Precursor Cells of Mechanocytes

Precursor Cells of Mechanocytes ALEXANDERJ. FRIEDENSTEIN lmmunomorphological Laboratory, The Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences, Moscow, USSR Introduction . . . . . . . . . . Clonogenic Precursors of Mechanocytes . . . . Determined and Inducible Osteogenic Precursor Cells . Presence of Mechanocyte Precursors in Blood . . . Interrelationship between Mechanocyte Precursors and Hemopoietic Cells . . . . . . . . . . . . . . . VI * Diploid Strains of Fibroblasts VII. Concluding Remarks . . . . . . . . References . . . . . . . . . . I. 11. 111. IV. V.

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I. Introduction

Fibroblasts, bone, cartilage, and reticular cells, designated mechanocytes (Willmer, 1960), constitute tissue cell populations in which cell production continues in postnatal life. Mechanocyte production is quite intensive in tissues of growing organisms; it can also be observed or can be stimulated in adult mammals. The renewal rate of fibroblasts of loose connective tissue has thus far not been properly determined. According to a study of thymidine-3H incorporation, fibroblasts are slowly renewed at the expense of dividing cells. If it is assumed that on repeated administration of t h ~ m i d i n e - ~the H linear increase in percentage of labeled cells continues up to the point where it reaches loo%, for rodents the turnover time of dermal fibroblasts will prove to be about 60 days, and of esophagus tunica propria fibroblasts about 125 days (Cameron, 1971). However, it is not certain whether or not the fibroblast population of a given tissue is homogeneous as far as the rate of renewal is concerned. The turnover rate of bone tissue mechanocytes was studied in detail in young animals (Owen, 1970). In 2-week-old rabbits, osteoblasts on the periosteal surface of long bones are renewed during a 3-day period at the expense of dividing osteogenic precursors, that is, preosteoblasts. The rate of bone mechanocyte proliferation decreases sharply with age. It becomes evident from the decrease in t h ~ m i d i n e - ~ H pulse-labeling indices. Thus in mice of various ages these indices are: 1week old, 8.5%; 5 weeks old, 2.7%; 8 weeks old, 0.8%; and 26 weeks old, 0.2% (Tonna, 1961). 327

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A functionally important category of mechanocytes includes stromal reticular cells of hemopoietic and lymphoid organs. The ambiguity of identification of this cell type on the basis of such criteria as argentophilia or phagocyte activity has been stressed more than once (Gall, 1958; Steinman and Cohn, 1975; Stuart, 1975), and the function of these cells has been the subject of numerous but not sufficiently founded speculations. In situ stromal mechanocytes are normally associated with reticular rather than collagenous fibers and are slightly different from mechanocytes of another location (Stuart and Davidson, 1971). Yet, in vitro they acquire the typical fibroblast character and synthesize collagen (Friedenstein et al., 1970). It has been established that reticular cells are the important component of hemopoietic and lymphoid tissues. In lymphoid organs they permit the recognition of antigens by immunocompetent cells (Hanna and Szakal, 1968; Nossal et al., 1968) and possibly participate in the cooperation of the T and B cells. Reticular cells are responsible for the microenvironment in hemopoietic organs, which affects the proliferation and differentiation of hemopoietic and lymphoid cells (Friedenstein et al., 1974a). According to studies utilizing a complete tritiated thymidine-labeling method complemented by thymidineJ4C administration (Haas et al., 1969), stromal mechanocytes retain their label for a very long time and are considered resting cells. Thus under normal steady-state conditions mechanocytes have no turnover or a very slow turnover rate compared with rapidly renewing cell types. However, situations in which intensive proliferation of mechanocytes is induced are well known. Among them are the formation of granulation tissue in inflammation and wound healing and the stimulation of osteogenesis in fracture repair (Tonna and Cronkite, 1964) and by estrogens (Simmons, 1968). The proliferation rate of mechanocytes in such cases is sharply accelerated when compared to their proliferation in the steady state. For example, after localized depletion of bone marrow (Maloney and Patt, 1969), stromal mechanocytes physiologically at rest were shown to be stimulated to proliferate (MeyerHamme et al., 1971). It is also well known that, on explantation in vitro of many tissues from adult animals and man, intensive proliferation of fibroblasts is observed. Thus several different lines of evidence point to the existence of precursors of mechanocytes in postnatal life. As for these precursors, that is, the cells whose division results in the formation of new mechanocytes, certain questions have to be answered. They involve the identification of these precursor cells and a study of their properties, origin, and distribution in various tissues and organs.

