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Human oocytes reprogram adult somatic cells to pluripotent stem cells

ABSTRACT: The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells, holding promise for autologous cell replacement therapy. Though reprogramming of somatic cells by nuclear transfer was first demonstrated more than 60 years ago, only recently have human diploid embryonic stem cells been derived after nuclear transfer of fetal and neonatal fibroblasts. Because of the therapeutic potential of developing diploid embryonic stem cell lines from adult cells of normal and diseased human subjects, we have systematically investigated the parameters affecting efficiency and developmental potential in their derivation. We found that improvements to the oocyte activation protocol, including the use of both a kinase and a translation inhibitor, and cell culture in the presence of histone deacetylase inhibitors enable development of diploid cells to the blastocyst stage. Developmental efficiency varied significantly between oocyte donors, and was inversely related to the number of days of hormonal stimulation required to reach mature oocytes, while the daily dose of gonadotropin or the total number of MII oocytes retrieved did not affect developmental outcome. The use of diluted Sendai virus in calcium-free medium during nuclear transfer improved developmental potential, while the use of concentrated Sendai virus induced an increase in intracellular calcium and caused premature oocyte activation. Using these modifications to the nuclear transfer protocol, we successfully derived diploid pluripotent stem cell lines from both postnatal and adult somatic cells of a type 1 diabetic subject. The goal of this experiment was to determine if human oocytes have the ability to reprogram a somatic cell genome in the absence of the oocyte genome. Our previous research had indicated that human oocytes can reprogram adult somatic cells if the oocyte genome remains present (Noggle et al. Nature 2011, doi:10.1038/nature10397). The data presented here is part of a new series of experiments aimed at obtaining diploid cells after somatic cell nuclear transfer into enucleated oocytes. In this experiment, adult somatic cells were transferred into enucleated oocytes and thereafter cultured in the presence of 240ng/ml scriptaid for 17 hours. Samples were cultured until cleavage stage and then collected for microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Dieter Egli

PROVIDER: E-GEOD-54849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The stem cell lines were generated according to the principle described in Noggle et al., Nature 2011, Oct 5;478(7367):70-5. doi: 10.1038/nature10397. Title: Human oocytes reprogram somatic cells to a pluripotent state. Abstract: The exchange of the oocyte's genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patient's genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange. The major difference to Noggle et al. are that these new stem cell lines are tetraploid rather than diploid. The main technical difference is the addition of cytochalasinB during artificial activation, preventing extrusion of the second polar body, thereby resulting in the retention of a diploid oocyte genome, rather than a haploid one. Adult somatic cells were transferred into non-enucleated oocytes and then activated in the presence of cytochalasinB. Addition of cytochalasinB inhibits extrusion of the second polar body, resulting in tetraploid eggs. The efficiency of development to the blastoycst stage is described in: Yamada et al., 2014, Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells, Nature. 2014 Jun 26;510(7506):533-6. doi: 10.1038/nature13287. Blastocysts developing from these were used for the derivation or pluripotent stem cell lines. Gene expression analysis was performed to demonstrate transcriptional reprogramming. These cell lines contain both somatic and oocyte genomes.

Project description:We provide proof that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Mouse ooplasts transplanted with 2 somatic cell nuclei simultaneously (double nuclear transfer) support preimplantation development and the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes at the same time and exhibit characteristics of true pluripotency. By assessing allelic expression of Oct4 in normal and double nucleus transplanted embryos throughout development we demonstrate that oocyte mediated reprogramming is embarked with a probabilistic component and that one reprogrammed genome can dominate the other during preimplantation development even if merged into the same nucleus. Animal handling and cell recovery: Oocytes from mice of the B6C3F1 strain were used for SCNT and ICSI. Cumulus cells (for SCNT) and sperm (for ICSI) were either from a B6C3F1 or OG2 donor the latter expressing an Oct4 promoter-driven GFP transgene. Additionally cells from pure C56Bl/6 or C3H mice were retrieved for SCNT. Female mice were sacrificed by cervical dislocation 14 h after hCG injection and oocytes retrieved from oviduct ampullae. Oocytes and cumulus cells were handled in Hepes-buffered CZB (HCZB) medium. Double nuclear transfer ICSI and embryo culture: Micromanipulations and embryo culture were performed at 30°C (room temperature). For SCNT oocytes were first enucleated (spindle-chromosome complex removed) before transfer of one two or three cell nuclei with a micropipette driven by a piezo actuator under Nomarski optics. The reconstructed oocytes were activated for 6 h in Calcium-free alpha-MEM supplemented with 10 mM SrCl2 and 5 micro-g mL-1 cytochalasin B. Intracytoplasmic sperm injection (ICSI) of one two or four into intact oocytes was used as the fertilization control condition using sperm from 8-week-old mice of B6C3F1 or OG2 strains. Embryos were placed in embryo culture medium and cultured at 37°C under 5% CO2. The activated oocytes were cultured in alpha-MEM at 37°C and under 5% CO2 until use. On day 5 after activation SCNT and ICSI blastocysts were processed for ESC derivation. Analysis of allelic expression of Oct4: RNA from single embryos was extracted using the ZR-RNA MicroPrep kit (Zymo Research). Complementary DNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) according to manufacturers instruction. A nested PCR was performed to amplify part of the Oct4 cDNA containing a BamH1 sensitive SNP. Primer sequences and PCR conditions are given in the supplementary methods. The PCR product was digested for 8h using 100 units of BamH1-HF (NEB) subsequently subjected to agarose gel electrophoreses and the results grouped into 3 categories (mainly C57Bl/6 balanced mainly C3H) according to the observed band patterns. Global transcriptome analysis by microarray: RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto a MouseRef-8 v2 expression BeadChip (Illumina) following the manufacturer instructions. After washing as recommended chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. Microarray data processing: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was Background correction performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics was performed with in-house developed functions in Matlab. 8 samples were analyzed. tNT#1: tetraploid nuclear transfer, p17, double sedimentation; tNT#2: tetraploid nuclear transfer, p19, double sedimentation; tNT#3: tetraploid nuclear transfer, p25, double sedimentation; tNT#4: tetraploid nuclear transfer, p29, double sedimentation; tNT#5: tetraploid nuclear transfer, p18, double sedimentation; tNT#6: tetraploid nuclear transfer, p25, double sedimentation; NT#1: nuclear transfer, double sedimentation; NT#2: nuclear transfer, double sedimentation;

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Human oocytes reprogram adult somatic cells to pluripotent stem cells

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