An Automated, High Platform for Induced Pluripotent Stem Cell Derivation, Characterization and Differentiation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE69868 | GEO | 2015/08/03
SECONDARY ACCESSION(S): PRJNA287007
REPOSITORIES: GEO
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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
Project description:Although reprogramming of somatic cells to generate inducible pluripotent stem cells (iPSCs) is associated with remarkable epigenetic changes, the role and mechanisms of epigenetic factors in this process remains poorly understood. Here we describe identification of Jmjd3 as a potent negative regulator of reprogramming. Jmjd3-deficient MEFs produced significantly more iPSC colonies than did wild-type cells, while ectopic expression of Jmjd3 markedly inhibited reprogramming. We further show that the inhibitory effects of Jmjd3 are produced through both histone demethylase-dependent and -independent pathways acting in concert. The latter pathway is entirely novel and involved Jmjd3 targeting of PHF20 for ubiquitination and degradation by recruiting an E3 ligase Trim26. Importantly, PHF20-deficient MEFs could not be converted to fully reprogrammed iPSCs, even with knockdown of Jmjd3, Ink4a or p21, indicating dominant effects of this protein on reprogramming. Our findings identify a previously unrecognized role of Jmjd3 in reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls the reprogramming process. This study was an examination of phf20 and wdr5 binding patterns in embryonic stem cells, using a ChIP-Sequencing methodology with antibodies for each of these factors in the cell lines indicated.
2013-12-06 | E-GEOD-43247 | biostudies-arrayexpress
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. 16 matched samples, two IVF-ESCs, five sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, the parental fibroblast line, and the sperm and oocyte donor were genotyped using the Illumina Omni5, which interrogates 4.3 million SNPs across the human genome. Additionally, matched samples from a patient with Leigh syndrome, a NT-ESC line, three iPSC lines, and the parental fibroblast line were genotyped using the Illumina Omni5.
2014-07-08 | E-GEOD-53141 | biostudies-arrayexpress
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but existing cell types have imitations. Human embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the “gold standard”, but are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) can be produced from somatic cells by forced expression of pluripotency-associated factors, but are prone to genetic and epigenetic aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming, or secondary to the reprogramming method, we employed an alternative approach by somatic cell nuclear transfer (SCNT). SCNT-based reprogramming to NT-ESCs is mediated by factors present in oocyte’s cytoplasm, thus mimicking early embryogenesis. We generated genetically matched pluripotent stem cells and conducted genome-wide genetic, epigenetic and transcriptional analyses. We discovered that unlike iPSCs, NT-ESCs have a low burden of de novo copy number variations (CNVs), reflecting superior maintenance of genetic stability. Moreover, DNA methylation and transcriptome profiles of NT-ESCs corresponded closely to those of IVF-ESCs. In contrast, iPSCs harbored methylation abnormalities including residual CpG methylation typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT with the potential to satisfy the clinical requirements for cell replacement therapies. Bisulphite converted DNAs of two IVF-ESCs, two sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast were hybridized to the Illumina Infinium HumanMethylation 450K Beadchip
2014-07-08 | E-GEOD-53060 | biostudies-arrayexpress
Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture. 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived.
2015-06-08 | E-GEOD-69632 | biostudies-arrayexpress
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. Duplicate cDNA libraries of two IVF-ESCs, three sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast line were sequenced using Illumina HiSeq 2000. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.
2014-07-08 | E-GEOD-53094 | biostudies-arrayexpress
Project description:We generated three kinds of genetically identical mouse reprogrammed cells: induced pluripotent stem cells (iPSCs), nuclear transfer embryonic stem cells (ntESCs) and iPSC-nt-ESCs that are established after successively reprogramming of iPSCs by nuclear transfer (NT). NtESCs show better developmental potential than iPSCs, whereas iPSC-nt-ESCs display worse developmental potential than iPSCs. We used microarrays to distinguish the gene expression differences among three pluriptoent stem cells and identified that imprinted genes had a similar expression pattern in iPSCs and iPSC-nt-ESCs. We sought to obtain genetic identical pluripotent stem cell lines in order to minimize genetic variations among different reprogrammed cells. To that end, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4 and c-Myc were isolated and used as donors for different reprogramming experiments. We generated iPSCs after plating MEFs in the presence of dox in ES culture conditions, and then derived ntESCs after transplantation of the nucleus from the same MEFs into enucleated oocyte. Furthermore, we successively reprogrammed iPSCs by means of NT and established a set of nt-iPSC lines. Pluripotent stem cells generated from different reprogramming strategies were for RNA extraction and hybridization on Affymetrix microarrays.
2011-05-31 | E-GEOD-24705 | biostudies-arrayexpress
Project description:Here, we determined the DNA methylation profiles of 12 human cell lines, including 2 ESC lines, 2 pESC lines,4 virally-delivered iPSC lines, 2 episomal-delivered iPSC lines, and 2 parent cell lines that iPSCs derived from using Illumina’s Infinium HumanMethylation450. The iPSCs exhibited a hypermethylation status similarly to ESCs but distinct differences from the parent cells. Genes of common methylation pattern between iPSCs and ESCs were regarded as critical factors for stemness, while differences existing between iPSCs and ESCs implied that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cell reprogramming. No significant variant was identified between virally- and episomal- systems. De novo differentiated methylation regions as the signature of particular stem cell lines were figured out in detail in this study. 2 ESC lines, 2 pESC lines,4 virally-delivered iPSC lines, 2 episomal-delivered iPSC lines, and 2 parent cell lines that iPSCs derived from
2014-05-29 | E-GEOD-57992 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4 and Myc (OSKM) into somatic cells. Though iPSCs are pluripotent, they frequently exhibit high variation in their quality as measured by chimera contribution and tetraploid (4n) complementation. Thus, improving the quality of iPSCs is an indispensable prerequisite for future iPSC-based therapy. Here we show that one major determinant for iPSCs quality is the selection of the reprogramming factors combination. Ectopic expression of Sall4, Nanog, Esrrb and Lin28 (SNEL) in MEFs efficiently generated high quality iPSCs as compared to other combinations of factors. SNEL-iPSCs produced approximately 5 times more efficiently “all-iPSC” mice compared to OSKM-iPSCs. While differentially methylated regions, transcript number of master regulators, establishment of ESC-specific super enhancers, and global aneuploidy were comparable between the lines, aberrant expression of 1,765 genes, trisomy of chromosome 8 and abnormal H2A.X deposition were frequently observed in poor quality OSKM-iPSCs. For high-quality iPSCs, H2A.X pattern of SNEL is most similar to that of ESC, OSK and OSKM have more devoid regions than SNEL iPSCs. Compare H2A.X deposition pattern of the OSKM 4-factor iPS cell lines (4N-), SNEL 4-factor iPS cell lines (4N+) with ChIP-Seq. The same background ES cell line as the control line.
2014-09-07 | E-GEOD-55731 | biostudies-arrayexpress
Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Expression analysis of parental somatic tissues of human iPSCs used for the present study.
2016-06-29 | E-GEOD-73629 | biostudies-arrayexpress