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Precursors of mechanocytes should first be searched for among mechanocytes, that is, among those capable of proliferation. The question arises whether or not all the proliferative mechanocytes in a given tissue exhibit equal reproductive potency. The assumption that in tissues with cell renewal special lines of stem cells can be distinguished was made by Maximov (1927) many years ago, but it was largely speculative. The meaning attached to this concept had not always been treated unambiguously, and the concept itself was defined in varing terminology. I n the following discussion stem cells are those that, on the one hand, have the advantage of self-replication for a period commensurable with the life-span of the organism and, on the other hand, recruit mature cells of the given tissue from among their descendants. A situation alternative to the existence of special lines of stem cells may arise if all proliferative cells in the given tissue have equal reproductive capacities, although some of them may temporarily be excluded from the pool of actually proIiferating cells. So far, the existence of stem cells has been unambiguously proved only for hemopoietic tissue (Till and McCulloch, 1961; for reference, see Metcalf and Moore, 1971). Hemopoietic stem cells have the capacity for extensive proliferation, resulting in renewal of their own kind; committed precursors with little or no self-replicative ability are constantly recruited from their number. The existence of mechanocyte stem cells is still open to discussion. Yet, the importance of the problem involves more than just terminology. Fibroblasts are one of the favorite subjects in the study of proliferative activity, cell quantity regulation, cell aging, and neoplastic transformation. Whether special cell lines responsible for self-maintenance are present in mechanocyte populations or whether all their proliferative cells possess equal reproductive capacities is relevant to the analysis of each of the problems mentioned. Precursors of mechanocytes may b e searched for, however, not only within their own populations but among other connective tissue cells as well (lymphocytes, hemopoietic cells, macrophages). That these cells can transform into fibroblasts is a widespread view (for reference, see Bloom and Fawcett, 1962). It is based on observations of changes in cell composition of tissues, for example, in regions of inflammation or after in vitro explantation. Usually these changes result in the replacement of lymphoid cells and of macrophages by fibroblasts. Transitional forms of cells (polyblasts) can be easily found in each of these cases. As Maximov (1927) noted, this points to the possibility of transformation of one cell type into another, although it fails to prove it. For example, if in the initial cell population a small number of special fi-

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broblast precursor cells is present (or is added by influx), as a result of their proliferation the gradual replacement of other cell types may take place. These precursors may have nothing in common with either lymphocytes or histocytes, despite the presence of cells that are intermediate in structure as compared to the above cells and fibroblasts. Generally, the search for transition forms is hardly a suitable method for precursor cell identification, especially if the concentration of the latter is low. Risks run in the subjective evaluation of transition cells are widely known, but in the study of mechanocytes this method is particularly unreliable. In mixed cell populations, especially in mice, to identify a definite cell as a mechanocyte or a macrophage is not always easy, even employing electron microscopy (Leibovich and ROSS,1975). For example, after a description of dendritic cells of lymphoid organs was given (Steinman and Cohn, 1973), their relation to mechanocytes or macrophages could not be established (Steinman and Cohn, 1975). It was mainly because of these difficulties that classic histology failed to solve the problems of histogenesis of connective tissue pertaining to its early precursors. Indicative are the conclusions at which Jacoby (1965)arrived in summarizing the results of works on the transformation of connective tissue cells, namely, that there is no clear-cut morphological proof of transformation of any cell type into macrophages or of reverse transformation of macrophages into any cell type, fibroblasts, for example. The problem of mechanocyte precursor cells involves the question of the degree of mosaicism of precursors as regards their differentiation potential. Different types of mechanocytes, although sharing several essential properties, synthesis of collagen being the main one, differ from each other. The same is true of the collagen they synthesize. The question arises whether each type of mechanocyte has its own committed precursors or whether the formation of any or several types of mature mechanocytes from common precursors is possible. The ectopic development of bone and cartilage often observed in postnatal life (Ostrowski and Wodarski, 1972) serves as an indication. The study of ectopic bone formation initiated by the works of Huggins (1931; Huggins and Sammett, 1933) to this day remains a valid approach to the analysis of mechanocyte precursor cells. The utilization of stable chromosome and antigenic cell markers, as well as cloning methods, provided new tools for the study of precursor cells. They have been successfully used for the study of hemopoietic precursors (Metcalf and Moore, 1971).

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The results related to the problem of mechanocyte precursor cells and obtained mainly through methods of marking and cloning are considered in the following discussion. 11. Clonogenic Precursors of Mechanocytes

Cells with high proliferating potency may be singled out, and the descendants of each may be obtained separately by cloning in vitro. In order to obtain data on the concentration of clonogenic cells in the tissue cell population, cloning must be carried out as early as the initial explantation. However, with mechanocytes this requirement cannot be easily met. Usually the isolation of mechanocytes is connected with the necessity for carrying out enzymic digestion of the ground substance in the course of which not every cell is released or, as the case may be, some of them prove damaged. Hemopoietic and lymphoid tissues are the exception. They dissociate into cells after soft mechanical treatment. Subsequent explantation in monolayer cultures may be used as a means of selective cloning of mechanocytes from hemopoietic tissues (Friedenstein et al., 1970) Active in vitro proliferation of mechanocyte precursors makes this possible, since their concentration in hemopoietic tissues is low as compared to that of other cells. As a result, the descendants of fibroblast precursors form discrete fibroblast colonies, while the basic body of explanted cells acts as a natural feeder. In fact, fibroblast colonies are readily formed in monolayer cultures of bone marrow, spleen, thymus, and lymph node cells of adult mice, rats, guinea pigs, rabbits, dogs, and humans (Friedenstein et al., 1970, 1974a,b, 1976a; Luria et al., 1972; Panasyuk et al., 1972) (Fig. 1). The formation of a colony starts on the third or fourth day, and it consists of several cells. By the tenth day some colonies reach 0.5-0.8 cm in diameter and contain several thousand cells. Between days 5 and 12 the number of colonies does not increase, although the size of many of them does. Cells of the colonies are typical fibroblasts (Fig. 2) and are characterized by the presence of fibrils in the cytoplasm and large nucleolar complexes in the nuclei. Cells in the colonies synthesize collagen, which is detected both histochemically and by the incorporation of labeled proline as oxyproline, into the proteins of cells comprising the colonies; these proteins are secreted into the medium. It has not yet been determined what type of collagen is synthesized in vitro by mechanocytes of hemopoietic tissue origin and whether or

