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Functional genomics assay for characterizing pluripotent stem cell utility and safety

A technology of pluripotent stem cells and pluripotent cells, which is applied in the field of functional genomics research to characterize the utility and safety of pluripotent stem cells, can solve the problems of gene expression markers that cannot be reproduced, and achieve cheap automation and suitable for automation Effect

Active Publication Date: 2013-12-18
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Conversely, it has also been reported that iPS cells can differentiate as efficiently as ES cells in some cases (Boland et al., 2009; Miura et al., 2009; Zhao et al., 2009), and published gene expression markers of iPS cells (gene expression signature) may not reproduce (Stadtfeld et al., 2010)

Method used

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  • Functional genomics assay for characterizing pluripotent stem cell utility and safety
  • Functional genomics assay for characterizing pluripotent stem cell utility and safety
  • Functional genomics assay for characterizing pluripotent stem cell utility and safety

Examples

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Embodiment 1

[0922] Variation in DNA methylation and transcription among hES cell lines

[0923] There are many properties in a given ES cell line that can affect its DNA methylation, transcription or differentiation propensity. These may include the genetic background of the cell line, the manner in which the cell line was cultured, selection pressure exerted by extended in vitro growth, or unexplained random noise. Before attempting to investigate the underlying underlying causes of variation in the performance of pluripotent stem cell lines, it is crucial to first determine the nature and extent of variation present in a large cohort of cell lines.

[0924] To study inter-line variation among pluripotent stem cell populations or pluripotent stem cell lines, the inventors obtained 19 human ES cell lines at low passage numbers (p15-25) and cultured them under standard conditions After several generations, DNA was collected for DNA methylation analysis and RNA for transcriptional profilin...

Embodiment 2

[0935] Causes and consequences of epigenetic and transcriptional variation in human ES cell lines

[0936] To begin to explore the causes and consequences of transcriptional and methylation variation between ES cell lines, the inventors used a "reference map" to quantify the level of variation in these measurements at each locus (Tables 4 and 5). This quantification allowed the inventors to determine the proportion of genes that were mutated, as well as the identity of genes with minimal or substantial changes. The resulting distribution is highly skewed, with only 16% of all genes accounting for 50% of the DNA methylation variation and only 28% of all genes accounting for 50% of the gene expression variation ( Figure 2A ). Consequently, most variation between cell lines was restricted to a subset of loci, suggesting that the characterization of genes in both categories may provide insight into why they change and whether their changes have any effect on a given cell line u...

Embodiment 3

[0943] Global patterns of DNA methylation and transcription were similar between hES cells and hiPS cells.

[0944] The inventor's "reference map" of variation in human ES cell lines allows the inventors to determine the number and identity of genes that deviate from normal in any new cell line by statistical comparison to a "reference corridor" of ES cells. With the aid of reprogramming of defined factors to produce human iPS cell lines for a variety of applications (Park et al., 2008b; Takahashi et al., 2007; Yu et al., 2007), there is a consensus on how to select the most suitable iPS cell line for a given purpose. gradually increasing demand. Mapping DNA methylation and transcriptional variation in iPS cell lines allows one skilled in the art to determine whether there are sites that are systematically different between reprogrammed cells and their ES cell counterparts. Furthermore, this will further help guide the selection of high quality iPS cell lines (similar to what...

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Abstract

The present invention generally relates set of reference data or "scorecard" for a pluripotent stem cell, and methods, systems and kits to generate a scorecard for predicting the functionality and suitability of a pluripotent stem cell line for a desired use. In some aspects, a method for generating a scorecard comprises using at least 2 stem cell assays selected from: epigenetic profiling, differentiation assay and gene expression assay to predict the functionality and suitability of a pluripotent stem cell line for a desired use. In some embodiments, the scorecard reference data can be compared with the pluripotent stem cells data to effectively and accurately predict the utility of the pluripotent stem cell for a given application, as well as any to identify specific characteristics of the pluripotent stem cell line to determine their suitability for downstream applications, such as for example, their suitability for therapeutic use, drug screening and toxicity assays, differentiation into a desired cell lineage, and the like.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C.119(e) to U.S. Provisional Patent Application Serial No. 61 / 384,030, filed September 17, 2010, and Provisional Application No. 61 / 429,965, filed January 5, 2011, The contents of which are incorporated herein by reference in their entirety. technical field [0003] The present invention relates to methods of characterizing stem cells, such as by high-throughput methods, and to the standardization and optimization of pluripotent cell lines for disease modeling, study of stem cell populations, and their use in the treatment of disease Selected methods and combinations. [0004] governmental support [0005] This invention was made in part with government support under Grant No. U01ES017155 under the NIH Roadmap Initiative on Epigenomics ("NIH Roadmap Initiative on Epigenomics"), awarded by the National Institutes of Health. The US Government has certain rights in this inven...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6881C12Q2600/158C12Q2600/154A61P1/04A61P21/00A61P25/00A61P25/28A61P35/00A61P3/08A61P37/02A61P43/00A61P9/04A61P3/10C12N15/1072
Inventor 凯文·C·埃根亚历山大·迈斯纳克里斯托弗·博克伊万耶洛斯·基斯基尼斯格里特·安妮·弗兰斯·维斯塔本
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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