Stem Cells: Properties and Prospective Medical ApplicationsR. A. Musina, Ye. Ye. Yegorov, and A. V. Belyavsky
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia e-mail: email@example.com
Received November 11, 2003
Abstract - The properties of some stem cells (SCs) that are most interesting in terms of their implications for medicine (embryonic, hematopoietic, and mesenchymal SCs) are considered. SCs are undifferentiated cells capable of both self-maintenance and differentiation into specialized cells. According to their origin, SCs are divided into embryonic and somatic ones. The former can be maintained in vitro for an inﬁnitely long time and can differentiate into all cells of adult organisms. The latter have a limited capacity for differentiation and, probably, a limited proliferative potential. The plasticity of somatic SCs, i.e., their capacity for context-dependent differentiation into “unrelated” cell types, is of considerable therapeutic importance, although some researchers doubt this capacity. It is assumed that most types of SCs differentiate by the stepwise hierarchical maturation mechanism, one of the steps being rapidly proliferating progenitor cells. The use of SCs in medicine is currently at the stage of preclinical trials. Although embryonic SCs are promising for medicine, there are serious limitations of their use in therapy in the near future. However, the ﬁrst clinical trials have demonstrated that the approaches involving autotransplantation of hematopoietic and mesenchymal SCs are effective for treating ischemia of extremities and the consequences of myocardial infarction. Obviously, the use of SCs in medicine promises dramatic progress in treating many severe diseases.
Key words: stem cells, pluripotency, differentiation, proliferative potential, plasticity, cell fusion, telomerase, cell therapy.
INTRODUCTIONResearchers have been attempting to understand how living organisms maintain homeostasis, how they recover after damage, and what mechanisms are involved in the replacement of damaged cells with healthy ones. Targeted use of self-maintenance mechanisms would make it possible to fundamentally change the existing approaches to the treatment of many diseases.
Stem cells (SCs) are crucial for the cell homeosta-mass of the mammalian blastocyst give rise to all cell sis of the body, primarily because their main role is to types of the adult body. This capacity is termed totipocompensate for the natural loss of cells fulﬁlling spe-tency. Note that the cells of the blastocyst inner mass cialized functions. According to the most generally hardly meet the deﬁnition of SCs, because they are accepted deﬁnition, SCs are a special group of undif-converted into cells of other types during embryogenferentiated cells possessing two fundamental proper-esis; therefore, in general, they lack the capacity for ties: they are capable of self-maintenance and differ-self-maintenance. To date, no evidence has been entiation into specialized tissue-forming cells. obtained that at least part of these cells are self-main-Another important property of SCs is their consider-tained and preserved in adult organisms. However, if able proliferative potential, which allows them to the blastocyst inner cell mass is cultured ex vivo under divide many times and to be maintained as a cell pop-special conditions, it is possible to obtain so-called ulation throughout the life of the multicellular body. embryonic stem cells (ESCs) . These cells are toti-Note, however, that ex vivo cell populations whose potent, because they can give rise to all tissues of the identity to in vivo cells has not been proved and often body if introduced into the blastocyst. In addition, is far from evident are also often referred to as SCs. ESCs can differentiate in vivo into many cell types The generally accepted necessary criterion for classi-originating from all three embryonic layers. For fying these cells as SCs is their normal functioning as example, mouse ESCs can differentiate in vitro into either SCs or differentiated cells after introduction various types of embryonic cells and many adult cell into living organisms.