FIG. 1. Fibroblast colonies in 12-day-old cultures. (a) Culture of guinea pig bone marrow cells. (b) Culture of guinea pig thymus cells. (c) Culture of guinea pig spleen cells. (d) Culture of mice thymus cells.

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FIG.2. Fibroblasts in 10-day-old cultures of rabbit bone marrow cells. (a) Fixed culture. (b) Living culture.

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not mechanocytes of different hemopoietic organs or of separate colonies within one culture differ in this respect. The size of colonies, their shape, and the type of packing of cells within a culture varies. This may indicate a difference in the quality of colony-forming cells, or be due to unrelated reasons. The majority of colonies are monolayer, but in large colonies multilayer strands of fibroblasts, especially densely packed in the center of colonies, are frequently formed, Morphologically fibroblast colony-forming cells (FCFC) appear after the first hours of explantation as large mononuclear cells. Later they begin to spread on the surface of the substrate, taking on an elongated shape with an oval, light nucleus with a discernible nucleolar complex. They undergo the first S period in vitro 20-60 hours after explantation. These data were obtained in an autoradiographic H study of 4-day cultures grown in the presence of t h ~ m i d i n e - ~during the first 4-60 hours. Subsequent cultivation took place on a medium supplemented with nonlabeled thymidine to prevent reutilization of the label. In cultures in which t h ~ m i d i n e - ~was H present for less than 60 hours but for more than 20 hours, fibroblast colonies were found to be either completely labeled or completely unlabeled but not mixed (Friedenstein et al., 1974a), which points to the clonal nature of colonies. Actually, results of typification of cells in colonies developing on explantation of a mixture of cells differing in chromosome markers, the linear dependence of the number of colonies on the number of cells explanted, and time-lapse cinematographic observations of living cultures all indicated that fibroblast colonies are cell clones (Friedenstein et d , 1970). The formation of colonies occurs only when explantation is performed at an optimal initial density; with an excessive number of explanted cells per unit of culture vessel surface, fibroblasts form a monolayer; with too low a number of explanted cells, no fibroblast colonies are formed. Stable efficiency of colony formation (when linear dependence of the number of colonies on the number of explanted cells holds) is characteristic of a given hematopoietic tissue. It is achieved on explantation with an initial density of l@-lO5/cm2 of living cells of lymphoid or hemopoietic tissue, or on addition of 5 x 105-106/cm2of irradiated bone marrow cells which may be used in the capacity of a supplementary feeder. With lower initial density (2.5 x lo4 cells/ml) the explantationof mouse bone marrow cells in monolayer cultures leads to the formation of macrophage colonies which survive for the first 4 days of cultivation (Gond et d,1975). In mouse bone marrow cultures explanted at cell densities necessary for

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fibroblast colony formation, macrophages grow as a thick net of cells occupying spaces between fibroblast colonies as well as populating them. In the presence of a standard feeder (irradiated bone marrow cells) the fibroblast colony-forming efficiency of cells from different hemopoietic organs exhibits the same differences as those observed on their explantation without a supplementary feeder. This indicates that full-value feeder action is achieved by the presence of a sufficient number of living or irradiated cells and is not the property of a certain category of cells; hence the stable efficiency of colony formation reflects the content of FCFC among the cells of a given hemopoietic organ. FCFC are highly resistant to the damage involved in explantation. In fact, when they are harvested with trypsin and passaged into a new vessel, several hours after adhesion of explanted bone marrow cells, the number of colonies formed is the same as in vessels with cells not subjected to passaging. Cloning in monolayer cultures may be employed for the comparison of FCFC concentration in different cell populations and for the estimation of changes in the FCFC content under the influence of various factors. The FCFC content in various hemopoietic organs differs considerably and undergoes noticeable age-related changes. Thus bone marrow in one femur of a guinea pig contains about 4 x lo3 FCFC on the tenth day after birth, that is, about 30 FCFC per lo5bone marrow cells; in guinea pigs of 160- to 216-gram weight these values are 4 x lo3 and 4.5 per lo5, respectively; in those of 230- to 320-gram weight, 19 x lo3and 20 per lo5, respectively; in 1-year-old guinea pigs with weights above 1 kg femoral bone marrow contains only about 2 X 103 FCFC, and their concentration is 2 per lo5bone marrow cells. In the spleen of 10-day-old guinea pigs there are 0.7 x lo3FCFC, and their concentration is 7 per 10’. These values are 2.5 x lo3and 1.5per lo5,respectively, in adult guinea pigs, and 2.3 x lo3and 0.5 per lo5in 1-year-old guinea pigs. The FCFC content in 10-day-old guinea pig thymus is 0.6 X lo3, and the FCFC concentration is close to 0.3 per lo5.In adult guinea pigs these values are 2 x lo3and 0.5 per lo5,and in 1-year-old guinea pigs the FCFC content in the thymus is not subject to determination. I n F1 CBA x C,,Bl adult mice the FCFC content is 0.2 x lo3 in the bone marrow of one femur, thymus, and spleen, and the FCFC concentration is 1.6 per lo5 in bone marrow, and 0.2 per lo5 in both thymus and spleen. By passaging cells from fibroblast colonies of bone marrow, spleen,

