. Similarly to bone marrow-derived MSCs, human adipose tissue-derived stem cells (ASCs) can give rise to a variety of cell types in vitro and in vivo; however, they have a propensity to differentiate into primarily mesodermal lineages. Even so, their capacity to differentiate into nonadipogenic mesodermal pathways seems to be restricted. Emerging DNA methylation profiles at adipogenic and nonadipogenic gene promoters in freshly isolated, cultured, or differentiated ASCs aim to provide an epigenetic explanation for this restrictive differentiation potential. A review of these studies indicates that human ASCs are epigenetically marked by mosaic hypomethylation of adipogenic promoters, whereas nonadipogenic lineage-specific promoters are hypermethylated. Surprisingly, in vitro differentiation toward various pathways maintains the overall methylation profiles of undifferentiated cells, raising the hypothesis that ASCs are at least epigenetically preprogrammed for adipogenesis. Novel attempts at reprogramming the epigenome of MSCs have been initiated to enhance the differentiation capacity of these cells.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Epigénesis Genética , Células Madre Mesenquimatosas/fisiología , Adipogénesis/fisiología , Cromatina/metabolismo , Metilación de ADN , Regulación de la Expresión Génica , Humanos , Regiones Promotoras GenéticasRESUMEN
Functional reprogramming of a differentiated cell toward pluripotency may have long-term applications in regenerative medicine. We report the induction of dedifferentiation, associated with genomewide programming of gene expression and epigenetic reprogramming of an embryonic gene, in epithelial 293T cells treated with an extract of undifferentiated human NCCIT carcinoma cells. 293T cells exposed for 1 h to extract of NCCIT cells, but not of 293T or Jurkat T-cells, form defined colonies that are maintained for at least 23 passages in culture. Microarray and quantitative analyses of gene expression reveal that the transition from a 293T to a pluripotent cell phenotype involves a dynamic up-regulation of hundreds of NCCIT genes, concomitant with down-regulation of 293T genes and of indicators of differentiation such as A-type lamins. Up-regulated genes encompass embryonic and stem cell markers, including OCT4, SOX2, NANOG, and Oct4-responsive genes. OCT4 activation is associated with DNA demethylation in the OCT4 promoter and nuclear targeting of Oct4 protein. In fibroblasts exposed to extract of mouse embryonic stem cells, Oct4 activation is biphasic and RNA-PolII dependent, with the first transient rise of Oct4 up-regulation being necessary for the second, long-term activation of Oct4. Genes characteristic of multilineage differentiation potential are also up-regulated in NCCIT extract-treated cells, suggesting the establishment of "multilineage priming." Retinoic acid triggers Oct4 down-regulation, de novo activation of A-type lamins, and nestin. Furthermore, the cells can be induced to differentiate toward neurogenic, adipogenic, osteogenic, and endothelial lineages. The data provide a proof-of-concept that an extract of undifferentiated carcinoma cells can elicit differentiation plasticity in an otherwise more developmentally restricted cell type.
Asunto(s)
Carcinoma/genética , Regulación de la Expresión Génica/fisiología , Células Madre/citología , Células Madre/fisiología , Transcripción Genética , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Daño del ADN , Genoma Humano , Humanos , Células Jurkat , Mesodermo/citología , Mesodermo/fisiología , Neuronas/citología , Neuronas/fisiología , Reacción en Cadena de la Polimerasa , Extractos de Tejidos/fisiologíaRESUMEN
Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal +CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Regulación de la Expresión Génica , Células Madre/citología , Células del Estroma/citología , Transcripción Genética , Tejido Adiposo/metabolismo , Antígenos CD , Antígenos CD34/biosíntesis , Adhesión Celular , Diferenciación Celular , Linaje de la Célula , Membrana Celular/metabolismo , Proliferación Celular , Células Cultivadas/metabolismo , Citoesqueleto/metabolismo , Regulación hacia Abajo , Endoglina , Citometría de Flujo , Humanos , Inmunohistoquímica , Antígenos Comunes de Leucocito/biosíntesis , Mesodermo/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Receptores de Superficie Celular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Distribución Tisular , Ingeniería de Tejidos , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
We report the differentiation of human adipose tissue stem cells (ATSCs) to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract. Reversibly permeabilized ATSCs were incubated for 1h in a nuclear and cytoplasmic extract of rat cardiomyocytes, resealed with CaCl(2), and cultured. Three weeks after exposure to extract, ATSCs expressed several cardiomyocyte markers including sarcomeric alpha-actinin, desmin, and cardiac troponin I, and displayed targeted expression of the gap junction protein connexin 43. Formation of binucleated and striated cells, and spontaneous beating in culture were also observed. A low proportion of intact ATSCs exposed to the extract also showed signs of alpha-actinin and connexin 43 expression. Additional evidence of differentiation was provided by induction of expression of nuclear lamin A/C, a marker of terminally differentiated cells, and a remarkable increase in cell cycle length. Together with our previous data, this study suggests that alteration of cell fate using cellular extracts may be applied to multiple cell types. Cell extracts may also prove useful for investigating the molecular mechanisms of stem cell differentiation.
Asunto(s)
Tejido Adiposo/citología , Técnicas de Cultivo de Célula/métodos , Miocardio/citología , Células Madre/citología , Actinina/metabolismo , Animales , Bromodesoxiuridina/farmacología , Diferenciación Celular , División Celular , Núcleo Celular/metabolismo , Células Cultivadas , Colorantes/farmacología , Conexina 43/metabolismo , Citoplasma/metabolismo , ADN Complementario/metabolismo , Femenino , Citometría de Flujo , Humanos , Cinética , Microscopía Fluorescente , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Factores de Tiempo , Xantenos/farmacologíaRESUMEN
The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73% P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL(-1); P < 0.005) and androstenedione (70 v. 16 ng mL(-1); P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17beta-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed 'oocyte capacitation'. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.
Asunto(s)
Envejecimiento , Líquido Folicular/química , Donación de Oocito/veterinaria , Oocitos/fisiología , Esteroides/análisis , Porcinos/fisiología , Androstenodiona/análisis , Animales , Blastocisto/fisiología , Estradiol/análisis , Femenino , Progesterona/análisis , Maduración Sexual , Testosterona/análisis , Donantes de TejidosRESUMEN
Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.