EMBRYONIC STEM CELLSThe bodies of adult animals, including humans, consist of more than 200 types of cells developing from three embryonic layers. The ability of cells of the same type to give rise to different types of cells and tissues is termed pluripotency. Pluripotency is one of the main characteristics of all SCs. Some cells of early mammalian embryos can differentiate into all types of cells and tissues. For example, the cells of the inner mass of the mammalian blastocyst give rise to all cell types of the adult body. This capacity is termed totipotency. Note that the cells of the blastocyst inner mass hardly meet the definition of SCs, because they are converted into cells of other types during embryogenesis; therefore, in general, they lack the capacity for self-maintenance. To date, no evidence has been obtained that at least part of these cells are self-maintained and preserved in adult organisms. However, if the blastocyst inner cell mass is cultured ex vivo under special conditions, it is possible to obtain so-called embryonic stem cells (ESCs) . These cells are totipotent, because they can give rise to all tissues of the body if introduced into the blastocyst. In addition, ESCs can differentiate in vivo into many cell types originating from all three embryonic layers. For example, mouse ESCs can differentiate in vitro into various types of embryonic cells and many adult cell types, including the cells of skeletal and cardiac mus- cles, hematopoietic cells, yolk sac cells, smooth muscle cells, adipocytes, chondrocytes, endothelial cells, melanocytes, neurons, glial cells, pancreatic islet cells, and primitive endoderm [2–11]. ESCs have a normal karyotype and can be cloned and maintained over many passages without changing their properties under controlled conditions, i.e., culturing on a carrier layer of embryonic fibroblasts and/or in the presence of leukemia inhibitory factor (LIF) . The so-called embryonic (primordial) germ cells isolated from the embryonic genital tubercle are similar to ESCs (primarily in totipotency) [13, 14]. ESCs from blastocysts have greater proliferative and differentiation potentials than the cells of the genital tubercle. In addition, the latter are difficult to obtain. The cells of embryonal teratocarcinomas are also similar to ESCs in some respects; however, their differentiation potential is reduced, compared to that of ESCs, and they are often aneuploid, which limits their clinical use [12, 15]. J.A. Thomson  was the first to obtain human ESCs from donor blastocysts in 1998 . About that time, J.D. Gearhart and coworkers  proposed a method for isolating embryonic germ cells from the genital tubercles of four- to five-week-old human embryos. Human ESCs are similar to mouse ESCs, although they differ in some respects; e.g., LIF has no effect on maintaining the totipotency of human ESCs in vitro [18, 19]. Classical ESC markers are alkaline phosphatase isozymes; the transcription factor Oct-4; a high telomerase activity; and some cell-surface markers, e.g., GCTM-2, TRA 1-60, SSEA-3, and SSEA-4  recognized by monoclonal antibodies to specific embryonic and tumor-specific antigens. The physiological roles of most markers, except for Oct-4, remains unclear. Studies with mouse ESCs and embryos demonstrated the crucial role of Oct-4 in maintaining the totipotency of early embryonic and germ-line cells . The differentiation of the cells of the inner mass is accompanied by a decrease in Oct-4 content; in turn, the change in Oct-4 synthesis rate in ESCs results in the loss of totipotency and the onset of differentiation [21, 22]. In addition to Oct-4, there are other transcription factors, mainly synthesized by undifferentiated ESCs, e.g., Nanog , which ranks high in the hierarchy of factors determining the undifferentiated state of ESCs, and Genesis . Recent advances in the techniques of gene-expression analysis made it possible to identify many other genes whose expression is characteristic of the undifferentiated state of ESCs. As expected, the sets of genes expressed in mouse and human ESCs have been found to overlap considerably . The totipotency of ESCs made them a suitable model system for studying the mechanisms underlying early developmental stages in mammals [26–28]. Culturing ESCs also allows their use as a test system for estimating the effects of various cytokines and growth factors on cell differentiation .
SOMATIC STEM CELLSThe SCs of adult organisms, or somatic SCs, are the cells of different types located in specialized tissues and organs. They have been found in the blood, bone marrow, skeletal muscles, the cornea and iris of the eye, tooth pulp, brain, spinal cord, blood vessels, liver, skin, gastrointestinal tract, and pancreas. The main function of somatic SCs is the permanent reproduction of mature specialized cells. Therefore, somatic SCs are present in all organs and tissues of the body throughout life, forming pools of reserve cells. Somatic SCs are markedly pluripotent. Except for rare exceptions, all SCs of adult organisms can give rise to cells of different types. Usually, the principle of colocalization of SCs and the effector function is met; this means that SCs differentiate into the cells of the effector organ or tissue in which they are located. However, there are exceptions. For example, hematopoietic SCs (HSCs), which can differentiate into all types of blood cells, are normally located almost exclusively in the bone marrow, which is not an effector organ of the circulatory or immune system. However, the rule of the colocalization of SCs and the effector function is complied with in early embryogenesis: at this stage, HSCs are located in the lumen of the aorta . This, as well as the presence of other SC types in the bone marrow and some data presented below, indicate that the bone marrow may be regarded as a special organ whose main function is to maintain the homeostasis of the circulatory and immune systems, bones, and, probably, some other system, rather than a mere part of the hematopoietic tissue.
The degree of pluripotency of various SC types varies considerably, from almost monopotent cells (the SCs of the cornea) to cells with wide differentiation spectra, such as hematopoietic, mesenchymal, and neural SCs [31–33].