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and thymus origin diploid strains of fibroblasts may be easily obtained, thus being the progeny of the initially explanted FCFC (Friedenstein et d., 1970, 1974a,b). It is obvious that complete differentiation of mechanocytes does not occur in monolayer cultures. Chondrogenic cells may serve as the example (Mayne et al., 1973); in specific in vitro environments they form progeny similar to fibroblasts and not to cartilage cells. Fibroblast strains of bone marrow, spleen, and thymus origin are morphologically almost indistinguishable (Friedenstein et aZ., 197413). In particular, neither osteoblasts nor osteocytes are formed in monolayer cultures of bone marrow fibroblasts. Yet it turned out that fibroblast strains of bone marrow and not those of any other origin differentiate into osteoblasts and osteocytes on retransplantation in vivo. I n fact, when fibroblasts from bone marrow strains are placed in a difhsion chamber, intensive osteogenesis takes place inside the chamber (Friedenstein et al., 1970). Still more interesting are the properties of these cells on explantation in an open cell system. Small fragments of bone with bone marrow were found under the kidney capsule where fibroblasts from bone marrow cultures had been grafted (Friedenstein et al., 1974a). Thus, in the case of in vitro cultivation the mechanocytes of bone marrow origin retain not only the capacity for bone formation but also the degree of organization necessary for the bone developed to take the form of a bone marrow organ, that is, to be identified by hemopoietic cells as suitable for repopulation. At the same time fibroblasts from spleen cultures on retransplantation into diffusion chambers form reticular tissue, and on regrafting under the kidney capsule form stromal tissue which is repopulated by lymphoid cells (Friedenstein et al., 1974a) (Fig. 3). Thus FCFC represent mechanocyte lines that are different in different hemopoietic organs. FCFC serve as precursors of stromal mechanocytes responsible for transferring the microenvironment operative in hemopoietic organs. Bone marrow stromal cells, as is known (Danis, 1957), possess pronounced osteogenic potency, and therefore bone marrow FCFC simultaneously serve as osteogenic precursor cells. Consequently, in adult life hemopoietic organs contain a bank of clonogenic precursors of stromal mechanocytes with varying differentiation potency. In the bone marrow and in the thymus FCFC comprise a fraction of slowly proliferating cells (Keiliss-Borok et al., 1972). In 6- and 14-dayold guinea pigs after a 72-hour t h ~ m i d i n e - ~labeling H 15 and 2% of FCFC are labeled, respectively; in adult animals all FCFC remain nonlabeled and are not killed as a result of “thymidine suicide” in in-

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FIG. 3. Experimental scheme for regrafting fibroblasts from bone marrow and spleen cultures (Friedenstein e t al., 1974a).

cubation with thymidine-H3 of high specific activity. In this respect they are distinctly different from precursors of macrophages, which comprise a fraction of rapidly proliferating cells (Valkman and Gowans, 1965; Van Furth and Cohn, 1968). FCFC are extremely adhesive cells. In the absence of serum, adhesion of 90% of FCFC from bone marrow and thymus cell suspensions takes 90 minutes, the majority of them adhering to the glass within the first 30 minutes (Friedenstein, 1973). Radiosensitivity of bone marrow and spleen FCFC in guinea pigs was determined by the suppression of colony formation after the irradiation of cellular suspensions (Friedenstein, 1973; Friedenstein et al., 1974a). It is characterized by a Do of 178 r and by an n of 1.4. FCFC of human bone marrow exhibit similar radiosensitivity. Radiosensitivity of mouse bone marrow FCFC has a Do of about 220 r and an n of about 1.4 (Friedenstein et al., 1976a). The same Do value was obtained by Metcalf (1972) for FCFC from the pleural cavities of mice. It is characteristic of clonogenic precursors of stromal mechanocytes that some of them are capable of surviving even after radiation doses as high as 2000 and 6000 r. The regeneration pattern of bone marrow FCFC after whole-body sublethal radiation (Friedenstein et al., 1976a) has a striking similarity

