by Academician Vladimir YARYGIN (Russian Academy of Medical Sciences), Corresponding Member Gennady SUKHIKH (Russian Academy of Medical Sciences), Konstantin YARYGIN, Dr. Sc. (Med.), Russian State Medical University; Igor GRIVENNIKOV, Dr. Sc. (Biol.), Institute of Molecular Genetics, Russian Academy of Sciences

The human body is composed of a huge number (-10 ¹³ ) of cells of different structural and functional types (about 250 in all) - myoblasts, epithelial, nerve (ganglion), immune cells, among others. All of them come from one single fertilized ovum, or egg cell. Dividing, it gives rise to a blastocyst (embryo vesicle) made up of spherical trophoblast and somatic (body) cells, the building bricks of a growing organism. All these body mass cells are identical, and they are also known as totipotent, or omnipotent, embryonic stem cells.

Now the stem cell is a progenitor of a tree of descendants - it is from this cell, which is at the foot of the stem, that the tree grows. Some daughter cells may be identical to the stem cell and go to build up the stem, while others may be specialized (myoblasts, epithelial, nerve cells and others) which go to form branches. Dividing, the stem cell produces at least one copy. This one, while dividing, may beget another stem cell (in what we call symmetric cell division). But it may also be a specialized cell (asymmetric division). This is how all specialized cells that form body tissues and organs come into being. However, stem cells, too, keep up in any body organ - as long as the organism is alive - to replace the spent, dead cells or else regenerate the tissue if it is impaired. Such physiological regeneration, whereby dead cells are substituted, proceeds at different rates. At one pole we have nerve cells which persist all through man's life. And at the other pole are intestinal epithelium cells, all of them replaced within hours.

The transformation of a stem cell into a specialized one occurs in a process known as differentiation. This is a process, mind you, not an event taking place at one particular moment. Upon its asymmetric divi-

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sion, the stem cell gives rise to a progenitor (precursor) cell. The division of progenitor cells may be asymmetric. Then one stem cell will finally produce specialized cells of two or even several morphofunctional types. By way of example we can cite hemopoietic ("blood-making") stem cells of the marrow. They give rise to hemopoietic progenitor cells "targeted" at the formation of red and white blood cells of all types.

All cells of the organism contain a complete set of genes; yet any gene can be activated only in embryonic stem cells. Specialized cells lose this capability. For instance, myoblasts (muscle cells) - under normal physiological conditions of course- will never change the spectrum of active (expressed) genes so much as to become nerve cells. Not the embryonic stem cells, though: given proper conditions and targeted impulses, they can produce any specialized cell. This property is called totipotency (omnipotence). Totipotent, omnipotent cells preserve some of their capability even in "adult" tissues. As shown by experiments, stem cells that are present in the adipose tissue or in the bone marrow may develop into muscle or epithelial cells if transferred into some other tissue and if acted upon by a specific set of chemical agents.

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With age, however, the spectrum of the stem cell's possible conversions shrinks. Say, embryonic stem cells undergo changes already in the course of the organism's intrauterine growth: from totipotent they become pluripotent (capable of producing many cell types, but not all of them); then they turn into oligo- and monopotent cells (those generating a small number of cell types or just one particular type, respectively). Yet another process, too, takes place in the course of ageing: the number of stem cells in body tissues goes down, which reduces their capacity for physiological regeneration and rehabilitation upon disease or injury.

There is evidence for the existence of two types of "adult" stem cells-specialized and universal ones. The former, different in different tissues, are under normal conditions capable of regenerating cells proper to a particular tissue-like, for instance, epithelial cells of the intestine or blood-making cells of the bone marrow. As to universal cells, these are identical in all tissues and pluripotent. They may be implicated in the regeneration of the "host tissue" and may be carried by blood into other, "foreign tissues" and cause their rejuvenation.

The headway made in the study of stem cells gives us hope that in the next few years it will become possible to treat many diseases by acting upon these cells in a patient's organs and tissues, or by transplanting stem cells of the host organism (but proliferated outside the organism), or else by taking stem cells from other individuals (in much the same way as we use blood transfusion). The United States, though leading in the experimental studies of stem cells in animals, is lagging behind in their practical medical applications due to legal, religious and ethical snags involved in cell transplantation. Now, the religious and ethical objections look rather odd and far- fetched to most Europeans.