Somatic SCs are very few and dispersed over tissues. In contrast to ESCs, whose origin is clear by definition, little is known about the origin of SCs in adult tissues. In addition, most pathways of SC differentiation into mature cells have not been studied sufﬁciently (except for hematopoiesis). Obviously, speciﬁc differentiation pathways depend on the type of SCs. By analogy with hematopoietic differentiation and on the basis of other data [31, 32], somatic SCs are generally assumed to meet the principle of stepwise hierarchical maturation that is strictly directed and irreversible. The least differentiated, primitive SCs are mainly quiescent. Their division yields more differentiated cells of intermediate type, which are termed progenitor cells. These cells form a rapidly proliferating population and are less pluripotent and more committed towards a deﬁnite type of mature cell. Progenitor cells give rise to differentiated cells via several generations of intermediate cells, which become increasingly mature from one generation to another.
As follows from the deﬁnition of SCs, somatic SCs are capable of self-maintenance; however, upon close consideration, this issue becomes intricate and ambiguous. Possibly there is no ideal reproduction with all properties of the parent SCs preserved, at least not in all SC types. It is conceivable that telomerase activity is one of important factors of reproduction. The maintenance of the normal structure of telomeres, which are shortened as a result of terminal underreplication is necessary for the long proliferation of any set of cells. Telomerase activity is lower in somatic SCs than in the cells with unlimited proliferation potential, such as ESCs and transformed cell lines [34, 35]. Apparently, the insufﬁcient telomerase activity leads to a gradual shortening of telomeres in somatic SCs. The proliferative potential of somatic SCs is correspondingly lower than that of ESCs, although it is still high. Recent data indicate that factors other than telomerase are important for SC self-maintenance. For example, gene Bmi-1 of the Polycomb Group family is necessary for the self-maintenance of hematopoietic, neural, and, probably, some other SC types [36, 37]. The mechanism of the Bmi-1 effect is likely to be related to suppressed expression of the genes encoding inhibitors of kinases from the CDK family, which serve as negative cell-cycle regulators.
STEM CELL NICHESNiche is one of the main terms in the biology of SCs. A niche is deﬁned as the combination of the cellular microenvironment and extracellular matrix that is speciﬁc for a given type of SC and can serve as a “shelter” for SCs for an indeﬁnitely long period of time . Note that a niche implies a close, long, active interaction with SCs. Therefore, the niche produces various factors promoting SC survival and the maintenance of their undifferentiated state and controls SC proliferation and differentiation. Thus, the exit from a niche is related to the transition to the irreversible differentiation of SCs. However, the causeand-effect relationship between these processes is vague; i.e., it is unclear whether the SC exit from the niche (including that resulting from the SC division and displacement of one of the daughter cells from the niche) is the primary event triggering differentiation or, conversely, spontaneous differentiation of the SC breaks its relationship with its niche. In this connection, it is interesting that the division of the SCs of the Drosophila germ line is spatially oriented, with the mitotic spindle being oriented towards the cells of the niche. As a result, only one of the daughter cells remains connected with the niche and retains the characteristics of a SC, whereas the other cell starts differentiation. The cell adhesion protein DE-cadherin and the component of the signal pathway Wnt-APC and Armadillo (β-catenin is its analog in vertebrates), which are located at the CS–niche boundary, are involved in this process.
Nevertheless, the effect of the niche on SCs should not be considered absolute. It is well known that some factors can disturb the relationship between hematopoietic SCs and their niches, which results in SC release into the blood ; however, the SCs subsequently return to their places, with their state and functions preserved. In addition, SCs produce large amounts of extracellular regulatory molecules  and, most probably, can affect the formation and properties of their microenvironment.
The study of SC niches is in its infancy, because the subject is too complicated. The niches of germ-line, intestinal epithelial, and epidermal SCs have been partly described , and some factors controlling the self-maintenance of SCs have been identiﬁed. For example, the important roles of the components of the signal pathways for some development regulators, such as Notch, Wnt, and Hedgehog, have been demonstrated [41, 42]. To date, the niche of HSCs has been studied in the greatest detail. Early osteoblasts lining the inner surface of bones proved to be an important or even the main component of this niche [43, 44], with an increase in the number of osteoblasts resulting in a proportional increase in the number of HSCs. Jagged 1 produced by osteoblasts and activating the Notch factor is important for maintaining HSCs. Of interest is the fact that the proteins N-cadherin and βcatenin concentrate in the zone of contact between HSCs and osteoblasts, which is very similar to the aforementioned organization of the interaction between the Drosophila germ-line SCs with their niches. These data indicate that the principles of SC– niche interaction are the same for different SCs and in different phylogenetic groups.
