Stem cells, cell therapy, the hope of a new regenerative medicine based on the replacement of damaged cells with new cells from embryos, cloning, or even so-called therapeutic cloning: everyone is talking about it and the newspapers do their best to enlighten opinion.

The revision of the bioethics law of 1994 which prohibits in its state any experimental manipulation on the human embryo is under construction and work in this respect will have to continue. These new perspectives offered by biology arouse both hope and fear and clash consciousness as technologies and discoveries that have changed our outlook on living things have done in the past.

The Academy of Sciences could not remain on the fringes of a social debate in which the biological sciences and the extraordinary development they know play a leading role. As it had done in 2001 in connection with a public health problem for which science remains the only remedy, Transmissible Spongiform Encephalopathies and in connection with a question which affects both health and society, longevity and aging.

Stem cells, capable of ensuring cellular homeostasis of tissues, exist in adults. Their role is to ensure cell renewal over the course of life. Those which are at the origin of blood cells have been known for several decades and cell therapy is commonly practiced to treat certain leukemias by bone marrow transplants.

During organogenesis, tissue formation is ensured by embryonic cells whose mode of operation is that of stem cells. They are the source of cells present in adult organs and ensure their sustainability.

What do we mean by stem cells?

  These are undifferentiated cells with the ability to self-renew while producing other cells that engage in one or more differentiation pathways. The modalities of their operation vary according to their nature and the context in which they are placed. In the course of the description which follows, the various types of known stem cells will be defined as well as the level where they are situated in the life cycle of the individual.

It is by studying certain adult tissues such as blood or skin that the concept of stem cells was advanced from the 1950s to the 1960s to account for the necessary renewal of the cells that constitute them. Stem cells that function when the different tissues are in place are also at work in the embryo, at the stage of organogenesis.

Finally, “embryonic stem cells” (ES cells for Embryonic Stem cells) derived from very early embryos in certain mammals, are an artificial production of biotechnology in that they extend in the form of permanent and indefinitely transplantable lines, a stage very ephemeral of embryonic development.

The history of embryonic stem cells begins with the study of particular testicular tumors, teratocarcinomas.

The historical reminder was the main theme of the communications of Martin Evans and Gail Martin. Their articles in 1981, for the first time, showed that it was possible to “derive”, from the internal cell mass of blastocyst 6, cell lines which, while remaining pluripotent, continue to multiply. Unlike commonly cultured cells, embryonic cells cultured under well-defined conditions do not burn out and can apparently be transplanted indefinitely.

This is not the case for cells from older embryos, fetuses or adults. Even those that lend themselves best to the conditions of in vitro culture, such as fibroblasts (from connective tissue), wear out after a certain number of transfers. Only cells that have undergone tumor transformation survive in culture indefinitely.

This capacity is accompanied by an alteration of their genome which is often manifested by an abnormal karyotype. This is not the case with embryonic stem cells which remain euploid and retain the power to differentiate into various cell types if they are placed in favorable culture conditions. The fact that embryonic development and tumor cells have certain characteristics in common has been stressed for a long time: both actively multiply and are able to migrate within the tissues of the embryo or the adult.

In the 1960s, the interest of certain biologists focused on testicular tumors encountered in humans and, with a particularly high incidence, in certain strains of mice, such as strain 129. These tumors contain various tissues , often well differentiated, (but organized in an anarchic way) which coexist with cells of the embryonic type; these tumors are transplantable. In fact, finely fragmented teratocarcinomas injected into the peritoneal cavity of a healthy mouse of the same strain cause the development of ascites tumors.

This phenomenon can even be obtained in certain cases by the intraperitoneal injection of a single cell coming from a teratocarcinoma thus demonstrating the existence of tumor stem cells. The line between the normal embryonic state and this type of tumor appeared even more blurred following the decisive experiment carried out in 1970 by the American biologist Leroy Stevens.

By grafting normal blastocysts into ectopic sites of the adult (under the kidney capsule or in the anterior chamber of the eye) he noticed that the embryos, far from continuing their development, form tumors similar to teratocarcinomas containing differentiated tissues and embryonic type cells called EC cells (for Embryonal Carcinoma cells). In the years 1970 to 1975, several laboratories transferred EC cells from spontaneous or experimental teratocarcinomas to in vitro culture7. The purpose of these cultures was to obtain embryonic cells in large quantities and to try to elucidate, on this material, what are the mechanisms responsible for their differentiation towards such or such cell type.

Clones of EC cells can be derived from individual cells (isolated in a pipette) and cultured in vitro in a culture medium conditioned by a layer of feeder cells. Martin Evans 1975.

In the early 1980s, Gail Martin noted that EC cells grown in vitro in a culture medium conditioned by a layer of feeder cells8 remained undifferentiated if they were transplanted frequently (every three days) and formed a single-cell layer. On the contrary, in prolonged cultures, cells formed aggregates which detached, floated in the medium, covered with a layer of epithelial cells and became “bodies embryoids ”in which various differentiated tissues appeared.