Surgeons, though not aware of it at the time, were the first to start transplanting human stem cells long ago. Say, in blood transfusion a small number of such cells gets in from the donor. As shown recently, the thus transplanted cells penetrate the recipient's tissues where they are differentiated. In leukosis (leukemia, or blood cancer) doctors make a wide use of blood transfusion and marrow transplants: they inject blood-generating cells-sound leukocytes (white blood cells) and lymphocytes (lymph cells), not affected by cancer; this improves the patient's condition.

Unfortunately marrow or blood rejection cases occur every now and then. Attempts are being made to minimize this hazard by selecting a donor in whom the structure of proteins is similar to that of the recipient. Drugs are also being administered to suppress the donor's immune response. But this procedure may pose another threat: the immune sys-

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tem enables the organism to eliminate pathogenes and cells with a mutated genome- the cells that may grow into malignancies someday. The problem of tissue incompatibility and rejection may be tackled by transplanting the host stem cells reproduced outside the patient's organism. Yet another important consideration: stem cells are not prone to malignant degeneration-their immune defenses are much stronger than those of specialized cells in protecting the genome. That is why stem cells are taken from the bone marrow of leukemia-afflicted patients and conserved in liquid nitrogen. Thereupon the marrow, including tumor cells in it, is suppressed by radiation or cytostatics (drugs arresting the growth and proliferation of cells). Next, hemopoiesis (blood production) and immunity are restored by transplanting the stem cells preserved in liquid nitrogen; a complete set of normal blood cells will thus be formed. Such substitutions are made not only in the case of leukemia, they are also practiced in treating other tumors to supplement radio- and chemotherapy.

We have cited examples of therapeutic uses of "adult" stem cells, i.e. those isolated from human tissues upon birth. And yet there are essential constraints on their application. First, the prohibitive costs. The point is that "adult" stem cells are present in quite insignificant amounts (fractions of one percent of the total number of cells in the tissue). Besides, they grow but poorly outside the organism; their isolation and reproduction is a laborious and costly process. Also, the potential of stem cells goes down with age. Cell preparations obtained from seniors cannot give birth to cells of definite types.

But the situation is different with cells isolated from embryos. Depending on their growth stage, they are; called embryonic or fetal cells. These have no drawbacks we have spoken about. Methods have been developed for sustaining such cells and for their multiplication outside the host organism (while "adult" stem cells are either differentiated or die after several cycles of reproduction in a culture medium). Therefore we can grow "lines" of appropriate human cells differing in a set of surface proteins that trigger an immune response in a host organism upon transplantation. Furthermore, we can now set up their banks in which one can find cells good for transplantation to any patient (by analogy with blood banks). All embryonic and fetal stem cells are toti- or pluripotent. Accordingly, we are hoping to develop techniques for substituting any specialized cells in the human organism.

In fact, any human individual born today may rely on a stock of stem cells close in their characteristics to embryonic cells and genetically identical with all other cells of this particular organism. For this purpose umbilical blood containing stem cells (or a sample isolated from it) could

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be collected during delivery and stored in liquid nitrogen up until the moment of transplantation. This service is now offered in many American and European obstetrical clinics. Work is underway for developing techniques of using "adult" stem cells as "spare parts" in the event of disease or injury. It is convenient to make use of such cell sources as hair follicles, extracted teeth or the adipose tissue obtained during lipoxacy (sucking-off subcutaneous fat with a special needle) in cosmetology.

To date the so-called degenerative illnesses, such as atherosclerosis, hypertension, cancer, diabetes and many others, are pre-eminent. Although there are quite a few treatment procedures, for the most part they can only alleviate the patient's condition and give no cure. Modern medication can have a positive effect on sick cells but cannot make them healthy. Say, should the tension of smooth-muscle cells in some body organ be high, it provokes a spasm and painful dysfunction. Spasmolytics give relief by relaxing the muscle. However, these do not heal the sickly cell, its pathological tension will keep upwards time and again. True, medicamental therapy has great achievements to its credit. Hypertensive patients taking medicines regularly can keep their arterial pressure within bounds and thus control their condition. In the United States, the hypertonic mortality rate has declined severalfold in the last fifty years or so. Still, the cells responsible for hypertension remain sick, and should the patient stop taking medication, the symptoms are sure to be back and worse.

