Stem Cell Grandchildren

Biologists have learned how to grow spermatozoa "in vitro"

Oleg Lischuk, N+1

Since the discovery of stem cells and their universal ability to develop into any other cells of the body, scientists have been thinking about obtaining germ cells from them. Such a discovery would revolutionize the treatment of infertility – any person, regardless of age, health status and even the presence of genitals, could have their own child in genetic terms. However, germ cells are so different from all the others that even the theoretical possibility of obtaining them "in vitro" caused reasonable doubts.

And so, on February 25, 2016, a publication in the journal Cell Stem Cell dispelled these doubts (Zhou et al., Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro). Chinese researchers have managed to obtain sperm cells from embryonic stem cells suitable for in vitro fertilization. In the experiment, the use of these cells for conception led to the appearance of healthy offspring capable of reproduction. Before that, no one had been able to grow functional germ cells outside the body.

A little bit of the school curriculum

Any multicellular animal reproducing sexually has two fundamentally different types of cells: germ cells, or gametes, and all other cells of the body, or somatic cells.

Somatic cells contain a paired (diploid) set of chromosomes – half from each parent (for example, a person has 46 chromosomes: 23 from the mother and 23 from the father). These cells multiply by division, which is called mitosis. It happens relatively simply: the DNA of the cell doubles, two paired sets of chromosomes are formed, then these sets diverge to different poles of the cell, after which a constriction is formed in it, dividing it in half. As a result, two identical cells are obtained, similar to the mother.

With germ cells, everything is more complicated – their precursors, primary germ cells, or gonocytes, have a paired set of chromosomes, and as a result, they should turn out to be eggs and spermatozoa with a single (haploid) chromosome set. Therefore, the process of their division (gametogenesis, which in the case of spermatozoa is called spermatogenesis) goes through several intermediate stages.

In vertebrates, gonocytes are formed from universal stem cells in the yolk sac of the embryo from about the sixth week of its development. As tissues and organs are formed, these cells migrate to the gonads (gonads), that is, in the male body – to the testicles. There they form a population of cells called spermatogonia. At the beginning of puberty, these cells begin to actively multiply by mitosis.

In this case, some cells differentiate into so-called first-order spermatocytes, which also have a double set of chromosomes. These cells, unlike spermatogonia, divide by meiosis, in which DNA doubling does not occur. As a result of the first division of meiosis, second-order spermatocytes are formed, carrying a single set of chromosomes. Then they undergo a second division of meiosis, similar to mitosis, yielding spermatids with a haploid set of chromosomes. These cells then differentiate into mature spermatozoa.

At each stage of this process, cells need a certain environment, surrounding cells and signaling factors that guide their division and development. Testicles, having a complex microscopic structure, provide the necessary conditions, but reproducing these conditions in the laboratory is an almost impossible task, especially at the last stages of spermatogenesis.

The staff of Kyoto University in Japan managed to come closest to solving it. In 2011, they were able to direct the differentiation of mouse embryonic stem cells into gonocyte–like cells (HPC), but for the subsequent stages of spermatogenesis they had to be planted in the testicles of adult mice - they failed to achieve meiosis "in vitro".

"Good stuff"

Scientists from the Chinese Academy of Sciences and their colleagues from Nanjing, Changsha, Hefei and Yangzhou used the achievements of their Japanese colleagues in their research. Using a "cocktail" of cytokines similar to the signaling molecules of early extraembryonic tissues, they differentiated mouse embryonic stem cells into epiblast-like cells (resembling a yolk sac) and further into HPC.

In order to create conditions close to the internal environment of the gonads, HPC was mixed in a nutrient medium with an equal number of epithelial cells obtained from the testicles of newborn mice. After that, various combinations of morphogens were added to the medium – substances that guide the differentiation of cells in the right direction and the formation of organs and tissues from them. As one of the researchers Xiaoyang Zhao (Xiao-Yang Zhao) noted, hundreds of experiments had to be done to get the right combination. As a result, the combination of morphogens KSR, BMP-2/4/7, activin A and retinoic acid triggered the process of spermatocyte meiosis.

However, having started meiosis, it is necessary to regulate its course. To do this, on the seventh day, morphogens were removed from the nutrient medium and a hormonal mixture was added: follicle-stimulating hormone, testosterone and bovine pituitary extract. Regarding the latter, an expert reproductologist from the Jackson Laboratory in Bar Harbor (Maine) Mary Ann Handel emotionally remarked: "God knows what's wrong with him? But it's probably a good thing."

Scheme of sperm development in vitro and in vivo. Quan Zhou et al., Cell, 2016

Nevertheless, this hormonal combination turned out to be the only one that ensured the correct course of all the key stages of meiosis, which was confirmed by immunochemical, cytological, genetic analyses, sequencing and PCR. The result was the appearance in the culture of sperm–like cells with a haploid chromosome set - in fact, immature sperm without a tail and with "extra" organelles.

These cells were used for fertilization by the standard procedure of intracytoplasmic sperm injection (ICSI), in which male gametes are injected into the egg with a glass microneedle. This led to the development of morphologically complete embryos, which were transferred to the mouse uterus for gestation. The born mice were no different from animals conceived naturally, and produced their own offspring.

A word to the skeptics

The publication of the Chinese made some scientists doubt the results obtained. So, the head of the Kyoto team that developed the method of obtaining HPC from stem cells, Minitori Saitou pointed out that the temperature in the incubator was maintained at 37 degrees Celsius, which can stop the development of sperm. He also noted that fluorescence microscopy in cells does not show proteins characteristic of HPC.

Niels Geijsen, a stem cell expert from Utrecht University in the Netherlands, noted that the successes of Chinese scientists "are amazing if [what is described in the article] really happened."

The Gold Standard

Despite the skepticism of a number of colleagues, the researchers note that their work meets all the criteria of the "gold standard" of proof of obtaining full-fledged germ cells "in vitro", which were formulated by the already mentioned Handel and colleagues in 2014. These criteria include the normal amount of DNA, the number and shape of chromosomes in cells at all stages of development, their correct divergence during meiosis, as well as the suitability of the resulting cells to produce reproducible offspring. Handel herself agreed that the work meets the "gold standard".

Mice conceived with sperm "from a test tube". Xiao-Yang Zhao, Jiahao Sha, Qi Zhou

At the current stage, the developed technique is a valuable platform for research of all stages and necessary conditions of spermatogenesis, as well as the significance of each specific factor in this process. Its possible clinical application is still very far away. First, first you need to make sure that the next generations of mice conceived with artificial sperm are healthy and reproduce the results on other animal models. Secondly, it is unclear whether such an approach will work in humans. Thirdly, an adult does not have embryonic stem cells, and whether induced pluripotent cells, which can be obtained from a mature organism, are suitable instead of them is a big question. Fourth, where to take the testicular cells of newborns. Fifth, it is hardly possible to obtain permission for such experiments and develop a legal basis for them in the modern world. And these problems are not the only ones.

Nevertheless, the first success inspired many scientists. "If it works in a mouse, there is no biological justification that it will be ineffective in humans. But we will have to find out the necessary conditions for this [environment] and guide the cells through this very subtle choreography," said Harvard stem cell expert George Daley.

Anyway, it's better to remember the names of Xiaoyang, Quan Zhou, Mei Wang and their colleagues. If the results obtained by them can be confirmed and reproduced, it will be easy to win a bet on who will become laureates of one of the next Nobel Prizes in medicine and physiology.

Portal "Eternal youth" http://vechnayamolodost.ru 29.02.2015