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to the recovery pattern of hemopoietic stem cells, established by Lajtha and Schofield (1969). For example, the initial loss resulting directly from irradiation of mice with 150 r is approximately 70% of control values for hemopoietic stem cells and 40% for FCFC. In each instance the recovery pattern is characterized by a marked secondary loss occurring over the 24 hours after esposure, and by a long postirradiation lag and dip lasting 4-6 days. It is followed by a return to normal values by about the fifteenth day. Bone marrow FCFC belong to stromal cells and are histogenetically independent of hemopoietic stem cells (see Section V). Similar patterns of postradiation recovery of these two cell lines may indicate that both of them are regulated by the same mechanisms, or that these two categories of bone marrow precursors-stromal precursor cells and hemopoietic precursor cells-are in some way correlated. The FCFC content changes under stimulation of hemo- and lymphopoiesis. Thus 2 hours after bleeding the number of bone marrow FCFC increases several times, and the number of lymph node FCFC goes up sharply on immunization, that is, 30-fold after 24 hours and 40-fold after 7 days (Friedenstein et al., 197413). These changes come short of proving that stromal cells of lymph nodes recognize antigens or that bone marrow stromal cells are sensitive to hypoxia caused by bleeding or to erythropoietin. Reactions of stromal cells may be the result of their interactions with immunologically competent cells and with erythroid precursors. The shifts in the FCFC number (30-fold in 24 hours in lymph nodes and severalfold in 2 hours in bone marrow) show that the FCFC number in hemopoietic organs may increase either through additional recruitment of cells of local origin or as the result of an influx of FCFC from without. FCFC are present not only in hemopoietic tissue. They can be observed among peritoneal fluid cells (Luria et al., 1972; Friedenstein et al., 197413) and among cells from the pleural cavity (Metcalf, 1972; Friedenstein et al., 1974b). It was found (Luria e t al., 1972) that, 1 month after immunization with complete Freund’s adjuvant, the concentration of FCFC among peritoneal fluid cells increases eight times. Fibroblast colonies are formed in vitro only by precursors that possess sufficiently high proliferative capacities. Thus the formation of a 50-cell clone requires not less than 6 cell doublings, and a colony of 1000 cells not less than 10 cell doublings. Diploid strains of bone marrow fibroblasts of guinea pig, rabbit, or human origin survive up to 26 passages (Miskarova et al., 1970; Panasyuk et al., 1972), which corresponds to approximately 50 cell doublings,

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Cells of not every fibroblast colony arising on initial explantation are capable of prolonged self-maintenance. Large and small colonies are different in this respect. In 10- to 12-day cultures the highest labeling indices are found in large colonies containing not less than lo3 cells; cells of these colonies give rise to diploid strains on passaging. Thus FCFC capable of forming large colonies seem to have a higher proliferative potency than FCFC that form small colonies. Yet, whether or not it is possible to judge the proliferation potency of FCFC by colony size is open to discussion. The size of the colony may be influenced by many factors on which the fate of cells formed at each cell division may depend. Some of them may be of a stochastic character, and therefore identical colony-forming cells may produce nonidentical colonies. Hence the heterogeneity of fibroblast colonies in regard to size does not prove FCFC heterogeneity in proliferation potency. In connection with this there exists the possibilitythat all mechanocyte precursors possess similar proliferation potency, and that all of them can behave as FCFC on explantation in vitro. In this case there are no grounds for singling out from among them a special category of stem cells. Yet available data show that at least for one category of mechanocytes the compartment of precursor cells includes not one but at least two kinds of cells, only one of which has self-maintenance ability. This category of mechanocytes is represented by ectopic bone tissue cells. 111. Determined and Inducible Osteogenic Precursor Cells

Few mechanocyte populations can be divided into groups in which cells differ in differentiation level and proliferation activity. In this respect bone tissue is a suitable subject for study (Owen, 1972). Maturing mechanocytes, that is, osteoblasts and osteocytes, in this tissue are limited topographically and are in many ways different from osteoprogenitor cells (Young, 1962). Bone formation occurs in the skeleton throughout life, because of the activity of periostal and endostal cells. It provides for the growth of bones and their remodeling, in the course of which renewal of bone tissue mechanocytes takes place. Morphologically distinguishable stages of osteogenesis involve proliferation of osteoprogenitor cells, preosteoblasts, which display fibroblast morphology but are characterized by high alkaline phosphatase activity (Pritchard, 1956), and for their transformation into osteoblasts, the majority of which are not subject to further division. Finally, osteoblasts become transformed into osteocytes embedded in calcified bone matrix. The number of di-

