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...

AI Technical Summary

Benefits of technology

The present invention provides methods for selecting and identifying pluripotent stem cells that have near-embryonic telomere length, telomerase activity, and expression levels of certain genes, which are useful for therapeutic purposes. These methods involve analyzing the telomere restriction fragment length, telomerase activity, and gene expression levels in the cells. The invention also includes compositions of cells produced and selected using the described methods, as well as kits and computer program products for performing these methods. Overall, the invention ensures the quality and safety of clinical-grade cell-based therapies derived from human embryonic stem cells or reprogrammed somatic cells.

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 cells useful in the treatment of degenerative diseases such as heart failure, Parkinson's disease, immune senescence and vascular disease has led to numerous studies of iPS cell pluripotency, though there is little research on the effects of transcriptional reprogramming on cellular aging, in particular on telomere length regulation.

Comparisons with established hES cell lines is additionally complicated by drift in TRF length during propagation in vitro (Rosler E S et al, 2004).

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