Project description:Inner cell mass (ICM) cells of two independent E3.5 blastocysts were profiled by transcriptome sequencing to assess the expression of extracellular matrix components.
Project description:A unique embryonic stem cells showing naïve state was established from primplantation mouse blastocyst but maintaind their self renew under FGF2 stimulus condition We used microarray to compare gene expression patterns of our pluripotent stem cell line (named FGF-ESC) with ESCs or EpiSCs in addition to inner cell mass from E3.5 preimplantation blastocyst as in vivo control. Total RNA was extracted from FGF-ESC, ESC, EpiSC and inner cell mass from E3.5 preimplantation blastocyst, and served for microarray analysis using Affymetrix Mouse Gene 2.1 Array
Project description:A unique embryonic stem cells showing naïve state was established from primplantation mouse blastocyst but maintaind their self renew under FGF2 stimulus condition We used microarray to compare gene expression patterns of our pluripotent stem cell line (named FGF-ESC) with ESCs or EpiSCs in addition to inner cell mass from E3.5 preimplantation blastocyst as in vivo control.
Project description:Induced pluripotent stem cells (iPSCs) hold promise for generating personalized xenogenic organs via development of cross-species chimeric animals. However, whether human and other primate iPSCs are capable of establishing cross-species chimeras remains unknown. Recognizing the ethical concerns of cross-species chimerism using human iPSCs, we explored the capacity for cross-species chimerism between distinct, non-human primates. Injection of either pig-tailed macaque iPSCs or chimpanzee iPSCs into the rhesus macaque blastocyst embryos demonstrated that these cells survive, proliferate, and integrate near the rhesus inner cell mass (ICM). Ectopic expression of BCL2 in pig-tailed and chimpanzee iPSCs greatly improved the success rate of establishing cross-species blastocyst chimerism. This study represents the first successful cross-species blastocyst chimerism between distinct, non-human primate species, and highlights critical factors that may be necessary to unlock the broad potential of primate iPSCs to form cross-species chimeras, with diverse applications for basic research and translational medicine.
Project description:Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited such as highly pure cell populations or early developmental stages of mammalian preimplantation embryo where only few hundred cells might be available. Numerous attempts have been made to reduce the number of cells needed for successful ChIP-seq experiment, however, the developed methods are often complex, laborious or not sensitive enough. Here we describe a new simple method called Restrictase Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables to obtain high quality genome-wide histone modification profiles from as few as 100 cells. The method is simple, cost-effective, takes one day to complete and can potentially be automated. We subsequently use the novel method to derive the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass (ICM) and trophoectoderm (TE) of bovine blastocyst stage embryos.
Project description:Aneuploidy has been well documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening (PGS) in a 24-hour protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy, and distinguishes certain meiotic from mitotic errors. In this study our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from seventeen couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e., one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of fifty-one (96.1%) inner cell mass (ICM) samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that trophectoderm karyotype is an excellent predictor of inner cell mass karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.
Project description:The pig is important for agriculture and as an animal model in human and veterinary medicine, yet, despite over 20 years of effort, it has proved a difficult species from which to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of LIF-dependent, so called naïve type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. These cells resemble mouse ES cells and are distinct from the FGF2-dependent, induced pluripotent cell type derived from porcine somatic cells. High throughput SNP chip genotyping was conducted on Illumina's Porcine SNP60 BeadChip (WG-410, a service provided by Geneseek, NE, http://www.neogen.com/GeneSeek/). The results exhibited that the two lines pluripotent stem cells from the inner cell mass of porcine blastocysts were porcine origin and genetically distinct. Porcine pluripotent stem cells were derived from the inner cell mass with transduction with human KLF4 by lentiviral transduction.
Project description:The inner cell mass in blastocyst is the origin of all the somatic and germ cells in mammals, and of pluripotent stem cells in vitro. As the conserved principles between pig and human, here we performed comprehensive single-cell RNA-seq for porcine early embryos from oocyte to early blastocyst. We show the specification of inner cell mass and trophectoderm in morula, and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and inner cell mass of early blastocyst, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe absence of dosage compensation with respect to X-chromosome in morula, and incomplete dosage compensation in early blastocyst. However, the dynamics of dosage compensation may be independent on the expression of XIST induced X-chromosome inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine pluripotent stem cells and improve the research in regenerative medicine.