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visions preosteoblasts undergo has not been established precisely; the duration of osteoblast presence on the bone surface prior to transformation into osteocytes is about 72 hours in a 2-week-old rabbit metaphysis (Owen, 1970). Bone formation may take place in postnatal life not only in the skeleton but also outside it (ectopically) in practically any organ (Bridges, 1959; Ostrowski and Wodarski, 1972). Ectopic bone is particularly readily induced under the influence of two osteogenic inducers, namely, transitional epithelium of the urinary tract (Maximov, 1907; Huggins, 1931; Friedenstein, 1968) and decalcified bone matrix (Urist, 1965). The very fact of bone induction in open cell systems, for example, subcutaneously or in muscles, does not testify to the transferral of initially nonosteogenic cells to the bone formation pathway; the inducer may provoke an influx of osteogenic precursor cells from the skeleton. Experiments with diffusion chambers allow this possibility to be excluded. In diffusion chambers bone may be induced in originally nonosteogenic cell populations obtained from spleen, thymus, peritoneal fluid, and blood (Friedenstein, 1968; Friedenstein and Lalykina, 1970). The most intensive bone formation is observed in chambers with thymus cells when transitional epithelium or decalcified bone matrix is added (Friedenstein and Lalykina, 1972) (Fig. 4). Each of the above inducible cell populations contains hemopoietic or lymphoid cells, as well as clonogenic precursors of mechanocytes which can be detected as FCFC by the in vitro colony assay method. It was shown that FCFC serve as a reacting system in osteogenic induction, that is, they are the inducible osteogenic precursor cells (IOPC). In fact (Friedenstein, 1973; Friedenstein and Lalykina, 1973), inducibility is lost after elimination of FCFC from a suspension of spleen and thymus cells by adhesion and, vice versa, csteogenesis is easily induced in fibroblast diploid strains arising from FCFC of spleen and thymus origin. Induced bone formation is preceded by an intensive proliferation of preosteoblasts (Friedenstein, 1968; Ioseliani, 1972), which then differentiate into osteoblasts and finally into osteocytes. A group of proliferating preosteoblasts and a group of osteoblasts and osteocytes are clearly discernible in ectopic bone and are practically the same as in skeletal bone tissue. Bone marrow is also present in fully developed ectopic bone (Friedenstein, 1968; Reddi and Huggins, 1975). Despite these similarities to skeletal bone tissue, induced bone differs from the former in one essential property, namely, the lack of self-maintenance in the absence of inducer (Friedenstein, 1968).

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FIG. 4. Bone formation in diffusion chambers with thymus cells. (a) Guinea pig thymus cells plus transitional epithelium cells (total preparation, alkaline phosphatase). (b) Rabbit thymus cells plus decalcified bone matrix.

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Bone induced by autotransplants of bladder mucosa is sustained for years, and the remodeling connected with osteogenesis continues. Bone induced by allogeneic transplants of bladder mucosa behaves differently. By the end of the third week allografted epithelium undergoes immunological resorption and the induced bone is also resorbed (Friedenstein, 1968). It should be noted that, by the time of epithelium resorption, the induced bone has a wide layer of osteoblasts and preosteoblasts belonging to recipient cells and consequently not subject to immunological resorption. Bone tissue induced by decalcified bone matrix exhibits similar behavior. It is gradually resorbed, as is the case with the implanted inducing material. The resorption time depends on the method of preparation and the shape of the implanted inducing material. In several cases induced bone has been sustained for over 2 years (Reddi and Huggins, 1975). It is natural to search for the reason for inducer dependence of ectopic bone tissue in the peculiarities of its precursor cells. However, bone marrow isolated from foci of induced osteogenesis is capable of forming new bone on transplantation into a diffusion chamber without addition of the inducer (Friedenstein et aZ., 1976b).Thus ectopic bone possesses inducer-independent osteogenic precursors. Yet their presence provides for only a short life-span of bone tissue; after several weeks only fragments of dead bone are found in the chambers, and 0steogenesis stops. Meanwhile, in chambers with bone marrow from skeletal bones, osteogenesis does not stop for many months. Differences between skeletal and induced bone tissue distinctly appear at the level of clonogenic precursor cells. The FCFC content of bone marrow of induced bones is the same as that of bone marrow of skeletal bones. Yet FCFC properties are not identical. FCFC of skeletal bone marrow are determined osteogenic precursor cells (DOPC) (Friedenstein, 1973); they give rise to fibroblast diploid strains which on retransplantation in vivo spontaneously undergo osteogenic differentiation. Even after 26 passages in vitro DOPC do not lose their osteogenic potency (Miskarova et aZ., 1970). FCFC of bone marrow from ectopic bones also give rise to fibroblast diploid strains in uitro. Yet even after the first passage these fibroblasts do not form bone on retransplantation in viuo unless the osteogenic inducer is added to them (Friedenstein et al., 197613). This shows that, as regards 0steogenic potency, FCFC of ectopic bones remain IOPC, as were the FCFC in inducible cell populations prior to the action of an inducer. Hence osteogenesis in ectopic bones is provided by two different kinds of precursor cells: (1)the clonogenic cells responsible for main-

