Project description:Clinical-grade human embryonic stem cells (hESCs) from 4 centres in the UK were cultured in self-renewal conditions Genomic DNA was isolated from low passage hESCs and submitted for SNP analysis using Illumina HumanCytoSNP-12 v2.1 BeadChip arrays Evaluation of molecular karyotype of multiple clinical-grade hESC lines.
Project description:We constructed a clinical-grade haplobank of 27 induced pluripotent stem cells (iPSCs) lines prepared in accordance with good manufacturing practice regulations from seven donors who were homozygous for one of the four most frequent human leukocyte antigen (HLA)-haplotypes in Japan. The haplobank could provide HLA-matched iPSCs lines to ~40% of the Japanese population. Since the first release in 2015, these iPSC lines have been used in more than 12 clinical studies. We performed rigorous quality control (QC) tests, including residual episomal vectors, genetic mutations in cancer-related genes, copy number alterations, karyotype, expression of markers of the undifferentiated state, morphology, identity (HLA typing and short tandem repeat analysis), sterility and endotoxin. Although the significance of most mutations in cancer-related genes is unknown, we excluded iPSC lines with such mutations to maximize the safety. The haplobank we have established here is an important step toward the clinical application of iPSCs in cell therapies.
Project description:Clinical-grade human embryonic stem cells (hESCs) from 4 centres in the UK were cultured in self-renewal conditions Genomic DNA was isolated from low passage hESCs and submitted for SNP analysis using Illumina HumanCytoSNP-12 v2.1 BeadChip arrays
Project description:Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to insure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but as of yet not from humans. Here we analyzed a large collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics such as self-renewal capacity and a pluripotency-specific molecular signature. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Intriguingly, we found that a haploid genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics, development and evolution. RNA sequencing analysis was performed on a total of 2 samples of in vitro fertilization (IVF) control embryonic stem cell lines.
Project description:Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to insure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but as of yet not from humans. Here we analyzed a large collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics such as self-renewal capacity and a pluripotency-specific molecular signature. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Intriguingly, we found that a haploid genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics, development and evolution. RNA sequencing analysis was performed on a total of 15 samples, including haploid and diploid human parthenogenetic embryonic stem cells at different differentiation states and cell cycle phases.
Project description:Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to insure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but as of yet not from humans. Here we analyzed a large collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics such as self-renewal capacity and a pluripotency-specific molecular signature. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Intriguingly, we found that a haploid genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics, development and evolution. Gene expression analysis was performed on a total of 2 samples of haploid and diploid human parthenogenetic embryonic stem cells in G1.
Project description:Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to insure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but as of yet not from humans. Here we analyzed a large collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics such as self-renewal capacity and a pluripotency-specific molecular signature. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Intriguingly, we found that a haploid genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics, development and evolution. Genome-wide DNA methylation profiling by Illumina Infinium HumanMethylation 450K Beadchip was performed on a total of 12 samples, including undifferentiated haploid and diploid human parthenogenetic embryonic stem cells in either G1 or G2/M, as well as 3 in vitro fertilization (IVF) control embryonic stem cell lines.
Project description:G-banding of human embryonic stem cells (hESC) has proved their predisposition to aneuploidy of chromosomes 12, 17 and X. Now, using array-based comparative genomic hybridization, we find that hESC also accumulate other recurrent chromosomal abnormalities, such as duplications of stemness genes, submicroscopic instability of 20q11.21 and the appearance of a derivative chromosome 18. Keywords: comparative genomic hybridization, genomic integrity of human embryonic stem cells Array-based comparative genomic hybridization was performed on 48 DNA samples from 17 human embryonic stem cell lines, all cultured in our laboratory under the same conditions. All lines were hybridized against DNA obtained from peripheral blood from donors with a known normal karyotype. No replicates were done from the same DNA sample, but, whenever possible the same stem cell line was analysed at later passages. All detected abnormalities were confirmed by FISH and/or G-banding.
Project description:<p>In this study we used next generation deep sequencing technologies to analyze the genomes of Harvard University Stem Cell lines 63 and 64. We performed 101-bp paired-end whole genome sequencing of the two cell lines using Illumina HiSeq platforms. The sequence reads obtained were analyzed for copy number and used for replication timing analysis. Our data suggests that read depth profiles can be used to map replication timing in Embryonic Stem Cells (ESCs). Further we observe that replication profiles are highly correlated across ESCs but distinct from those of other cell types such as Lymphoblastoid Cell Lines (LCLs). These results demonstrated that read depth data from whole genome sequencing can be used to study variation in replication timing within the human population and across different cell types. Whole genome sequences from HUES63 and HUES64 used for this study are being submitted.</p>