STEM CELL PLASTICITYMany recently published studies deal with a new fundamental property of somatic SCs, namely, the ability of the SCs that give rise to the cells of a given tissue to differentiate into cells of other, “unrelated” tissues (even those originating from other embryonic layers) under certain conditions. This property was termed plasticity, and the differentiation into the “uncharacteristic” cell type is often called transdifferentiation or, more correctly, transdetermination. For example, bone marrow SCs of mesodermal origin are capable of differentiating into hematopoietic cells . These results raised a kind of euphoria among researchers studying SCs and brought about a new concept postulating that all somatic SCs possess an extremely wide plasticity and, provided the necessary microenvironment conditions, can differentiate into cells of any type . However, later publications showed that the turn to the new SC paradigm would be premature.
Currently, the plasticity of SCs is heatedly debated. Specialists have divided into two parties, one of which denies SC plasticity, at least in the form described in the aforementioned studies. Several facts agree with this point of view. First, the reproduction of these studies has not conﬁrmed the plasticity of some SC types, e.g., HSCs [50, 51]. In addition, some data, namely, the possibility of SC fusion with mature cells, other SCs, and progenitor cells, cast doubt on the published evidence for SC plasticity. This fusion was ﬁrst obtained when coculturing ESCs with neural progenitors  and bone marrow cells  in vitro, which was accompanied by the epigenetic reprogramming of somatic cell nuclei. Later experiments with the mouse model of type tyrosinemia demonstrated that the therapeutic effects of the injection of bone marrow cells were accounted for by the fusion of these cells with hepatocytes . Finally, recent studies demonstrated that the transplanted bone marrow-derived cells fused with Purkinje cells in the cerebellum, as well as with hepatocytes and cardiomyocytes [55, 56]. It is true that some hepatocytes; cardiomyocytes; and, probably, Purkinje cells are normally polyploid; hence, the fusion of these cells may be a normal physiological process.
In addition to cell fusion, one more explanation for the observed phenomena was recently proposed. It was hypothesized that bone marrow contained SCs or progenitor cells of other differentiation pathways, such as muscular, neural, and hepatocytic ones. These cells are similar to HSCs with respect to the set of surface markers and migrate to the bone marrow along the gradient of the chemotaxis factor chemokine SDF-1 .
There are grounds to say that the presumed plasticity of SCs contradicts the traditional notions on the properties of HSCs and the entire paradigm of the unidirectional, irreversible differentiation of SCs. However, the plasticity principle itself does not violate the dogmas of cellular developmental biology. Experiments on cloning amphibians and, later, mammals showed that the nuclei of most somatic cells not only retain the complete set of genes necessary for development, but also are capable of epigenetic reprogramming upon being transferred into the egg cell cytoplasm. There are examples of the transdetermination of Drosophila imaginal disks depending on the microenvironment. Regeneration in amphibians involves dedifferentiation and cell lineage switching . Finally, it is well known that the destiny of cells may be changed, and even whole organs may be formed, as a result of the expression of single genes; the most striking example is the formation of ectopic eyes in the regions of the overexpression of the regulatory gene Pax6 in Drosophila . These data demonstrate that the intracellular action of single key regulatory factors can easily overcome epigenetic control. The effects of extracellular factors on the cell epigenetic state, which is a necessary condition in the model of plasticity without cell fusion, is necessarily indirect. However, it is a priori possible that there are sets of extracellular stimuli that are strong enough to overcome the epigenetic barriers that separate SC types from one another. The differentiation of cultured mesenchymal SCs into cardiomyocytes  and neurons  induced by 5-azacytidine and retinoic acid, respectively, which has been obtained in numerous experiments, indicates that such stimuli do exist, at least for ex vivo systems of SC culturing. In these experiments, the contact and fusion of SCs with the respective differentiated cells were excluded.