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tenance of the mechanocyte cell population; the participation of these cells in osteogenesis is inducer-dependent; (2) the committed osteoprogenitor cells which are the inducer-independent descendants of clonogenic cells. In other words, a separate category of selfmaintained cells from which the immediate osteogenic precursors originate may be singled out in ectopic bone tissue and regarded as stem cells. There are reasons to believe that the osteogenic precursors present in skeletal bones, that is, DOPC and preosteoblasts, are also two different kinds of cells, although both of them are inducerindependent. One may speculate that the existence of bone tissue always requires continuous recruitment of immediate osteogenic precursors. Skeletal bones arising as a result of the action of inducers operating in embryogenesis possess a line of DOPC capable of recruiting immediate osteogenic precursors without additional inductive stimuli. Bone induction in postnatal life is obviously different from embryonal induction in that the latter occurs at the level of self-maintained precursors and therefore leads to the emergence of an osteogenic cell line maintained throughout life, while the former occurs at the level of osteoprogenitor cells with limited proliferative capacity and amounts to recruitment of immediate osteogenic precursors from IOPC or to their storage, but not to the appearence of new DOPC. Therefore ectopic bone can exist only as long as the inducer is present. The formation of additional amounts of skeletal bone tissue often takes place in postnatal life, for example, during callus formation in fracture repair. Whether it is provided by immediate osteogenic precursors alone, or whether DOPC (or IOPC) are also involved, is not known. Only one case has been studied from this angle so far, namely, bone formation in the bone marrow cavity after curettage. It was found that after 11 days the number of FCFC in curettaged mouse femur (Wilson e t al., 1974) increases fivefold, and in distant bone marrow parts, fourfold. This observation is of particular interest, since after mechanical removal of the femoral bone marrow no stimulation of proliferation of cytokinetically resting stromal cells in distant bone marrow parts is observed (Meyer-Hamme et al., 1971).

IV. Presence of Mechanocyte Precursors in Blood Whether or not precursor cells for mechanocytes circulate in blood is a moot question. Facts indicating such a possibility have been known for a long time.

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They were obtained by the first workers who dealt with in vitro explantation of blood cells (Carrel and Ebeling, 1922; Timofejewski and Benevolenskaja, 1926). A more complete description of fibroblast development in plasma clot cultures of cells with a buffy coat was given by Maximov (1927/1928). Later his observations were confirmed by many investigators (for reference, see Luria, 1972). It has been shown that fibroblasts in human placental blood cultures synthesize collagen (Malek et al., 1973) and that fibroblast strains capable of prolonged passaging can be obtained from human peripheral blood (Paul, 1958). It is thus beyond doubt that cells from the blood may yield fibroblasts in vitro or in diffusion chambers (Stirling and Kakkar, 1969). Until recently, however, it was assumed that these facts are not proof of the circulation of precursor cells of mechanocytes but result from stray fibroblasts introduced during blood sampling. Actually, walls of blood vessels are always traumatized during blood sampling. Since connective tissue is their necessary component, it is easy to presume that connective tissue fibroblasts are present in the material under analysis and thus simulate the presence of precursor cells of fibroblasts among circulating blood cells (Ross and Lillywhite, 1965; Kalus et al., 1968). At present, however, results allowing the exclusion of such a possibility have been obtained. By the in vitro colony assay method one can determine the number of clonogenic precursors of fibroblasts present in an analyzed cell suspension. If equal volumes of blood are taken from a donor in such a way that severe trauma is inflicted on a blood vessel wall (multiple punctures) in the first case and less severe trauma (a single puncture) in the second case, the number of FCFC in the first sample must be greater than in the second if their presence is due to blood contamination. Yet tests did not confirm this assumption. In the cultivation of guinea pig blood the number of fibroblast colonies did not depend on the number of punctures (Luriaet al., 1971).This result shows that the presence of precursors of fibroblasts is not due to blood contamination, but that they actually circulate in blood. In guinea pigs one fibroblast clone is formed per lo5 leukocytes, that is, at least one FCFC is present. Approximately the same FCFC concentration is found in rabbit blood. For human leukocytes corresponding data are not available, since stable fibroblast colony formation on explantation into monolayer cultures has not been achieved. The morphological nature of FCFC and the organ from which they enter the blood are unknown. It is worthwhile mentioning here the observation that, in cell cultures from arterial and venous blood, fibroblasts grow in different numbers. This is obviously connected with

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peculiarities of FCFC circulation and not with differences in the structure of vascular walls. What happens to fibroblast precursors is also far from clear; they may reside in the tissues and participate in the formation of mechanocytes, or die in the bloodstream or in a definite organ. As a result of this ambiguity the circulation of FCFC in blood cannot serve as evidence of the exchange of mechanocyte precursors between tissues and organs. Yet it makes the very possibility of it accessible to study.