These differentiation capabilities of EC cells can also be expressed in vivo, as Ralph Brinster (Philadelphia) showed in 1974. When injected into a normal mouse blastocyst and recognizable from host cells by a genetic marker, EC cells (derived from a teratocarcinoma) were shown to be able to participate in the differentiation of virtually all tissues of the mouse. As in the original tumor (teratocarcinoma) these cells lose their malignant character when they differentiate, but, unlike their haphazard behavior in the tumor, they integrate perfectly with the normal tissues of the host embryo.

These cells, however, do not appear to be able to be incorporated into the germ line of the mouse, which remains chimeric only at the level of its somatic cells. Although EC cells were used by several laboratories in the 1960s and 70s to study cell differentiation, they were not really satisfactory experimental material. Their tendency to acquire tumor properties in culture made them unreliable and the ideal for researchers in developmental biology would have been to have cells similar to those of normal embryos but which would have been endowed with the same properties as EC cells. that is, those of self-renewal indefinitely as stem cells while retaining the ability to differentiate into various cell types. After Brinster’s experience, it took more than 6 years to develop the long-term culture of stem cells from normal embryos.

Chimerical mice

Gail Martin set out to cultivate the internal cell mass of blastocyst mouse embryos after it was rid of the trophectoderm using an anti-mouse antibody in the presence of complement (a process known as immunosurgery). She had the idea of ​​using a medium conditioned by EC cells to increase the number of embryonic cells. The success rate of these experiments (which involved outbred swiss mice) was low: approximately one in 100 embryos provided a stable ES cell line.

At the same time, Martin Evans in England pursued similar work and used another method to increase the number of embryonic cells initially placed in culture, that of deferred implantation: if one performs an oophorectomy shortly after fertilization, one prevents the implantation of the embryo in the uterine wall. The blastocyst embryo therefore remains in the cavity of the uterus and its size increases.

Mr. Evans uses strain 129 mice and achieves a much higher success rate. It was then revealed that this strain 129, which is also distinguished by a high incidence of teratocarcinomas, lends itself particularly well to the establishment of stable ES cell lines. As with EC cells, the essential condition which allows these cells to continue to divide while remaining undifferentiated and pluripotent is to remain in a monolayer. If these cells become abundant, they form aggregates and differentiate. ES cells are distinguished from EC cells from teratocarcinomas by the fact that they are euploids while EC cells tend to become aneuploids and thereby drift into malignancy.

ES cells injected into a blastocyst generate chimeric mice like EC cells but, unlike them, they become incorporated into the germ line of the host. The nourishing cell layers used by these pioneers were soon replaced by a “growth factor”, LIF (Leukemia Inhibitory Factor), which proved to be a powerful differentiation inhibitor for mouse ES cells. Other substances can also have this effect like oncostatin M or CNTF (factor discovered for its trophic effect on certain neurons).

Do the differences in mammalian species lend themselves to the establishment of ES cell lines?

It is not so. As we have just seen, from the work of the pioneers clear differences appeared, in this respect, between the various breeds of mice, the strain 129 being particularly suitable for these experiments. Some species have so far shown resistance to the establishment of ES cell lines; this is the case for cattle and rats. In 1995, ES cells were obtained from blastocysts of a primate, the Rhesus monkey, by researchers at the Wisconsin Primate Research Center in the United States.

In 1998, the same group published that human blastocysts also lent themselves to this evolution in culture but under conditions different from those which are favorable to the culture of mouse ES cells. Indeed, the differentiation of human ES cells is not inhibited by LIF, oncostatin M or CNTF as is the case in mice. The medium developed by J. Thomson allows the culture population to double in 20 to 40 hours. Human cells that retain high activity of the telomerase enzyme are, overall, more difficult to grow and handle than those of mice.

However, their differentiation capacities tested in vitro are satisfactory. Many cell types have so far been obtained in culture from human ES cell lines such as neurons, glia, hepatocytes, muscle and various epithelia. Several strategies have been developed to screen differentiated cells for possible transplantation for cell therapy. It should be emphasized that the injection in adults of pluripotent stem cells generally causes the occurrence of malignant tumors, these must therefore be carefully separated from the differentiated cells and excluded from the inoculate whose purpose is the replacement of injured cells. or dead. In conclusion, this still preliminary work raises great hopes for the use of ES cells for cell therapy in medicine.

Another source of stem cells is the germ cells of the fetal gonads.

Very early in the course of embryonic development certain cells escape the inductions which could direct their differentiation towards a particular cell type. They remain in an undifferentiated state and are, during organogenesis, animated by a particular behavior: they migrate to the gonadal blanks, lodge there and constitute a reserve of cells intended to perpetuate the species. These germ cells are intended, when the time comes, to differentiate into male (sperm) or female (oocyte) gametes according to their genetic equipment.

Morphological sexual differentiation

It was first shown in mice that the germ cells extracted from the gonad and placed in culture under adequate conditions can proliferate and provide cell lines quite comparable to ES cells derived from blastocysts (they are called cells EG to distinguish them from ES cells and indicate that they come from germ cells). In 1998, John Gearhart obtained human embryonic stem cells from germ cells extracted from the gonads of fetuses resulting from abortions.