Human iPSCs derived under feeder free conditions displays an unqiue cell cycle and DNA replication genotype
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ABSTRACT: Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells under feeder-layer conditions. These feeder-derived iPSCs generated in different labs exhibit greater variability than between different traditional embryo derived hESC lines. For that reason, it is important to develop a standard and defined system for deriving autologous patient stem cells. We have generated iPSCs under feeder-free conditions using Matrigel coated vessels in chemically defined medium, mTeSR1. These feeder-free derived iPSCs are in many ways similar to feeder-derived iPSCs and also to hESCs, with respect to their pluripotent gene expression (OCT4, NANOG, SOX2), protein expression (OCT4, NANOG, SSEA4, TRA160) and differentiation capabilities. We conducted a whole genomic transcript analysis using Affymetrix Human Gene 1.0 ST arrays to elucidate the important differences between traditional feeder-derived iPSCs and feeder-free derived iPSCs. We reveal that feeder-free iPSCs have over-represented terms belonging to DNA replication and cell cycle genes which are lacking in feeder-derived iPSCs. Feeder-free iPSCs are in many aspects more similar to hESCs including; apoptosis, chromatin modification enzymes and mitochondrial energy metabolism. We have also identified potential biomarkers for fully reprogrammed iPSCs (FRZB) and partially reprogrammed iPSCs (POTEG, MX2) based on their expression trends across all cell types. In conclusion, feeder-free derived iPSCs is transcriptomically more similar to hESCs than feeder derived iPSCs, in many biological functions. For each cell sample, 2 or 3 biological replicates were obtained.
Project description:Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells under feeder-layer conditions. These feeder-derived iPSCs generated in different labs exhibit greater variability than between different traditional embryo derived hESC lines. For that reason, it is important to develop a standard and defined system for deriving autologous patient stem cells. We have generated iPSCs under feeder-free conditions using Matrigel coated vessels in chemically defined medium, mTeSR1. These feeder-free derived iPSCs are in many ways similar to feeder-derived iPSCs and also to hESCs, with respect to their pluripotent gene expression (OCT4, NANOG, SOX2), protein expression (OCT4, NANOG, SSEA4, TRA160) and differentiation capabilities. We conducted a whole genomic transcript analysis using Affymetrix Human Gene 1.0 ST arrays to elucidate the important differences between traditional feeder-derived iPSCs and feeder-free derived iPSCs. We reveal that feeder-free iPSCs have over-represented terms belonging to DNA replication and cell cycle genes which are lacking in feeder-derived iPSCs. Feeder-free iPSCs are in many aspects more similar to hESCs including; apoptosis, chromatin modification enzymes and mitochondrial energy metabolism. We have also identified potential biomarkers for fully reprogrammed iPSCs (FRZB) and partially reprogrammed iPSCs (POTEG, MX2) based on their expression trends across all cell types. In conclusion, feeder-free derived iPSCs is transcriptomically more similar to hESCs than feeder derived iPSCs, in many biological functions.
Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.
Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs. Six samples were analyzed. The gene expression profile of four iPS clones were compared to the H9 human embryonic stem cell line and the parent endothelial progenitor cell line.
Project description:Transient expression of two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research. Experiment Overall Design: Gene expression profiles measured using human genome Affymetrix Gene Chip arrays were grouped by hierarchical clustering, and correlation coefficients were computed for all pair-wise comparisons
Project description:Transient expression of two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.
Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by the introduction of the transcription factors Oct4, Sox2, Klf4 and cMyc using various methods. Here, we describe a new approach for the derivation of murine iPSCs using a polycistronic non-viral inducible vector integrated into pseudo attP sites via the C31 integrase-mediated site-specific recombination and subsequent vector excision by Cre recombinase. The pluripotency of the derived iPSCs was proved by in vitro and in vivo tests. The derived transgene-free iPSCs reactivated the endogenous pluripotency genes like e.g. Oct4, Sox2 and Nanog and the global gene expression profiles of iPSCs lines are highly similar to ESCs and distinct from parental murine fibroblasts. We demonstrated the differentiation potential of iPSCs by generation cells of the three germ layers as well as we successfully created germline chimeric mice from transgene-free iPSCs. In this study, we presented an efficient method for the generation of transgene-free iPSCs using dual-recombinase technology.
Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by the introduction of the transcription factors Oct4, Sox2, Klf4 and cMyc using various methods. Here, we describe a new approach for the derivation of murine iPSCs using a polycistronic non-viral inducible vector integrated into pseudo attP sites via the C31 integrase-mediated site-specific recombination and subsequent vector excision by Cre recombinase. The pluripotency of the derived iPSCs was proved by in vitro and in vivo tests. The derived transgene-free iPSCs reactivated the endogenous pluripotency genes like e.g. Oct4, Sox2 and Nanog and the global gene expression profiles of iPSCs lines are highly similar to ESCs and distinct from parental murine fibroblasts. We demonstrated the differentiation potential of iPSCs by generation cells of the three germ layers as well as we successfully created germline chimeric mice from transgene-free iPSCs. In this study, we presented an efficient method for the generation of transgene-free iPSCs using dual-recombinase technology. expression data of iPSCs/ESCs/MEFs
Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs. Total RNA obtained from EF1a-control-, OE-NANOG-, OE-OCT4- or OE-SOX2-transduced hESCs.
Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs.
Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs.