“For the discovery that mature cells can be reprogrammed to become pluripotent”. Many of us may know the succinct summary of the Nobel Prize-winning discovery shared jointly by Sir John Gurdon and Shinya Yamanaka. Especially Gurdon’s seminal papers are an inspiration for my research and provide examples of a standard of science that should hold true as much today as they did more than half a century ago.

This is what I learned from Gurdon’s early papers:

  1. 1.

    Ask a clear question of fundamental importance. It was controversial at the time whether differentiated cells contain the same set of genes as the early totipotent embryo. Gurdon’s experiments were driven by curiosity as to whether the nucleus of a differentiated cell could support development of other cell types.

  2. 2.

    Choose your experimental system wisely. Early nuclear transplantation experiments showed promise in reprogramming nuclei from blastula but not from later stage embryos of the frog Rana pipiens (Briggs et al., 1952). Eggs from this frog species were limited owing to seasonal egg laying. Instead, Xenopus laevis could be stimulated to lay eggs throughout the year and proved to have better developmental potential (Gurdon et al., 1958).

  3. 3.

    Overcome technical challenges. Extraction of the meiotic chromosomes from X. laevis eggs was difficult and could be overcome by UV irradiation to fragment DNA (Gurdon et al., 1958).

  4. 4.

    Use unambiguous markers for a ‘clean’ readout. To unequivocally demonstrate that the frog emerging from the somatic cell nuclear transfer (SCNT) experiments was derived from the nucleus of a differentiated donor cell, and not from the nucleus of the host egg, donor nuclei were derived from a natural mutant whose cells harboured one rather than two nucleoli (Gurdon et al., 1958).

  5. 5.

    Use a functional assay to assess the result. SCNT experiments demonstrated that nuclei from intestinal epithelial cells can give rise to fertile adult frogs (Gurdon et al., 1966). Therefore, a key conclusion was that differentiated nuclei retain genes necessary for generating all cell types, including functional gametes. Importantly, the work also implied that the egg cytoplasm can reprogramme differentiated nuclei back to a totipotent state.

differentiated nuclei retain genes necessary for generating all cell types

Surprisingly, 60 years on, we are still largely in the dark about the mechanisms that enable reprogramming to a totipotent chromatin state. Epigenetic modifications likely contribute barriers to reprogramming (Hoermanseder et al., 2017). We also think that certain transcription factors — by serving as pioneer factors that enable opening of chromatin and facilitate embryonic genome activation — have important roles in overcoming these barriers to reprogramming. However, how chromatin is efficiently reprogrammed by potential pioneer factors only hours after fertilization and what signals and additional molecular factors mediate this transition to totipotency remain questions of fundamental importance.