Rejuvenation and Reprogramming- First Works
Last update: Oct 2020
Gurdon's Experiments on Xenopus laevis
Both rejuvenation and cellular reprogramming were concepts put forward by John Gurdon in 1958, after his experiments using Xenopus laevis (African frog). Speaking in terms of Waddington's landscape, rejuvenation happens when a cell goes back up its differentiation path, gradually becoming more immature, and finally reaching the pluripotent state at the top of the hill.
In short, John Gurdon managed to clone a frog by transplanting the nucleus of an intestinal epithelial cell (which is a somatic cell) from feeding tadpoles into an enucleated egg. Those developed into normal feeding tadpoles. The procedure was further improved to produce fertile adult frogs. Let us look at it in more detail.
Figure 1. Cell reprogramming
When investigating organism development, scientists often use amphibians. This is because of the large size of their eggs and embryos, and their resistance to microsurgery.
John Gurdon decided to use African frogs for his experiments because, unlike other model amphibians, they can be stimulated to produce fertile eggs all year round. Also, they have a short (under 1 yr) life cycle, which makes it easier to study its mutants.
Now, let's look at the experimental procedure (Fig.2). First, chromosomes located on the surface of unfertilized wild type (2-nu strain) frog egg were damaged with UV light, enucleating the eggs. In the meantime, intestinal epithelial cells were extracted from a 1-nu strain feeding tadpole. A nucleus of one of the epithelial cells was injected into an enucleated egg that developed into an embryo. In several cases, the embryo developed into a feeding tadpole. Gurdon and his associates then improved on this procedure, managing to produce mature, fertile frogs.
However, how did Gurdon know that the embryo and the resulting tadpole or frog were the outcomes of successful nuclear transplantation? After all, it is possible that UV light failed to destroy the chromosomes initially present in the unfertilized egg. Precisely to avoid such confusion, 1-nu strain nucleus and 2-nu strain eggs were used in the experiment. This serves as an important nuclear marker. 1-nu mutant cells' nucleus contains only one nucleolus instead of one to two in wild type. This means we can take a sample from any tissue of the tadpole, look at in under the microscope, and determine whether the animal is of 1-nu or 2-nu strain by merely looking at the appearance of the nucleus. Thus, we can readily determine whether the nuclear transfer has been successful.
Figure 2. Nuclear transplant from Xenopus laevis tadpole tissue
After this successful experiment, Gurdon and Laskey also tried to transplant nuclei from various adult frog tissues such as skin, which were further down the developmental path compared to tadpole tissues. However, they were only able to produce tadpoles and no adult frogs using this procedure (Fig.3).
Gurdon and his associates' experimental results hinted towards the existence of something reversing differentiation present in the egg cytoplasm. Notably, it disproved the theory that the cell cannot move back up its developmental landscape. It seemed that cell fate could be reversed.
To sum up this story, in 2012, Gurdon received the Nobel Prize in Physiology or Medicine, jointly with Shinya Yamanaka. Interestingly, he got it mainly thanks to a single author paper he published in 1962.
Figure 3. Nuclear transplant from adult frog tissue (partially successful)
Dolly
After cloning frogs, scientists attempted to clone a sheep, demonstrating that epigenetic memories of mammal somatic cells can also be erased. The work was carried out at The Roslin Institute in Scotland by a team led by Professor Sir Ian Wilmut and resulted in the birth of Dolly- the first mammal to be cloned from an adult cell.
The procedure was similar to that of frog cloning, with few additional steps (Fig.5). The cells for cloning came from mammary glands of an adult Finn Dorset (white) female sheep. The enucleated egg cells came from Scottish Blackface (brown-black) species.
Figure 4. Dolly with Sir Ian Wilmut
In order for the nucleus to function correctly inside of the host egg, the mammary cells were starved so that the cell cycle can be halted, and cells enter a "dormant" state before fusion with an unfertilized egg cell. Mammary gland cell was then fused with egg cell using electrical pulses, followed by cell division and embryo formation in some cases. Embryos were when transferred to 13 surrogate Scottish Blackface sheep, and one of them became pregnant. After a standard gestation period of 5 months, Dolly was born in 1996. One could immediately tell the cloning has been successful, as she had the white face of a Finn Dorset nucleus donor, instead of the black face of the Scottish Blackface egg donor.
