Cloning and stem cells

Cloning and stem cell research grew from a single question in developmental biology: when a cell specialises into, say, a skin or gut cell, does it permanently lose the genetic information needed to make other cell types? Experiments addressing that question led to cloned frogs, Dolly the sheep, embryonic stem cells and, eventually, the reprogramming of adult cells. Reproductive biology has been central to the story, because cloning depends on eggs and embryos, and because stem cells may one day help to restore fertility.

Can a specialised nucleus make a whole animal?

In 1962 John Gurdon replaced the nucleus of a frog egg with the nucleus of a mature, specialised cell from the intestine of a tadpole. The egg developed into a functional tadpole, and later repetitions produced adult frogs. The result showed that a specialised cell nucleus still contains all the information needed to build an entire organism. It was greeted with scepticism at first, but was confirmed by others and launched the technique that eventually led to the cloning of mammals.

Dolly and the cloning of mammals

The technique, now called somatic cell nuclear transfer, involves removing the chromosomes from an unfertilized egg and introducing the nucleus of a donor cell. In 1997 researchers reported the birth of lambs derived from three cell populations, from an adult mammary gland, a fetus and an embryo. The lamb from the adult cell, Dolly, confirmed that specialisation does not involve irreversible changes to the genetic material. A key step was inducing the donor cells to become quiescent before transfer, a method that had already produced the first offspring from a differentiated cell line derived from an embryo.

Since then, more than 20 mammal species have been cloned, but efficiency remains very low. In mice, only 1 to 2% of cloned embryos transferred to surrogate mothers reach term, and clones often show abnormal placentas, and in some cases obesity, immune deficiency, breathing problems and early death. The main obstacles are epigenetic: chemical marks on DNA and its packaging proteins that record a cell’s specialised identity are not fully erased by the egg. A particular modification of histone proteins, H3K9me3, acts as a general barrier to reprogramming, and errors in imprinting and in X-chromosome regulation impair development after implantation. Learning to overcome these barriers allowed the first monkeys to be cloned in 2018, more than 20 years after Dolly.

Proposed applications include the propagation of valuable livestock, the rescue of near-extinct animal species and “therapeutic cloning”, the derivation of patient-matched stem cells from cloned embryos, first achieved with human cells in 2013. Each raises its own scientific and ethical debates.

Embryonic and induced pluripotent stem cells

Stem cells are defined by two properties: they can renew themselves and they can give rise to functional specialised tissues. Embryonic stem cells are derived from the inner cell mass of the blastocyst, the early embryo described on the page on early embryo development and implantation; human embryonic stem cells were first grown in the laboratory in 1998, building on earlier work with mouse embryos. Adult, or somatic, stem cells reside in particular tissues and organs and replenish their specialised cells; some remain inactive until the body needs them for repair.

In 2006 Shinya Yamanaka showed that mature mouse cells could be reprogrammed into pluripotent stem cells by introducing only a few genes. These induced pluripotent stem cells (iPSCs) behave much like embryonic stem cells and can be made from human cells, opening new ways to study disease without using embryos. Gurdon and Yamanaka shared the 2012 Nobel Prize in Physiology or Medicine for the discovery that mature cells can be reprogrammed to become pluripotent.

Germline stem cells and gametes in a dish

Reproductive biology has its own stem cells. In the adult testis, spermatogonial stem cells continuously renew themselves and produce the cells that become sperm. In 1994 researchers showed that such stem cells, taken from donor mice and injected into the seminiferous tubules of sterile males, could repopulate the testes and produce mature spermatozoa. Spermatogenesis from the donor cells looked normal under the microscope, and the authors suggested the method could also serve as a tool for biomedical science and biotechnology. It opened new avenues for studying how sperm production is sustained, a subject covered on the page on the testis and spermatogenesis.

More recently, in vitro gametogenesis has aimed to recreate the whole development of germ cells from pluripotent stem cells in culture. Beyond its potential for treatment, the approach is valued as a way to understand mammalian germ cell development and to model infertility in the laboratory. Mouse stem cells have been turned into functional eggs and sperm. Human pluripotent stem cells have so far been guided only to early stages, namely early oocytes and precursors of spermatogonia. Using laboratory-made gametes for human reproduction would require careful legal and ethical discussion.

What is established and what is still debated

Several conclusions are secure. Differentiated cells retain a complete genome; mammals can be cloned from adult cells; embryonic stem cells and iPSCs can be maintained and differentiated in culture; and transplanted spermatogonial stem cells can restore sperm production in mice. The open questions are just as important:

• why reprogramming by nuclear transfer remains so inefficient, and how far epigenetic errors in clones can be corrected;
• whether cloning can make a meaningful contribution to the conservation of endangered species;
• whether human eggs and sperm made from stem cells could ever be shown to be safe;
• how societies should regulate research that blurs the line between cells and embryos.