The researchers who believe that SC plasticity actually exists refer to the aforementioned ex vivo experiments and the studies where the destiny of transplanted bone marrow cells was changed, apparently, without their fusion with the cells of the target organ [62–65]. Advocates of SC plasticity are gradually reaching the following consensus: the plasticity actually exists, but it typically occurs in the cases of organ or tissue injury. For example, the degree of incorporation of the cells injected into the lungs and muscles markedly increases when the organs are injured [65, 66]. According to this notion, at least two conditions are necessary for plasticity to be expressed in vivo. First, the injured organ must secrete factors that mobilize SCs, i.e., cause their release from natural niches into the blood, and promote their migration toward, and colonization of, the injured organ. SDF-1 is probably one of these factors . Second, SCs must enter an environment that can induce SC differentiation along a new lineage. The existence of at least two signals involved in the migration and colonization stages was demonstrated in experiments on the damage-induced differentiation of bone marrow cells into myocytes . It may even be supposed that the factors produced by an injured organ can affect the epigenetic state of SCs, weakening the barriers between the developmental programs of different SCs and sensitizing them to new sets of extracellular differentiation signals. In general, this plasticity indicates a second important function of somatic SCs (whose existence has not been convincingly demonstrated so far), namely, functional restoration of injured organs, which involves, among other factors, SCs anatomically remote and located in places well protected from injury, primarily, in the bone marrow. To conclude this issue, note that the scientiﬁc community has not yet come to terms about SC plasticity; however, there is no denial that there are mechanisms that allow SCs to overcome epigenetic barriers and change their destiny through either fusion with the cells of the target organ or direct effects of the cell environment or other signals. Therefore, SCs can be included in the structure of “unrelated” organs and tissues, which is the main mechanism of the therapeutic effect of these cells.
HEMATOPOIETIC STEM CELLSHSCs were found earlier than other somatic SCs and are currently the best studied type of SCs. Experiments with mice demonstrated that pluripotent bone marrow HSCs may be divided into two types: more primitive cells capable of self-maintenance for a long time and ensuring long-term but slow restoration of hematopoiesis, and more differentiated cells ensuring rapid but short-term restoration. The SCs that give rise to only lymphoid or only myeloid cells rank lower in the hierarchy [67, 68]. SCs more mature than the lymphoid and myeloid ones are the progenitor cells committed toward deﬁnite differentiation lineages.
Mouse HSCs have the surface markers Thy1.1, c-Kit, and Sca-1 and lack some markers that are characteristic of mature blood cells (Lin negativity) . The CD34 synthesis rate in the most primitive HSCs is lower than in more differentiated cells of mice . The phenotype of human HSCs is poorly studied, because it is impossible to analyze directly repopulation and hematopoiesis restoration in this case. There is evidence that human HSCs are CD34-, CD45-, c-Kit-, and Thy1-positive and CD38- and Lin-negative [70, 71].
Adult human HSCs are normally located in the spinal cord; however, small amounts of them are also found in the peripheral blood. The umbilical blood and placenta are rich in HSCs . Cells phenotypically identical to HSCs have been reported to possess plasticity. For example, they can differentiate into myocytes, cardiomyocytes, and liver cells and participate in the regeneration of the respective tissues after injury [62, 73, 74]. After HSC transplantation, these cells were found in the lung and intestinal epithelia and the skin of the recipient . Recent data demonstrated that HSCs were able to differentiate into endothelial cells; i.e., they exhibited hemangioblast activity [75, 76]. On the other hand, as noted above, there is evidence against the plasticity of HSCs.
HSC self-maintenance also remains an open question. Studies on the kinetics of hematopoiesis in mice using cell marking with retroviral vectors demonstrated that hematopoiesis was maintained mainly due to a small number of relatively short-lived cell clones sequentially replacing one another . Moreover, it is well known that the sequential transplantation of the bone marrow to irradiated mice resulted in a progressive exhaustion of the HSC function . The production of telomerase in HSCs is necessary but not sufﬁcient to counteract this process . There are several possible explanations to these data; however, the simplest one is that HSCs can undergo only a limited number of cell divisions, and hematopoiesis is maintained because some cells from the bone marrow pool of HSCs regularly enter the cell cycle and start irreversible differentiation. Moreover, it is well known that HSCs are extremely difﬁcult to propagate, or even maintain, in vitro for even a short time. Nevertheless, certain progress has been made in the ﬁeld recently, mainly related to studying the molecular mechanisms controlling HSC functions. Several research groups used hybridization with “gene chips” to analyze the “molecular signature” of HSCs, i.e., the repertoire of the genes whose expression is considerably higher in HSCs than in differentiated cells. The comparison of the gene sets obtained by two research groups [80, 81] showed that they contained about 600 common genes, which corresponded to a 65% overlap . However, attempts to identify genes common to different types of SCs or, using the generally accepted slang, “stemness” genes yielded discouraging results. Although the sets of genes identiﬁed by the two research groups contained approximately 200–300 such genes, they, in general, hardly overlapped . Note, however, that the comparison was made between HSCs isolated from the body and SCs cultured ex vivo (ESCs and neural SCs). The molecular programs operating in these cases may have been considerably different. In addition, the resultant sets of genes are far from complete, so the stemness genes, which are most likely to encode rare mRNAs, may have been left out accidentally.
