Unknown,Transcriptomics,Genomics,Proteomics

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Somatic cell nuclear reprogramming by mouse oocytes endures beyond reproductive decline

ABSTRACT: The mammalian oocyte has the unique feature of supporting fertilization and normal development while being able of reprogramming the nuclei of somatic cells towards pluripotency and occasionally even totipotency. Whilst oocyte quality is known to decay with somatic aging it is not a given that different biological functions decay concurrently. In this study we tested whether oocyte reprogramming ability decreases with somatic aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT) at levels similar to ooplasts of young donors. Despite differences in transcriptome between ooplasts of old and young mice gene expression profiles of the resultant SCNT blastocysts were very similar. Furthermore albeit results showing global down-regulation of apoptosis-related genes in climacteric ooplasts and the reported beneficial effect of inhibiting p53 on transcription factor-induced (iPS) pluripotency reprogramming capacity of young ooplasts was not affected by specifically blocking p53. Our observations strongly suggest that the outcome of oocyte-induced reprogramming is determined by the availability of intrinsic reprogramming factors tightly regulated throughout aging as well as intracellular and environmental checkpoints during pre-implantation development towards selection of the successfully reprogrammed embryos. While oocytes are not regenerated but rather last for life we further propose that these cells can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction. Total mRNA was isolated for samples from young and climacteric mice (pools of 20 ooplasts and 20 SCNT blastocysts in duplicate) using a RNeasy Mini Kit (Qiagen Valencia CA USA) as described by the manufacturer (no DNase treatment). RNA concentrations as well as purity and integrity check were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit (Agilent Technologies Palo Alto CA USA). RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies Ismaning Germany) was used to amplify the total mRNA with two rounds of amplification according to the manufacturerâ??s instructions. Amplified RNA was eluted with 15 ÂµL RE Buffer included in the RiboAmp HS Plus Amplification Kit. RNA concentrations were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit. Turbo Labeling CY3 Kit (MDS Analytical Technologies) was used to label 3 Âµg of amplified RNA with Cy3. Concentration and frequency of incorporation were measured with NanoPhotometer (Implen). Microarray wash and detection of the labeled RNA on GeneChips were carried out according to the manufacturerâ??s instructions (Agilent Technologies). Gene expression profiling was performed using Agilent Whole Mouse Genome Oligo Microarrays (4 x 44k each array containing 41174 features). Array image acquisition and feature extraction was performed using Agilent G2505B Microarray Scanner and Feature Extraction software v.9.5 (Agilent Technologies). 6 samples were analyzed. YoungOoplasts: young ooplasts 1 biological rep ClimactericOoplasts: climacteric ooplasts 1 biological rep YoungBlastocysts: young blastocysts 2 biological rep ClimactericBlastocysts: climacteric blastocysts 2 biological rep

ORGANISM(S): Mus musculus

SUBMITTER: Marcos Araúzo-Bravo

PROVIDER: E-GEOD-23654 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mammalian oocyte has the unique feature of supporting fertilization and normal development while being able of reprogramming the nuclei of somatic cells towards pluripotency and occasionally even totipotency. Whilst oocyte quality is known to decay with somatic aging it is not a given that different biological functions decay concurrently. In this study we tested whether oocyte reprogramming ability decreases with somatic aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT) at levels similar to ooplasts of young donors. Despite differences in transcriptome between ooplasts of old and young mice gene expression profiles of the resultant SCNT blastocysts were very similar. Furthermore albeit results showing global down-regulation of apoptosis-related genes in climacteric ooplasts and the reported beneficial effect of inhibiting p53 on transcription factor-induced (iPS) pluripotency reprogramming capacity of young ooplasts was not affected by specifically blocking p53. Our observations strongly suggest that the outcome of oocyte-induced reprogramming is determined by the availability of intrinsic reprogramming factors tightly regulated throughout aging as well as intracellular and environmental checkpoints during pre-implantation development towards selection of the successfully reprogrammed embryos. While oocytes are not regenerated but rather last for life we further propose that these cells can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction. Total mRNA was isolated for samples from young and climacteric mice (pools of 20 ooplasts and 20 SCNT blastocysts in duplicate) using a RNeasy Mini Kit (Qiagen Valencia CA USA) as described by the manufacturer (no DNase treatment). RNA concentrations as well as purity and integrity check were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit (Agilent Technologies Palo Alto CA USA). RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies Ismaning Germany) was used to amplify the total mRNA with two rounds of amplification according to the manufacturerâ??s instructions. Amplified RNA was eluted with 15 ÂµL RE Buffer included in the RiboAmp HS Plus Amplification Kit. RNA concentrations were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit. Turbo Labeling CY3 Kit (MDS Analytical Technologies) was used to label 3 Âµg of amplified RNA with Cy3. Concentration and frequency of incorporation were measured with NanoPhotometer (Implen). Microarray wash and detection of the labeled RNA on GeneChips were carried out according to the manufacturerâ??s instructions (Agilent Technologies). Gene expression profiling was performed using Agilent Whole Mouse Genome Oligo Microarrays (4 x 44k each array containing 41174 features). Array image acquisition and feature extraction was performed using Agilent G2505B Microarray Scanner and Feature Extraction software v.9.5 (Agilent Technologies).

Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. 16 matched samples, two IVF-ESCs, five sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, the parental fibroblast line, and the sperm and oocyte donor were genotyped using the Illumina Omni5, which interrogates 4.3 million SNPs across the human genome. Additionally, matched samples from a patient with Leigh syndrome, a NT-ESC line, three iPSC lines, and the parental fibroblast line were genotyped using the Illumina Omni5.

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Somatic cell nuclear reprogramming by mouse oocytes endures beyond reproductive decline

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