Marmoset cloning breakthrough yields stem cells from cloned embryos for the first time

The common marmoset is increasingly used in biomedical research because it is small, breeds quickly and can be genetically modified. However, generating and preserving specific genetic lines is inefficient. Somatic cell nuclear transfer (SCNT), which reprograms an adult cell nucleus inside an enucleated egg, has worked in mice, cattle, pigs and cynomolgus monkeys. In marmosets, cloned embryos have previously stalled before the morula stage, with no blastocysts or offspring reported.

The team first reproduced the baseline problem. Using adult fibroblasts (GFP-transgenic or wild-type) or cumulus cells as donors, fused with enucleated in vitro matured oocytes, SCNT embryos treated only with TSA arrested at or before the 8-cell stage. This coincides with marmoset zygotic genome activation, pointing to incomplete reprogramming. In cloned 1-cell embryos, H3K9me3 accumulated strongly in the pseudo-pronucleus. Injecting mouse Kdm4d mRNA removed this signal globally.

Adding Kdm4d to TSA allowed blastocyst formation at 14.5% per pronucleus-stage embryo with GFP fibroblasts (10 of 69). With cumulus cells the rate was only 2.4%. The blastocysts showed a NANOG-positive inner cell mass and CDX2-positive trophectoderm, and GFP signal confirmed the nuclear donor origin. Adding the G9a inhibitor as a third treatment gave similar blastocyst rates (14.9% for GFP fibroblasts, 13.6% for wild-type fibroblasts), but the authors report it improved blastocyst quality. Only the triple-treatment blastocysts yielded ntESC lines: 4 from 10 GFP blastocysts (40%) and 3 from 17 wild-type blastocysts (17.6%). Without Kdm4d, no blastocysts formed.

Per the abstract, the ntESCs had normal karyotypes and were pluripotent, with germ-layer differentiation capacity. Nuclear DNA matched the donor cells and mitochondrial DNA matched the recipient oocytes, as expected from SCNT. Transcriptome comparison with fertilization-derived ESCs found abnormally expressed genes, some line-specific and some shared across lines. Translation-associated and ribosomal genes were notably dysregulated, suggesting partial resistance to reprogramming.

This work provides the first route to preserving and engineering marmoset genotypes via ntESCs, for example from valuable or hard-to-breed transgenic founders. It also identifies remaining epigenetic barriers to optimize. Important caveats are that no cloned marmoset offspring were reported, blastocyst efficiency is still modest, cumulus cell donors performed poorly, sample sizes are small, and ntESCs show transcriptional deviations from normal ESCs. The full text available for this summary was truncated, so the later results (G9a inhibitor effects, pluripotency, genomic and transcriptomic analyses) are described from the abstract and highlights.