In addition, researchers from several laboratories found that the overexpression of some regulatory genes, such as HoxB4, Hes-1, and AML1-ETO, induced HSC propagation in vitro and in vivo [84–86]. This allows us to hope that the regulatory factors identiﬁed will be used for HSC culturing and modiﬁcation. However, there is evidence  that these genetic manipulations may alter the properties of HSCs.
MESENCHYMAL STEM CELLSThe Russian researcher A.J. Friedenstein discovered mesenchymal stem cells (MSCs) in the 1960s [88, 89]. Recently, they have attracted special attention due to some their properties that are promising for clinical use. One of these properties is the wide differentiation spectrum and the possibility of long-term culturing.
The main source of MSCs is the bone morrow; these cells are progenitors of adipocytes, chondrocytes, osteoblasts, and the bone marrow stroma. Notwithstanding numerous studies, most aspects of MSC biology, including their ontogeny, location in the bone marrow, and functions, remain unknown. Data on these issues are often contradictory, because different researchers use different approaches to MSC cultur-ing, estimation of their differentiation potential, and their capacity for self-maintenance. In addition, MSCs are difﬁcult to identify because of their heterogeneity. Friedenstein, having demonstrated the presence of a cell population fundamentally different from the hematopoietic one in the bone marrow, described their variation with respect to size, morphology, proliferative capacity, alkaline phosphatase synthesis, and osteogenic capacity in vivo . Afterwards, bone marrow MSCs were demonstrated to be capable of in vitro differentiation into various cell types [91–93].
Currently, MSCs are most often regarded as pluripotent cells morphologically similar to ﬁbroblasts that are present in the adult bone marrow and can proliferate like undifferentiated cells and differentiate into the cells of various tissues, including bones, cartilage, fat, tendons, muscles, and bone marrow stroma [32, 60, 94–98]. One of the characteristic features of these cells is the capacity of cultured MSCs for adhesion to plastic, which allows bone marrow MSCs to be easily separated from other cells, e.g., hematopoietic ones.
MSCs or MSC-like cells capable of differentiation into the cells of various mesenchymal tissues under certain conditions are also present in other organs and tissues of mesodermal origin [99, 100], although, evidently, they are more numerous in the bone marrow. Normally, the peripheral blood does not contain noticeable amounts of MSCs . Bone marrow MSCs are often called stromal cells [102, 103]. Some authors doubt whether this generalized approach is justiﬁed, since it remains to be determined whether bone marrow MSCs, stromal cells, and MSC-like cells of other mesodermal tissues are essentially the same cells or they are different cell populations similar in differentiation capacity [104, 105].
Published data indicate that bone marrow MSCs are characterized by an extremely wide plasticity (although, as noted above, there are several possible mechanisms of this phenomenon). MSCs can give rise to some neural elements, hepatocytes, cardiomyocytes, and lung epithelial cells [66, 102–109]. However, cultured MSCs may lose some of their pluripotency , apparently, because of the heterogeneity of their population, which normally includes progenitor cells with a limited differentiation potential . Some of the mechanisms of the unusually wide MSC plasticity were determined recently. For example, the comprehensive analysis of gene expression both at the population level and in individual MSCs showed that MSCs contained not only transcripts characteristic of adipocytes, chondrocytes, myocytes, osteoblasts, and stromal cells, but also those typical of endothelial, epithelial, and glial cells [111, 112].
Bone marrow MSCs and their descendants are closely related to hematopoietic cells. Apparently, their main function is to provide conditions for normal hematopoiesis . MSCs are known to produce certain cytokines and growth factors necessary for the differentiation of hematopoietic cells The list of cytokines includes such important hematopoietic factors as interleukins-6, -7, -8, -11, -12, and -14; granulocyte, macrophage, and granulocyte–macrophage colony-stimulating factors; LIF; ligand Flt-3; and SCF [104, 114]. MSCs are also closely related to endothelial cells and osteoblasts.
