Methods for telomere length and genomic DNA quality control and analysis in pluripotent stem cells

Abstract

The generation of clinical-grade cell-based therapies from human embryonic stem cells or cells reprogrammed to pluripotency from somatic cells, requires stringent quality controls to insure that the cells have long enough telomeres and resulting cellular lifespan to be clinically useful, and normal gene expression and genomic integrity so as to insure cells with a desired and reproducible phenotype and to reduce the risk of the malignant transformation of cells. Assays useful in identifying human embryonic stem cell lines and pluripotent cells resulting from the transcriptional reprogramming of somatic cells that have embryonic telomere length are described as well as quality control assays for screening genomic integrity in cells expanded and banked for therapeutic use, as well as assays to identify cells capable of abnormal immortalization,

Description

INTRODUCTION[0001]In the 19th century August Weismann introduced the parsimonious theory that heredity and the perpetual regeneration of the body in the life cycle stem from an immortal continuum of germ-line cells (Weismann, 1891; McLaren, A 1992, 2001). As a corollary, he postulated a dichotomy of cell fates in metazoans. Germ-line cells were theorized to possess a replicative immortality (though punctuated with alternating meiotic and mitotic events) while somatic cell lineages a finite replicative capacity. The mortal phenotype in somatic cell types, in turn, was implicated in the finite capacity for tissue regeneration and progressive onset of age-related degenerative disease in the human soma over time.[0002]Leonard Hayflick's demonstration that human somatic cells age in vitro (Hayflick & Moorhead, 1961; Hayflick, 1965; Hayflick, 1992) enabled the use cultured somatic cells as an experimental model of senescence and consequently the discovery that telomeric DNA could function...

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Benefits of technology

[0008]The generation of clinical-grade cell-based therapies from human embryonic stem cells or cells reprogrammed to pluripotency from somatic cells, requires stringent quality controls to insure that the cells have long enough telomeres and resulting cellular lifespan to be clinically useful, and normal gene expression and genomic integrity so as to insure cells with a desired and reproducible phenotype and to reduce the risk of the malignant transformation of cells. Assays useful in identifying human embryonic stem cell lines and pluripotent cells resulting from the transcriptional reprogramming of somatic cells that have embryonic telomere length are described as well as quality control assays for screening genomic integrity in cells expanded and banked for therapeutic use, as well as assays to identify cells capable of abnormal immortalization.

[0009]Aspects of the present invention include methods of selecting/identifying cell lines of pluripotent stem cells, e.g., induced pluripotent stem cells (iPS cells) derived by reprogramming somatic cells with tra

Problems solved by technology

In addition, initial experiments demonstrated that while it was possible to remodel human somatic cell nuclei into pseudo pronuclei, embryonic development generally ceases before blastocyst formation in current protocols, making current studies of human telomere length regulation during reprogramming problematic (Cibelli, J. B. et al, 2001).

The attraction to facile, cost-effective, and ethically non-problematic means of producing a host of transplantable patient-specific cel

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