Stem cell transplants stimulating the natural rejuvenation of cells could help overcome certain age-related diseases aggravated by unhealthy life styles and characterized by an imbalance between the wear of cells and the regenerative potential of tissue. Cell transplantation could have a great future in traumatology as a remedy of speeding up the rehabilitation of impaired tissues. On this very basis effective methods of rejuvenation and longevity can be developed.

So stem cell uses hold bright promises in medicine. Some countries have been at it for a number of years-Russia, too: it has specialized clinics in Moscow, St. Petersburg, Pushchino and Novosibirsk. Hundreds of transplantations have been carried out with much success. The results have been quite good for some diseases. Stem cell transplants we are making have proved very effective in healing deep burns and eye retina injuries. We have obtained positive data in our attempts to cure optic nerve pathologies. Transplanting fetal (embryonic) liver cells to patients afflicted with hepatocirrhosis, we can restore in part the functions of the sick tissue to a tolerable condition or, in grave cases, we give our patients at least a chance to live long enough till their turn of liver transplantation.

It is common knowledge that myocardial infarction is caused by a spasm or obstruction of arteries sustaining the heart muscle (myocardiums). The sickly vessels have to be removed, with blood circulation restored by means of bypass surgery (shunting). But this is not enough to restore the dead part of the cardiac muscle. If the infarction zone is large, the contractile function of the heart is impaired, and the patient is unable to live a normal life. A stem cell transplant can restore, in part or in full, the structure and functions of the cardiac muscle. Here we use a preparation of marrow and embryonic or fetal stem cells. This suspension is injected into the blood flow or directly into the heart muscle along the boundary of necrosis (this procedure is made during bypass surgery). Such relatively simple surgeries are practiced in Belgium, Israel and in the United States as well as in some of Russia's clinics. A more extensive use of these techniques, we hope, will bring down the fatality rates in cardiovascular diseases, the scourge of advanced countries and Number One killer.

Stem cells of osteoblasts (precursors of bone cells), if injected into the fracture zone, speed up the process of knitting consolidation. In a similar way, chondroblasts (precursors of cartilage cells), introduced into an articular bursa, stimulate regeneration of the cartilage on the surface of the joint.

Russian doctors have also achieved encouraging results in using stem cells to treat neural diseases caused by injuries of the brain and spinal marrow; this is also true of ischemic insult, amyotrophic lateral sclerosis and Alzheimer's disease.

Embryonic (fetal) stem cells are very good in the case of premature ageing. Switzerland, for instance, runs clinics with a good record in this field-when sheep or pig cells are injected to patients. True, here we cannot speak of genuine "rejuve- nascence". But it is certain that the process of tissue degeneration is accelerated, and some of the functions of the body and brain are normalized. Most likely, it will become possible to slow down the ageing of tissues and organs by active renovation of cell elements as well as by sustaining proper blood supply and normalizing hormonal regulation. What we need is to improve the transplantation techniques.

Russia's doctors, unlike their colleagues in many Western countries, have amassed great practical experience in the application of new technologies based on the transplantation of stem cells or their preparations. Our medics are going ahead with their work. In May 2002 the Presidium of the Russian Academy of Medical Sciences (RAMS) endorsed a comprehensive program in this field, "New Cell Technologies for Medicine". To enforce action on it, Russian Academy of Sciences (RAS), RAMS, and the federal Health Ministry have instituted a joint research council to coordinate efforts of the medical community. A specialized commission on cell and tissue preparations and associated technologies has been set up within the framework of this program.

Unfortunately none of the countries has yet an effective state-run system to evaluate the efficiency and safety of treatment procedure involving cell technologies. This is a new

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and difficult problem. What we need is a legal code to regulate work in this area of medicine. Lawmakers and public health officials in Russia and elsewhere are facing difficulties in this respect. In some countries like the United States a prohibitive approach still prevails, while in others (Sweden and Israel) attempts are being made to give the green light to new technologies.

The gestation stage of every new method of cell therapy should proceed in four stages. To begin with, a cell preparation should be safe and effective. Adequate production technologies should be developed accordingly. The preparation thus obtained must then be tested on animals or cell culture models of a disease. The next, third, stage should include clinical tests on a small number of patients. And should broad clinical tests prove a success, the preparation can be recommended on an official basis for the treatment of a particular pathology. Additional research and tests are needed before extending this very preparation in treating other disease.

