Abstract
Mus musculus is the only known species from which embryonic stem cells (ESC) can be isolated under conditions requiring only leukemia inhibitory factor (LIF). Other species are non-permissive in LIF media, and form developmentally primed epiblast stem cells (EpiSC) similar to cells derived from post-implantation, egg cylinders. To evaluate whether non-permissiveness extends to induced pluripotent stem cells (iPSC), we derived iPSC from the eight founder strains of the mouse Collaborative Cross. Two strains, NOD/ShiLtJ and the WSB/EiJ, were non-permissive, consistent with the previous classification of NOD/ShiLtJ as non-permissive to ESC derivation. We determined non-permissiveness is recessive, and that non-permissive genomes do not compliment. We overcame iPSC non-permissiveness by using GSK3B and MEK inhibitors with serum, a technique we termed 2iS reprogramming. Although used for ESC derivation, GSK3B and MEK inhibitors have not been used during iPSC reprogramming because they inhibit survival of progenitor differentiated cells. iPSC derived in 2iS are more transcriptionally similar to ESC than EpiSC, indicating that 2iS reprogramming acts to overcome genetic background constraints. Finally, of species tested for ESC or iPSC derivation, only some M. musculus strains are permissive under LIF culture conditions suggesting that this is an evolutionarily derived characteristic in the M. musculus lineage.
Highlights
Induced pluripotent stem cells are derived from differentiated cells that have been reprogrammed into an undifferentiated embryonic stem cell (ESC)-like state1. induced pluripotent stem cells (iPSC) offer a potentially unlimited source of patient-specific embryonic stem cells (ESC)-like cells that could be readily used for both research and therapeutics
We evaluated the effect of genetic background on iPSC derivation using Oct[4], Klf[4], Sox[2], and Myc in five classical laboratory strains (129S1/SvImJ, NOD/ShiLtJ, A/J, C57BL/6J, and NZO/H1LtJ) and three wild derived strains (WSB/EiJ, PWK/PhJ, and CAST/ EiJ), that are the eight parental strains of the CC19
Previous knowledge indicates that the NOD/ShiLtJ background is non-permissive to ESC formation and instead forms cells that more closely resemble the epiblast stem cells (EpiSC) state of mouse development[17,18]
Summary
Induced pluripotent stem cells (iPSC) are derived from differentiated cells that have been reprogrammed into an undifferentiated embryonic stem cell (ESC)-like state1. iPSCs offer a potentially unlimited source of patient-specific ESC-like cells that could be readily used for both research and therapeutics. The ability to derive naïve ESC from the inner cell masses (ICM) of pre-implantation blastocysts appears restricted to select permissive strains of mice. Examples of such permissive strains come from the 129S1/SvImJ background from which ESC were first derived. Even more unfavorable is the NOD/ShiLtJ genetic background, which has been found to be non-permissive to ESC derivation in standard conditions and instead readily forms EpiSC-like cells[18]. The inability of two of these strains to form iPSC under standard conditions is predictive of a larger genetic background effect that could re-appear in offspring populations of the CC or the DO, thereby severely limiting the ability to conduct genetic research in cell-based systems. We characterized the non-permissive phenotype; and in response to the need to reprogram cells from diverse backgrounds, developed a novel approach to overcome the limitations imposed by non-permissive genetic backgrounds on iPSC derivation
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