Figure 5. Cloning of Dolly the Sheep
One year later, DNA analysis revealed that Dolly's telomeres were shorter than that of a normal one-year-old sheep. Dolly's DNA came from an adult cell whose telomeres were shorter than that of a "younger cell", and they were probably not fully renewed during her development. Scientists expected this telomere shortening to adversely impact the animal's health, making it appear older than it actually is. However, after extensive health screens, no conditions related to aging were discovered.
Dolly produced six offspring over her lifetime. After her last birth in 2000, she and other sheep at The Roslin Institute contracted a virus that causes lung cancer. Later, she was diagnosed with and successfully treated for arthritis. She was euthanized at 6 years of age because of painful lung tumors. Typically, sheep live up to 10 years, but her shorter lifespan may not necessarily be the result of her being a clone.
Fusing Somatic Cells with Pluripotent Stem Cells
Tada and his associates demonstrated another way to rejuvenate a cell besides transferring a somatic cell nucleus into an enucleated egg. He and his team managed to reprogram somatic cells by fusing thymocytes (progenitors of T cells inside the thymus) with embryonic stem (ES) cells. The resulting ES-thymocyte cell hybrid showed a different gene expression profile to thymocytes. For example, Oct4-GFP transgene, which is normally repressed in thymocytes, became active 2 days after cell fusion. Hybrid cells exhibited pluripotency in vivo, contributing to all three (endoderm, ectoderm, and mesoderm) primary germ layers of chimeric embryos. It was thus theorized that ES cells could remove some epigenetic memories of somatic cells, reverting them to ES cell state.
“REPROGRAMMING FACTORS”
Taken together, the experimental results of Gurdon, Wilmut, and Tada suggested that both egg cytoplasm and pluripotent stem cells contain some kind of "reprogramming factors" that are capable of erasing the epigenetic memories of somatic cells. This allows those cells to go back up their developmental path. The next section describes the search for one of those "reprogramming factors".
Sources
Content
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[2] Gurdon J. B. The cloning of a frog. Development 2013 140: 2446-2448; doi: 10.1242/dev.097899
[3] Scientific American. 2012. Transplanted Nuclei and Cell Differentiation, by Sir John B. Gurdon. https://www.scientificamerican.com/article/transplanted-nuclei-and-cell-differentiation-gurdon/. [Accessed 5 May 2020].
[4] Dolly the Sheep. 2020. The Life of Dolly. https://dolly.roslin.ed.ac.uk/facts/the-life-of-dolly/index.html. [Accessed 5 May 2020].
[5] Judith L. Fridovich-Keil. 2020. Dolly. https://www.britannica.com/topic/Dolly-cloned-sheep. [Accessed 5 May 2020].
[6] Katarina Zimmer. 2017. Dolly's Cloning Likely Didn't Cause Premature Aging. https://www.the-scientist.com/the-nutshell/dollys-cloning-likely-didnt-cause-premature-aging-30585. [Accessed 5 May 2020].
[7] Tada M., Takahama Y., Abe K., Nakatsuji N., Tada T. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol. 2001 Oct 2;11(19):1553-8.
[8] British Society for Immunology. 2020. T-cell development in thymus. https://www.immunology.org/public-information/bitesized-immunology/immune-development/t-cell-development-in-thymus. [Accessed 5 May 2020].
Graphics
Figure 1: Proteintech, (2020), The Waddington’s epigenetic landscape model https://www.ptglab.com/news/blog/cell-fate-commitment-and-the-waddington-landscape-model/ [Accessed 5 May 2020].
Figure 2,3 : Julianna Carpenter, (2015), Transplantation Experiments: Each cell contains a full set of genetic instructions https://slideplayer.com/slide/4693591/ [Accessed 5 May 2020].
Figure 4: National Museums Scotland, (2018), Sir Ian Wilmut https://www.thetimes.co.uk/article/sir-ian-wilmut-creator-of-dolly-the-sheep-has-parkinsons-the-disease-she-offered-hope-of-curing-0zhbkm2xq [Accessed 5 May 2020].
Figure 5: Encyclopædia Britannica, (2020), Dolly the sheep; cloning https://www.britannica.com/topic/Dolly-cloned-sheep [Accessed 5 May 2020].