Project description:Mapping deletions of Chr7 in human iPSC derived from patients cells through reprogramming Two-condition experiment, normal diploid iPSCs vs del7q-iPSCs
Project description:Mapping deletions of Chr7 in human iPSC and ESCs derived from patients cells through reprogramming or chromosome engineering Two-condition experiment, normal diploid hPSCs vs del7q-hPSCs
Project description: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. Gene expression analysis was performed on a total of 5 human cell lines, including an isogenic set of 3 nuclear-transfer embryonic stem cell lines and their parental neonatal fibroblast cell line, as well as a fourth nuclear-transfer embryonic stem cell line, which was derived from adult fibroblasts from a type 1 diabetic subject.
Project description:Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation towards a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification towards lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in fetal lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.
Project description:The Purpose of this series of experiments is to identify copy number variations, duplications, and deletions in human induced pluripotent stem (hiPSC) cell lines. Genomic DNA of different human induced pluripotent stem cell lines are extracted and hybridized to NimbleGen CGH microarray, using genomic DNA of hESC H1 or H9 as common reference. Difference between different stem cell lines will be revealed.
Project description:The purpose of this series of experiments is to identify copy number variations, duplications, deletions and regions of homozygosity in human induced pluripotent stem (hiPSC) cell lines. Genomic DNA of human induced pluripotent stem cell lines are extracted and hybridized to Agilent aCGH+SNP microarray, using genotyped Agilent Reference DNA as common reference. Differences and similarities between fibroblast and stem cell lines will be revealed.