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Induced pluripotent stem cells can be generated in bulk culture

ABSTRACT: Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). However, successfully reprogrammed cells are highly proliferative and escape from cellular senescence - it is therefore conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency and size was higher when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs had indistinguishable in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs – this is of relevance for standardization and automation of cell culture procedures

ORGANISM(S): Homo sapiens

PROVIDER: GSE42807 | GEO | 2013/06/03

SECONDARY ACCESSION(S): PRJNA183495

REPOSITORIES: GEO

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Induced pluripotent stem cells can be generated in bulk culture

Project description:Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). However, successfully reprogrammed cells are highly proliferative and escape from cellular senescence - it is therefore conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency and size was higher when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs had indistinguishable in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs M-bM-^@M-^S this is of relevance for standardization and automation of cell culture procedures Gene expression profiles of induced fibroblasts (M-bM-^@M-^\iFM-bM-^@M-^]) from three different donors which were either clonally derived (M-bM-^@M-^\CM-bM-^@M-^]) or bulk-cultured (M-bM-^@M-^\BM-bM-^@M-^]) were compared

2013-06-03 | E-GEOD-42807 | biostudies-arrayexpress

An Automated, High Platform for Induced Pluripotent Stem Cell Derivation, Characterization and Differentiation

Project description:Induced pluripotent stem cells (iPSCs) have become an essential tool for both modeling how causal genetic variants impact cellular function in disease, as well as being an emerging source of tissue for transplantation medicine. Unfortunately the preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes that limit the scale and level of reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming that enables automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. Using this platform, we demonstrate that automated reprogramming and the pooled selection of pluripotent cells results in high quality, stable, iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single colony-subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines. Two independent human fibroblast lines were reprogrammed, using modified mRNA, into induced pluripotent stem cells (iPSCs). The genomic stability of several cell lines was evaluated using SNP arrays. Three iPSCs originating from one fibroblast line were tested at passages 8 and 20, with two of these derived as picked (clonal) lines and the third being a pooled population. Five iPSCs originating from a second fibroblast line were tested at passages 8 and 20, with three of these derives derived as picked (clonal) lines and two derived as pooled populations. The iPSCs were compared against the original parental fibroblasts.

2015-08-03 | E-GEOD-69868 | biostudies-arrayexpress

An Automated, High Platform for Induced Pluripotent Stem Cell Derivation, Characterization and Differentiation

2015-08-03 | GSE69868 | GEO

Expression data in human oral mucosa fibroblasts (hOFs), dermal fibroblasts, and hOFs-derived iPS cells

Project description:To define the characteristics of human oral mucosa fibroblasts (hOFs), we analyzed the gene expression of hOFs compared with that of human dermal fibroblasts (hDFs), and that of hOF-derived induced pluripotent stem cells (hOF-iPSCs). In this dataset, we found that 5,738 probes and 5672 probes were differently expressed in hOFs and hDFs respectively, compared with those in hOF-iPSCs at more than 2-fold levels. In contrast, only 434 probes are differently expressed between hOFs and hDFs.

2016-04-21 | GSE56805 | GEO

Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

Project description:Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors.

2023-11-14 | GSE230647 | GEO

Expression data from human iPSC-derived corneal epithelial cells

Project description:We successfully induced corneal epithelial cells from human iPSCs. Then, we perfomed global expression analysis using microarray to compare the character of hiPSC-derived corneal epithelial cells with that of the other kinds of cells. Total RNA was obtained from human iPSCs (hiPSCs), human iPSC-derived corneal epithelial cells (hiCECs), human corneal limbal epithelial cells (HCECs), human oral keratinocytes (HOKs), human dermal fibroblasts (HDFs) and six weeks-differentiated hiPSCs (hiPSC-derived ocular surface ectoderm, OSE) using the QIAZol reagent. A microarray analysis using Sure Print G3 human 8x60K slides (Agilent technologies) was performed at Takara Bio (Shiga, Japan).

