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Takahashi–Yamanaka reprogramming experiment

The Takahashi–Yamanaka reprogramming experiment was a 2006 study that converted differentiated mouse fibroblasts into pluripotent stem cells using four defined transcription factors. Kazutoshi Takahashi and Shinya Yamanaka introduced Oct3/4, Sox2, Klf4, and c-Myc into embryonic and adult fibroblast cultures and recovered cells that behaved like embryonic stem cells: they formed teratomas containing all three germ layers, and they contributed to the germline of chimeric mice. A 2007 follow-up generated the same cells from adult human dermal fibroblasts using the same four factors. The result showed that a small, molecularly specified set of inputs can reset a differentiated cell toward an embryonic-like state.12

Background

Two lines of work had established that the differentiated state is reversible. Nuclear-transfer experiments showed that the nucleus of a differentiated cell can direct development once it sits in an egg, which located the reprogramming activity in the egg cytoplasm.[citation needed] Embryonic stem cells grown in culture supplied a second route, and cell-fusion experiments showed that an ES-cell cytoplasm can reactivate embryonic genes in a somatic nucleus. The open question was whether a defined set of molecules could do the same job, since the egg and the stem-cell cytoplasm each contain thousands of factors. The experiment tested whether the reprogramming activity could be captured in a small combination of transcription factors.

Design

Takahashi and Yamanaka began from a list of genes enriched in embryonic stem cells and known to support pluripotency, and introduced candidate factors into mouse fibroblasts with retroviral vectors. They selected for colonies that reactivated a marker of the pluripotent state, then withdrew factors to find the minimal combination. The screen converged on four transcription factors: Oct3/4, Sox2, Klf4, and c-Myc. The resulting cells, called induced pluripotent stem (iPS) cells, were then tested for the defining properties of pluripotency: growth as colonies with ES-cell morphology, expression of ES-cell genes, differentiation into derivatives of all three germ layers, teratoma formation, and germline transmission in chimeras. The 2007 study repeated the procedure with adult human dermal fibroblasts and the same four factors.12

Result

The four-factor combination produced iPS cells from mouse embryonic and adult fibroblasts. The cells matched embryonic stem cells in morphology, proliferation, surface markers, gene expression, and the epigenetic state of pluripotency genes, and they differentiated into cell types from all three germ layers. In the mouse study, the cells also entered the germline, which demonstrated that reprogramming restored full developmental potential. The human follow-up generated iPS cells from adult fibroblasts with the same factors, and those cells matched human embryonic stem cells across the same assays, including telomerase activity and teratoma formation.12

What it established

The experiment defined a molecular route to pluripotency:

  • Four transcription factors, delivered together, convert differentiated fibroblasts into pluripotent cells.
  • The reprogrammed state is stable and functionally equivalent to the embryonic stem-cell state by the assays available.
  • Pluripotency can be reached through defined factors, which makes the process experimentally controllable and reproducible.

The result is the molecular counterpart to nuclear transfer. It turned “reprogramming” from a property of egg cytoplasm into a defined regulatory input, and it made patient-specific stem cells a practical research direction. c-Myc is one of the four factors and also an oncogene, which tied the reprogramming network to the same growth-control machinery studied in cancer, while p53 acts as a barrier that limits reprogramming efficiency. The observation that reprogramming resets epigenetic marks also connected the experiment to later work on partial reprogramming and the epigenetic clock.

Limits and later work

The original protocol delivered the factors with integrating retroviruses, which raised the risk of insertional mutations, and c-Myc contributed oncogenic potential. Later work replaced the retroviruses with non-integrating delivery and dropped c-Myc, at the cost of lower efficiency. The experiment produced a pluripotent state in a dish, and translating the result into regenerative medicine required controlling differentiation, avoiding tumor formation from residual undifferentiated cells, and scaling the process. Partial reprogramming, in which the same factors run briefly to rejuvenate cells while preserving their identity, grew directly out of this work and became a candidate aging intervention.

See also

References

Footnotes

  1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–676. doi:10.1016/j.cell.2006.07.024. ↩↩2↩3
  2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–872. doi:10.1016/j.cell.2007.11.019. ↩↩2↩3