Up until recently we in Russia had no forum for broad specialist discussion of a range of theoretical and practical problems bearing on cell technologies and their introduction. To remedy this situation the Russian State Medical University sponsored a conference on stem cells and prospects of their use in public health (2003). It brought together many leading experts of this country and of the newly independent states, i.e. the former Soviet republics, what we call "the near abroad".

In their reports Professor Iosif Chertkov (RAMS Hematological Research Center), RAS Corresponding Member Levon Chailakhian (RAS Institute of Theoretical and Experimental Biophysics) and Academician Nikolai Nikolsky (RAS Institute of Cytology) reviewed the research situation. RAMS Corresponding Member Gennady Sukhikh reported on successful works done at his laboratory and by associated teams at RAS research institutes in studying and developing methods for cultivation of fetal stem cells. Two communications came as a follow-up to this report. Professor Maria Alexandrova (N.K. Koltsov Institute of Growth Biology, RAS) told about her work on transplanting human neural stem cells to rats, a procedure that normalized their behavior upon experimentally induced hypoxia. And Dr. Yekaterina Chentsova (Helmholz Research Institute of Eye Diseases, Moscow) said that by applying the same method she had achieved partial restoration of the impaired eye retina in a rabbit. Professor Ruben Chailakhian (RAMS Institute of Epidemiology and Microbiology) spoke on studies of "adult" stem cells of the bone marrow. RAS Corresponding Member Leonid Korochkin reported on the principles of manipulating the genotype of stem cells, of much importance for gene engineering uses.

A few communications were concerned with the mechanisms of differentiation and de-differentiation of stem cells. One dealt with the proteinome of precursor cells (i.e. what proteins in particular they synthesize)-it was presented by a research team of the RAMS Research Institute of Biomedical Chemistry.

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Two interesting reports were made on methods for lifetime monitoring of transplanted cells, which is of the utmost importance for understanding the effects of cellular transplantation and the biology of stem cells in general. To identify transplanted cells in a recipient's organism, we should mark, or label them somehow. As a rule, we introduce "reporter genes", for their product is easily recognizable. At definite time intervals after transplantation, small pieces of the patient's tissue are extracted by a special needle (biopsy) so as to see whether the labeled (i.e. transplanted) cells are present and in what numbers, to what extent each of them is differentiated, how they interact with other cells, etc. Also, female cells may be transplanted into the male organism, and in this way we can do without labeling. Other procedures are likewise possible. But our uppermost task is to develop noninvasive techniques, that is without biopsy. This task can be tackled with the aid of positron emission tomography. In that case a gene is injected into cells selected for transplantation; the gene's expression product, a surface receptor protein, tightly binds substances of some definite class which are not present in the human organism under normal conditions. After transplantation, a substance of this class is injected, it is labeled by the isotopes fluorine-18 or carbon-11, both emitting positrons. This substance is bound to the cells. Consequently, positron emission tomography shows in which particular tissues these very cells stay at the moment. Since the half-life of the two above isotopes allows to make several observations a day, it is possible to watch the fate of the transplanted cells in dynamics and not traumatize our patient. Both isotopes are harmless if administered at concentrations used by us.

There were many communications on cell transplantations to treat various diseases and injuries. Thus, Academician V. Shumakov (RAS and RAMS), Academician L. Bokeria (RAMS) and RAS Corresponding Member Yu. Belenkov (RAS Cardiological Research Center) promulgated quite hopeful results on cellular therapy in treating diseases of the myocardium; and Professor S. Smirnov (Sklifosovsky First- Aid Institute) told the audience about the successful use of cell preparations in healing burns and skin injuries. Other speakers pointed to the expediency of applying in clinical practice the

results achieved by these leading surgeons. Staff members of the RAMS Hematological Center summed up the clinical record of bone marrow transplantation in rehabilitating hemopoiesis in cancer patients.

As a matter of fact, Russia holds a leading place in the techniques of regeneration of organs and tissues on the basis of innovative technologies involving cell transplants, that is we are on the cutting edge of the medical science and practice. Our doctors can give substantial aid to patients otherwise doomed to death or incapacitation. But since all contemporary research procedures are quite expensive and there is a good deal of muddle about the "ethics" of certain clinical methods (like therapeutic cloning and the use of human blasto-cytes and abortive material for cell transplants), further headway in this field depends on how soon a reliable legal base will be created (rules and regulations, property rights, and so on) and on adequate financing.

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