Project description:This study established the transcriptome of self-renewing, naïve induced pluripotent cells (iPSCs). Reprogramming was performed using Oct4, Sox2, cMyc and Klf4 expression together with SV40LT in bovine embryonic fibroblasts. Colonies were selected and passaged and validated for pluripotency (silencing of exogenous factors, internal markers and differentiation potential). Total RNA was isolated from iPSC colonies (passage 8) derived from three independent reprograming events. Using Poly(A) capture, mRNA was isolated and cDNA libraries prepared using Illumina TruSeq RNA sample Prep Kit and sequenced using Illumina HiSeq 4000. This dataset should serve as a baseline for understanding bovine pluripotency regulation via transcriptional control and signaling.
Project description:Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, but genetic instability is a major concern. Embryonic pluripotent cells also accumulate mutations during early development, but how this relates to the mutation burden in iPSCs remains unknown. Here, we directly compared the mutation burden of cultured iPSCs with their isogenic embryonic cells during human embryogenesis. We generated developmental lineage trees of human fetuses by phylogenetic inference from somatic mutations in the genomes of multiple stem cells, which were derived from different germ layers. Using this approach, we characterized the mutations acquired pre-gastrulation and found a rate of 1.65 mutations per cell division. When cultured in hypoxic conditions, iPSCs generated from fetal stem cells of the assessed fetuses displayed a similar mutation rate and spectrum. Our results show that iPSCs maintain a genomic integrity during culture at a similar degree as their pluripotent counterparts do in vivo.
Project description:Pluripotent stem cells (PSCs) have been successfully developed in many species. However, generating bovine induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs by overexpression of lysine-specific demethylase 4A (KDM4A) and repro-gramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined me-dia of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of in-tegration into extra-embryonic tissues. Finally, 24.6% cloning efficiency could be achieved in nu-clear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.
Project description:Orangutans are an endangered species whose natural habitats are restricted to the Southeast Asian islands of Borneo and Sumatra. For potential species conservation and functional genomics studies, we derived induced pluripotent stem cells (iPSCs) from cryopreserved skin fibroblasts obtained from captive orangutans. We report the gene expression profiles of iPSCs and skin fibroblasts derived from orangtuans. The overall goal was to evaluate gene expression biomarkers of pluripotency in iPSCs and skin fibroblasts derived from PBD-ZSD patients and healthy controls. Dermal fibroblast cultures from 2 orangutans were reprogrammed into iPSCs by transfection with retroviruses designed to express the human OCT4, SOX2, KLF4 and c-MYC cDNA. Fibroblasts and iPSCs were cultured in 1:1 ratio of DMEM:F12 medium supplemented with 20% KOSR (knock-out serum replacement) at 37°C with 5% CO2 until confluence for RNA extraction. The overall goal was to evaluate gene expression biomarkers of pluripotency in iPSCs and original fibroblast cultures.
Project description:Orangutans are an endangered species whose natural habitats are restricted to the Southeast Asian islands of Borneo and Sumatra. For potential species conservation and functional genomics studies, we derived induced pluripotent stem cells (iPSCs) from cryopreserved skin fibroblasts obtained from captive orangutans. We report the gene expression profiles of iPSCs and skin fibroblasts derived from orangtuans.
Project description:Embryonic stem cells are pluripotent and possess the ability to differentiate into numerous lineages during the developmental process. In similarity to embryonic stem cells, human induced pluripotent stem cells (iPSCs) possess the potential to differentiate into multiple lineages making them an excellent research tool. We generated iPSCs from multiple donors and also differentiated iPSCs from these donors into human neural progenitor cells (NPCs). We used human transcriptome arrays to detail the programme of gene expression underlying NPC induction and identified distinct classes of up-regulated genes during this process. Total RNAs were extracted from human induced pluripotent stem cells and induced pluripotent stem cell-derived neural progenitor cells. Their gene expression profiles were investigated using the Affymetrix GeneChip Human Transcriptome Array 2.0 platform.
Project description:Pathogenic NOTCH1 mutations are linked to congenital heart defects. To pinpoint how NOTCH1 deficiency affects cardiac development, we generated homozygous NOTCH1 knockout (N1KO) human induced pluripotent stem cells (iPSCs). We then performed high-throughput RNA-seq to profile differential gene expression in cardiomyocytes (iPSC-CMs) and endothelial cells (iPSC-ECs) derived from wild type (WT) and N1KO iPSCs.
Project description:HBV-KMT2B integrated human induced pluripotent stem cells (KMT2B-Int iPSCs) vs heterozygous mutated KMT2B iPSCs (KMT2B-HT iPSCs) vs homozygous mutated KMT2B iPSCs (KMT2B-KO iPSCs) vs their original iPSCs (KMT2B-WT iPSCs)