It has been demonstrated that some MSC properties may vary depending on culturing time and other conditions . For example, the biological characteristics and phenotype of MSCs sometimes depend on the plating density. There are data that cells grow considerably more rapidly if they are plated at low concentrations [104, 115, 116]. Cell density may also affect the expression of many genes ; culture conditions may determine the direction of MSC differentiation and their proliferative potential [116, 118]. According to published data, bone marrow MSCs are a mixture of morphologically and functionally different cell types. However, it is unclear whether this diversity merely reﬂects different maturation stages and/or culture conditions or there actually are different types of MSCs in the same organism. In this connection, of special interest are the studies performed in the laboratory headed by C.M. Verfaillie [118, 119] on culturing so-called pluripotent progenitor cells derived from human and mouse bone marrow. For isolating and culturing this cell population, combined media, various growth factors, and adhesion factors (e.g., ﬁbronectin) are used. Under controlled culture conditions, the characteristics of the pluripotent progenitor cells are close to those of ESCs: these cells also synthesize telomerase and Oct-4, have an almost unlimited proliferative potential, and can differentiate not only into mesenchymal, but also into visceral–mesenchymal, neuroectodermal, and endodermal cell types. These cells can differentiate in vitro into the tissues derived from all the three embryonic layers. Moreover, if these cells are introduced into the early blastocyst, they give rise to the majority of somatic tissues . However, it is still unclear whether they are the most primitive bone marrow MSC type, and even whether any cells in the bone marrow in vivo actually possess these properties, or they result from epigenetic changes occurring in MSCs in the course of long-term culturing.
Data obtained by another research group  shed some light on this problem. They systematically analyzed the properties of bone marrow-derived pluripotent SCs with the use of various methods, including the procedure described in the study  performed by Verfaillie and coworkers. Cells isolated and cultured using three substantially different methods are ﬁbroblast-like cells similar in proliferative potential and identical in morphology. The cells also have practically the same sets of surface markers. More than 95% of the cells in each population cultured carried CD105/endoglin, an MSC marker. These cells exhibited similar differentiation potentials when culturing under the conditions of targeted differentiation into the nerve, cartilaginous, muscular, and endothelial tissues. The authors concluded that the bone marrow-derived cell populations cultured by different methods were essentially the same and had almost no morphological or functional differences. To date, these are the only data indicating the inherent and functional unity of bone marrow MSCs.
MEDICAL APPLICATIONS OF STEM CELLSAs knowledge of SCs is accumulated, new prospects of fundamental and clinical studies become evident. Different types of SCs are used in genetic and embryological research, studying the functions of growth factors and cytokines, pharmacology, toxicology, and transplantology. The pluripotency of SCs and their capacity for self-maintenance and migration, as well as switching developmental programs and con-text-dependent differentiation (irrespective of their mechanisms), are their crucial advantages over differentiated cells in terms of their implications for regenerative medicine. Detailed knowledge of SC biology is important for the use of these cells in regenerative medicine for restoring injured tissues via cell transplantation or the creation of so-called bioartiﬁcial tissues. Studying SCs is also necessary for another branch of regenerative medicine, namely, the stimulation of in vivo regeneration using the natural potential of SCs located in the tissues involved .
The available data allow us to assume that many if not all SC types can be used in regenerative medicine. It is important to determine the priority uses of different types of SCs for treating different diseases. Apparently, human ESCs, which are totipotent and can be propagated in culture, are especially prospective for regenerative medicine. To date, substantial experience has been accumulated in the use of ESCs in animal models for treating diseases related to the destruction or dysfunction of speciﬁc cell types. Examples of these diseases are insulin-dependent diabetes mellitus (type I diabetes) related to the dysfunction of pancreatic islet cells and Parkinson’s disease in which dopaminergic neurons in a certain cortical zone are damaged. Experiments with animals have demonstrated that the transplantation of ESCs is effective for treating various chronic diseases, including spinal injuries, Purkinje cell degeneration, liver cell damage, Duchenne’s disease, and osteogenesis disturbances . Note, however, that ESCs cannot be used successfully until the problem of graft–recipient histocompatibility is solved . The proposed radical solution to this problem, namely, the so-called therapeutic cloning, involves the transplantation of the recipient’s cell nucleus into an egg cell. This makes the entire procedure extremely complicated and expensive, although the resultant ESCs are genetically almost identical to the recipient’s cells. Note, however, that ESCs introduced into the body may produce teratomas. Therefore, it is necessary to initiate differentiation and reject noncommitted ESCs before the transplantation, which complicates the procedure. Nor should ethical problems concerning the necessity to destroy the blastocyst for obtaining an ESC line be disregarded. Finally, reports on various pathologies in the animals obtained using nuclear transplantation raise concerns about the possible harmful effects.
