Despite the high profile of nuclear transfer in mammals, the basic cell biology in this cloning process is not well understood. Initially, it was believed that as early embryo cells develop and begin to differentiate into various types of tissue, the totipotency of the nucleus is lost forever. Nonetheless, the occasional cloning success shows that some differentiated cells can regain their totipotency, and their nuclei can repeat all the developmental events initiated in the fertilized egg. This works better with the nuclei of embryo or fetal cells, in that a greater number of healthy, live offspring are produced by nuclear transfer involving embryo or fetal cells than adult cells. It makes sense: These “younger” nuclei are likely to be less differentiated. But what is actually happening to dedifferentiate any of these cells, whether embryonic or adult, is very poorly understood. The original and incorrect thinking was that adult cells that no longer divide, such as neurons (your brain cells or nerve cells), would be poor candidates for use in cloning, whereas cells that continually divide (such as your skin cells) would be good candidates. But the studies of Dolly show that the opposite is true. Forcing cells to be quiescent, to stop their metabolism, and to slow down, by reducing the protein content of the culture medium, seems to enhance the possibility of nuclear reprogramming.

Therefore, when the Honolulu group decided to use mice, they chose nuclei from adult cells that have intrinsically very low, if any, turnover, without “forcing them to slow down by nutritional deprivation.” The three adult cells they studied were Sertoli cells (the supporting cells of the testicular tubules), neurons (brain cells), and cumulus cells (the cells that surround and nourish the ovulated egg). These three types of adult cells (like the adult cells in the Dolly experiment that went into the G-0/G-1 cycle phase via culture in a protein-deficient medium) are known always to be in the G-0 phase in the normal adult. All of these cells were used immediately for injecting adult nuclei into enucleated eggs of mice, with no prior in vitro culturing or any other prior manipulation.

In July 1998, approximately a year and a half after Wilmut’s historical paper, Yanagimachi’s group published their results in Nature (see figure below). Using adult Sertoli cells from the testes, they were able to get embryos to develop 40 percent of the time, but less than 1 percent developed into a fetus and none of them survived. Using neurons from the adult brain, they were able to obtain cloned embryos approximately 20 percent of the time, but again, less than 1 percent developed to a fetus and none survived. Using the nucleus from cumulus-cells, i.e., the cells that surround the mature ovulated egg and nourish it (the female counterpart to the male testicular Sertoli cell), out of a total of eight hundred cloned embryos that resulted from an adult cumulus-cell injection, only seventeen fetuses developed, and ten females survived to birth and were apparently healthy. This means that only about 1 percent of normal-appearing cloned embryos in these mouse studies using a specific type of adult cell (the cumulus cell) survived into normal adulthood. Similarly, in a third study, Yanagimachi reported that 5 out of almost 300 such embryos developed into normal females, again slightly over 1 percent.

These few healthy-appearing cloned mice were in turn cloned with the same technique for six further generations, always using the cumulus cell as the adult nucleus. The success rate declined with each subsequent generation. In one of the strains of mice, in fact, they could not clone beyond the fourth generation, and in the other strains studied, the success rate dramatically declined with each generation until in the sixth generation there was only 1 out of 1,000 that developed into a healthy live mouse.

The most fascinating but understated aspect of this monumental study of mouse cloning in Honolulu was that they could only clone females. In fact, up to that point it was absolutely impossible to clone males. Dolly was, of course, a female, and the only adult cells that seemed capable of reprogramming in Wilmut and Campbell’s study were cells that originated from the female reproductive tract. Remember, even Dolly came from the breast cells of a pregnant ewe.

Up until the end of 1999, none of the cloning experiments using adult somatic cells either in the sheep, the mouse, or the cow had utilized adult male cells. All of the cells that were successful in cloning, such as mammary gland cells, cumulus cells, or ovuductal cells, came from the female reproductive system, raising the question of whether males could be cloned at all from adult cells. This was accomplished with great difficulty by an amazing tour de force, once again by the Honolulu group. Yanagimachi’s team used the same technique for enucleation of metaphase-II eggs that he had already described, and the only difference now was that instead of trying neurons, Sertoli cells, or cumulus cells, he used cells that were cut from the tail tips of the mice, dispersed and cultured for one week under standard conditions, and then cultured for three to five days in a protein-poor medium. Just like in the Dolly study, he starved these cells into quiescence. Although roughly half of these injected eggs developed into embryos, only 3 out of 274 (1 percent) developed to full delivery, but two of those three died within one hour of birth. The one remaining male mouse baby developed into a normal, fertile adult male. Out of more than seven hundred cloning attempts using male mouse tail-tip cells, one male (0.14 percent) was obtained. Although there were many pregnancies, there was a high rate of abortion throughout the pregnancy, a high stillbirth rate, and frequent perinatal deaths, indicating that nuclear reprogramming in the vast majority of cases was not normal.

The Honolulu team (as well as Wilmut) had shown that contrary to the previous dogma, mammals could be reproductively cloned from adult somatic cells, but the extremely low success rate, as well as the failure to achieve this with the use of most adult cells, nonetheless indicated that there were many regulatory differentiating factors and developmental checkpoints about which we hadn’t a clue. We still did not understand very well what causes a cell to differentiate and to dedifferentiate or how to control it. There had to be myriad other processes occurring that we did not understand which led to poor embryo development, miscarriage, and stillbirths. The actual success rate for reproductive cloning is so low as to make it preposterous, from a medical point of view, to even conceive of doing this in humans, regardless of one’s ethical views about the morality or immorality of DNA replication in a subsequent individual.

Thus, the proper scientific message to take home from the cloning experiments in animals is that we need to learn much more about DNA in adult cells. Through such basic knowledge, perhaps we will eventually understand DNA replication, both abnormal and normal, enough to deal better with cancer, aging, degenerative diseases, and various genetic illnesses. But the notion that it would be easy to perform cloning, at least at the time of this writing, remains naive.

However, there is one aspect to cloning that is much more exciting than the absurd goal of trying to create a genetic copy of an adult animal. That is the concept of therapeutic cloning, which is inextricably tied to the issue of stem cells. I will conclude this chapter with a section on stem cells so that you can understand the full circle we’re now traversing. With PGD via <a href="https://glowing.com/glow-fertility-program">IVF</a> you can avoid the transfer of a genetically diseased or defective embryo. You can select out the few embryos that are actually healthy and thereby dramatically reduce the risk of having a severely handicapped child, something all mothers fear. PGD simply means picking the healthy embryos for replacement, and not replacing the diseased ones. Using stem cell technology, the possibility exists for actually curing, with gene therapy, a person or embryo that already has a genetic disease.