Methods of using ZSCAN4 for rejuvenating human cells
A cell, object technology applied in the field of rejuvenation of human cells using ZSCAN4
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Examples
Embodiment 1
[0368] Example 1: Zscan4 Expression Corrects Chromosomal Abnormalities in Mouse Embryonic Stem Cells
[0369] This example describes the discovery that expression of Zscan4, either by synthetic mRNA encoding Zscan4 or by a Sendai virus vector expressing Zscan4, can correct chromosomal abnormalities in mouse embryonic stem (ES). This example also demonstrates that synthetic mRNA encoding Zscan4 and Sendai virus vectors expressing Zscan4 can be used as biologics.
[0370] Materials and methods
[0371] cell culture
[0372]The MC1ZE murine ES cell line was previously reported (Amano et al., Nat Commun, 2013; 4:1966). MC1ZE cells were used as typical murine ES cells, which showed poor karyotype (ie, only about 20% of cells carried euploidy) due to long-term culture (passage number 33), and integration of exogenous genes. Cells were cultured at 37°C in 5% CO2 in complete ES medium as previously described (Amano et al., NatCommun, 2013; 4:1966.): DMEM (Gibco), 15% FBS (Atlanta...
Embodiment 2
[0389] Example 2: Effect of Zscan4 biological products on mouse embryonic stem cells
[0390] It has previously been shown that forced expression of exogenous murine Zscan4c from a plasmid vector integrated into the murine genome induces the expression of a unique set of genes including Tmem92, Stra8 and the endogenous Zscan4 gene (Amano et al., NatCommun, 2013; 4: 1966).
[0391] This example demonstrates that a Zscan4 biologic (eg, expressing Zscan4, either by a synthetic mRNA encoding Zscan4 or by a Sendai virus vector expressing Zscan4) exerts the same function in murine embryonic stem (ES) cells.
[0392] Materials and methods
[0393] cell culture
[0394]The MC1 murine embryonic stem (ES) cell line was previously used as a typical murine pluripotent stem cell to demonstrate the function of the exogenously introduced Zscan4c gene, which is integrated into the murine genome (Amano et al., NatCommun, 2013; 4:1966.) . MC1 cells were cultured at 37°C in 5% CO2 in compl...
Embodiment 3
[0405] Example 3: Effect of Zscan4 biological products on human induced pluripotent stem cells
[0406] The genes SERPINB4, DNMT3L, and DUX4 were identified as marker genes that were upregulated when the murine Zscan4c or human ZSCAN4 genes were overexpressed by a transgene-based gene expression system.
[0407] This example demonstrates that Zscan4 biologics (e.g., expressing Zscan4—either by synthetic mRNA encoding Zscan4 or by a Sendai virus vector expressing Zscan4) can be produced in a manner similar to that of the transgene-based Zscan4 overexpression system used in murine pluripotent stem cells. way to act on human iPS cells. This example also demonstrates that synthetic mRNA encoding human ZSCAN4 and Sendai virus vectors expressing human ZSCAN4 can be used as therapeutic biologics for the treatment of human pluripotent stem cells.
[0408] Materials and methods
[0409] cell culture
[0410] Human foreskin fibroblasts were cultured on mitotically inactivated murin...
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Abstract
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