Clinical applications of HSCs are mainly related to blood diseases. In leukemias and lymphomas, bone marrow transplantation is a widespread method of treatment. Recently, the umbilical blood and peripheral blood after the so-called HSC mobilization from bone marrow (see above) by, e.g., granulocytic colony-stimulating factor, have been used for this purpose. HSCs can also be used for treating coronary disease and myocardial infarction [74, 124].
Bone marrow-derived MSCs are of considerable interest for regenerative medicine [104, 125]. The considerable differentiation and proliferative potentials of MSCs demonstrated in experiments with animals indicate that MSCs offer wide opportunities for scientiﬁc and practical uses; these cells can probably compete with ESCs in solving many medical problems. MSC autotransplantation seems the most promising at the current stage of the development of medical science, because it precludes both ethical and immune problems involved in transplantation from the donor to the recipient. To date, considerable experience has been accumulated in bone marrow transplantation. One of the most important ﬁelds of MSCs application is bone repair, the more so as spontaneous repair of this tissue is slow because of the small amount of blood vessels . Various tissue-engineering constructions based on organic scaffolds with incorporated MSCs are usually used for treating bone injuries [126–131]. The potential of MSCs makes it possible to use them for treating hereditary degenerative diseases of the skeleton. For example, MSCs have been clinically tested as a means for treating osteogenesis imperfecta caused by a mutation of the type I collagen gene in children .
Due to their capacity for differentiation into vascular endothelial cells, skeletal muscle cells, and cardiomyocytes, MSCs can be used for treating ischemic diseases . Coronary disease and myocardial infarction as its consequence are the most prevalent diseases in industrially developed countries. Peripheral artery disease results in the necessity of 150 000 surgical amputations per year in the United States alone . Cerebral ischemia and stroke most often lead to more or less severe disability. These diseases are caused by a complete or partial occlusion of vessels due to various diseases, mainly atherosclerosis, which results in an insufﬁcient blood supply and tissue damage. One of the possible approaches to their treatment is to stimulate the formation of new blood vessels (collaterals) bypassing the damaged ones. Numerous studies with animals demonstrated that bone marrow-derived and cultured MSCs are currently the best material for transplantation to patients with coronary disease, myocardial infarction, peripheral artery disease, and cerebral ischemia [135–141]. The ﬁrst reports on successful preclinical trials of the bone marrow-derived autologous multinuclear cell fraction as a remedy for myocardial infarction and peripheral artery disease were published recently [142–144]. Note, however, that the rate of MSC survival upon transplantation is low, especially after culturing [145, 146], which makes it necessary to search for more effective ways of MSC incorporation into the injured tissues.
The use of SCs is currently regarded as the most promising strategy of cell therapy. Of interest is the possibility of using these cells as primary recipients of recombinant DNA ex vivo in gene therapy of hereditary and acquired diseases. This approach may considerably reduce the risk entailed in direct injection of DNA to humans . There is strong evidence that the combined gene–cell therapy is not only safer, but also more effective than the use of these approaches separately for treating various hereditary and degenerative diseases.
The use of SCs in clinical medicine is in its infancy. Apparently, many ethical and scientiﬁc problems must be solved to allow their application on a large scale. In our opinion, the most important questions are as follows:
- Do all SCs pass through the progenitor phase before differentiation, and what are the stages of this process in different SCs?
- What mechanisms determine differentiation or make SCs remain undifferentiated?
- How do SC properties change during in vitro culturing?
- Do adult SC normally possess plasticity in vivo, or is it a mere artifact of in vitro culturing? Is there such thing as in vivo SC plasticity unrelated to cell fusion?
- What signals regulate SC proliferation, differentiation, and presumed plasticity?
- What factors stimulate SC migration into injured organs and tissues? How universal is this mechanism?
- What mechanisms determine the incorporation of transplanted cells into the structure and functional activity of organs and tissues? How can these mechanisms be regulated?
Although the list of unsolved problems is impressive, the common opinion of researchers and practitioners is that the study of the biology of SCs is necessary for a deeper understanding of the natural mechanisms of regeneration and homeostasis, and practical applications of SCs have excellent prospects and will undoubtedly have a strong effect on the future development of medicine.
We thank S. Sharifullina for her expert help in preparing the manuscript.
This study was supported by the Russian Foundation for Basic Research, project nos. 03-04-48786 and 02-04-49196.
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