2016-03-09 | E-GEOD-73971 | biostudies-arrayexpress

Effects of XPC overexpression on global methylation

Project description:The study examines the impact of XPC overexpression on DNA demethylation in human dermal fibroblasts (HDFs) and during the early phase of somatic cell reprogramming (pre-iPSCs). We profiled the 5mC status in wild type and XPC gain-of-function HDFs and pre-iPSCs on a genome-wide scale by MeDIP-seq. XPC overexpression results in a loss of DNA methylation in both HDFs and pre-iPSCs. In XPC gain-of-function cells, not only does DNA methylation occur in fewer regions compared to control WT cells, but overall methylation levels are lower than in WT cells.

2017-05-22 | GSE92445 | GEO

Conversion of murine extraembryonic trophoblast stem cells into induced pluripotent stem cells

Project description:Extraembryonic trophoblast stem cells (TSC) can be converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4 and cMyc. TSC lines were derived from mice harboring a doxycycline inducible Oct4 allele and an Oct4-GFP reporter that has been demonstrated to be activated in cells upon acquisition of pluripotency. Oct4-GFP-positive blastocysts were collected at 3.5 dpc and transduced with lentiviruses encoding doxycycline inducible Sox2, Klf4 and cMyc transgenes (4FTSC). 4FTSC lines were passaged 10 times to establish a population of constantly growing, self-renewing TSCs in the presence of FGF4 and fibroblast conditioned media. To induce lineage conversion, 4FTSCs were cultured under ESC/Lif conditions and doxycycline. After 28 days, several colonies displaying ESC-characteristic dome-shaped colony morphology and bright Oct4-GFP fluorescence could be detected. The 4FTSC-derived colonies were isolated mechanically, dissociated by trypsinization, and plated onto MEFs in ESC medium without doxycycline demonstrating the independence of exogenous factors. They will be called TSC-iPSCs (Trophoblast stem cell derived induced pluripotent stem cells). To examine if the extraembryonic lineage-specific mRNA profile was overcome, the gene-expression profiles of TSC-iPSCs and their parental 4FTSCs were analyzed by microarray analyses and compared to control ESCs.

2011-02-01 | E-GEOD-25872 | biostudies-arrayexpress

Global gene expression analysis of peripheral blood cells, pluripotent cell lines, and iPS-derived T cells

Project description:Exhaustion of antigen-specific T cells represents a major challenge to adoptive immunotherapy against many types of cancer and viral infection. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8+ T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells (T-iPSCs) were then redifferentiated into CD8+ T cells that had high proliferative capacity and elongated telomeres. To confirm that T-iPSCs were compatible to other embryonic stem cells (ESCs) but different from T cells, and that redifferentiated T cells were compatible to T cells but different from natural killer (NK) cells, we analyzed and compared the gene expression profiles of the samples by cDNA microarray. These analyses revealed that our T-iPSCs and redifferentiated T cells were essentially the same as their counterpart pluripotent stem cells and T cells, respectively. We generated human T-iPSC lines from peripheral blood T cells of healthy donors. To confirm that established T-iPSCs were typical iPSCs, the gene expression profile of one T-iPSC clone derived from a healthy person (TkT3V1-7) was compared to those of peripheral blood T cells (CD4+ T cell and CD8+ T cell) and ESCs (KhES3). We also established T-iPSCs (H254SeVT-3) from a T-cell clone (H25-#4) which was derived from an HIV-1-infected patient. H254SeVT-3 was redifferentitated into functional T cells (reT-1 and reT-2.1) in vitro. The gene expression profiles of reT-1 and reT-2.1 were compared to those of H25-#4 and peripheral blood NK cells to clarify that the redifferentiated T cells carried the same characteristics as the original T-cell clone.

2012-12-25 | E-GEOD-43136 | biostudies-arrayexpress

Global gene expression analysis of peripheral blood cells, pluripotent cell lines, and iPS-derived T cells

2012-12-25 | GSE43136 | GEO

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