Induced pluripotency leapfrogs ahead

Somatic cells derived from differentiated tissues can be reprogrammed back to a pluripotent state. However, multiple barriers exist to prevent such extreme dedifferentiation from occurring in vivo, which explains the inefficient induction of pluripotency by ectopic coexpression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in somatic cells. In PNAS, Ganier et al. (1) report a method that uses frog eggs to boost transcription factor-mediated formation of induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) (Fig. 1).

Fig. 1.

Generation of M-phase egg extract-induced pluripotent stem (M-iPS) cells. This elegant approach for inducing pluripotency in mouse somatic cells uses amphibian mitotic egg extracts to condition permeabilized somatic cells synchronized in G1 phase, allowing efficient chromatin remodeling and nuclear reprogramming by OSKM transcription factors.

Transplantation of mouse somatic nuclei into enucleated amphibian eggs (or oocytes) is known to result in efficient reprogramming of gene expression, with concomitant induction of the pluripotent-specific transcription factor Oct4 (2). Progressing on from this seminal work, Ganier et al. (1) eliminate the stochasticity observed with OSKM-mediated nuclear reprogramming by marinating MEF nuclei in Xenopus egg extracts, synergizing the degree of pluripotency that each technique confers on its own.

The first requirement for this innovative approach at inducing pluripotency is that amphibian extracts must be obtained from mitotic eggs, which are uniquely permissive for efficient chromatin remodeling. Incubation in interphase egg extracts does not produce an equivalent effect. A second requirement is that donor somatic nuclei in permeabilized cells are not mitotic but instead synchronized in G1 phase. In contrast to egg extracts, somatic cells that have exited mitosis but not yet initiated DNA replication are more competent than mitotic cells for chromatin remodeling and induction of pluripotency by OSKM. The resulting “M-iPS cells,” so named owing to the prior conditioning with mitotic or M-phase egg extracts, produce adult chimeras when injected into blastocysts and successfully transmit through the germline to the next generation of mice.

So why is the cell cycle specificity of egg extracts and somatic nuclei so critical for efficient nuclear reprogramming? Incubation of somatic nuclei in mitotic egg extracts is thought to provide important factors that induce chromatin condensation and would be inactive during interphase, cleansing G1-phase somatic DNA from any bound transcription factors (3). This resets the transcriptional program for pluripotency induction by OSKM, setting a pristine landscape for transcriptional activity and repression at distinct loci throughout the genome.

A fascinating aspect of the study by Ganier et al. (1) is that amphibian egg extracts can prime mammalian somatic cells for nuclear reprogramming by OSKM. The implications of these findings are immense because amphibian eggs and oocytes can be obtained cheaply and in large amounts, whereas mammalian equivalents cannot. So, does the cross-species effect also extend to human somatic cells, and hence is the generation of human M-iPS cells possible using a combination of OSKM overexpression and amphibian remodeling extracts?

Overall, the work by Garnier et al. (1) further highlights the crucial role of the cell cycle in both nuclear reprogramming and maintenance of a pluripotent state. Although significant advances have been made in transcription factor-mediated induction of pluripotency, inherent differences between the cell cycles of somatic cells and pluripotent cells will partly account for its poor efficiency. Pluripotent cells proliferate much faster than somatic cells because of a truncated G1 phase, and these unique cell cycle characteristics are orchestrated by noncanonical molecular events (4–8). Consequently, key proteins that are essential for cell cycle control are also required for pluripotency, as exemplified by the small nuclear protein geminin, which coincidentally was identified through two independent screens in Xenopus laevis (9, 10). Oscillations in geminin levels maintain genomic integrity in proliferating somatic cells, but geminin is regulated differently in pluripotent cells, where its sustained presence throughout the cell cycle maintains the expression of the core stem cell factors Oct4, Sox2, and Nanog (8). The Xenopus–mammalian iPS system reported by Ganier et al. (1) will provide an invaluable resource to precisely determine which factors present in amphibian mitotic egg extracts promote nuclear reprogramming in mammalian somatic cells overexpressing OSKM. These might include critical cell cycle regulators that are also important for stem cell identity, such as geminin.