V. Interrelationship between Mechanocyte Precursors and Hemopoietic Cells The possibility of transformation of lymphocytes, monocytes, and macrophages into fibroblasts and other mechanocytes has been discussed more than once. Since all the above-mentioned cell types have been shown to be descendants of hemopoietic stem cells (for reference, see Metcalf and Moore, 1971), the question consequently is reduced to whether hemopoietic stem cells or their progeny serve as precursors of mechanocytes. This problem has been studied rather thoroughly as far as stromal mechanocytes of hemopoietic and lymphoid organs are concerned. Stromal mechanocytes are most closely associated with hemopoietic cells, and it is for hemopoietic and lymphoid tissues that this situation can be easily created when parenchyma cells carry a marker different from that of stroma cells. When fragments of bone marrow, spleen, thymus, or lymph node are transplanted heterotopically (i,e., subcutaneously, under the renal capsule or into the anterior eye chamber), the growth of stromal tissue is observed first, and then the graft becomes repopulated by hemopoietic or lymphoid cells (Danis, 1957; Dukor et al., 1965).As a result, a small organ similar to the parent one is formed at a new site. These successive changes are clearly seen in the transplantation of bone marrow fragments under the kidney capsule. Transplantation may be repeated with the extraction of bone marrow from the established heterotopic transplant and regrafting to a new recipient. Osteogenic tissue is formed at a new site after each passage, and then the graft is repopulated by hemopoietic cells. When a transplantation series begins with grafting of bone marrow derived from a whole femur of a mouse and passages are performed every 1.5-3 months, four successful graftings are possible, after which neither bone tissue nor bone marrow is formed at the site of regrafting (Friedenstein and Kuralesova, 1971) (Fig, 5).Thymus, spleen, and lymph nodes are sustained for two to three passages.

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I1

Ill

FIG.5. Experimental scheme for demonstrating the persistence of donor stromal cells in bone marrow heterotopic transplants. (I) Syngeneic serial grafting. (11) Semisyngeneic transplantation with regrafting into initial donor line. (111) Transplantation from male donor with regrafting into female immunized against male transplantation antigens.

In order that the transplantation be successful, it is necessary that the donor tissue be immunologically compatible with the recipient; it

is successful in syngeneic and semisyngeneic transplantation, but not in allogeneic transplantation. As compared to transplants of other tissues, heterotopic grafts of hemopoietic and lymphoid organs have an important peculiarity. It was found that established heterotopic transplants of bone marrow, spleen, lymph node, and thymus are composed of recipient cells but not of those of the donor. This applies to both hemopoietic and lymphoid cells (hemopoietic stem cells included), and macrophages (Green, 1964; Dukor et al., 1965; Friedenstein et al., 1968; 1976c; Didukh and Friedenstein, 1970). What actually happens when fragments of hemopoietic organs are grafted is a transfer of their typical microenvironment. This microenvironment is recognized by hemopoietic cells of the recipient, and they populate the graft. However, if heterotopic transplantation is carried out semisyngeneically (with a parent line donor and an F1hybrid recipient), retransplantation into the line of the initial donor proves successful (Friedenstein et al., 1968), although F, cells cannot survive in the initial donor

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line. Thus successful heterotopic transplantation is provided not b y hemopoietic cells but by other cells remaining unreplaced b y recipient cells in established semisyngeneic grafts (Fig. 5). The cells responsible for transferring the microenvironment of hemopoietic tissues are stromal mechanocytes. This follows from the results of retransplantation in vivo of fibroblasts from bone marrow and spleen cultures (Friedenstein et al., 1974a). Accordingly, stromal cells including clonogenic stromal precursors are the only cells of donor origin retained in established heterotopic bone marrow transplants, as has been proved by antigenic and chromosome cell markers (Friedenstein e t al., 1968, 1 9 7 6 ~ ) . Thus in heterotopic transplants tissue chimerism arises. This phenomenon is of particular interest in analysis of the interrelationship between mechanocytes and hemopoietic cells. Serial grafting of hemopoietic or lymphoid tissue causes intensive proliferation of stromal mechanocytes to occur on each regrafting. It was found that regraftings are successful only as long as the line of the stromal cells of the initial donor can sustain itself, in a new recipient (Friedenstein and Kuralesova, 1971). Accordingly, in experiments with bone marrow retransplantation, when the final recipient (female immunized against male antigens) was chosen so that the only immunologically compatible cells were cells of the intermediate recipient (female), while cells of initial donor (male) were incompatible, retransplantation was found to be unsuccessful (Friedenstein and Kuralesova, 1971) (Fig. 5). Consequently, not only are stromal mechanocytes not completely replaced, but they are not replenished by recipient hemopoietic cells at all. No transformation of hemopoietic cells into stromal mechanocytes has been observed in complete bone marrow radiochimeras either. Stromal mechanocytes even of long-term radiochimeras are not replenished by donor cells (Friedenstein and Kuralesova, 1971), in contrast to macrophages and hemopoietic and lymphoid cells. As an example, bone marrow mechanocytes may be considered. During the first period after irradiation bone marrow of radiochimeras cannot be successfully transplanted heterotopically. However, by the end of the first month transplantability is restored, but only if the grafts are made into the recipient line and never into the donor line (Friedenstein and Kuralesova, 1971). Correspondingly, FCFC from bone marrow of radiochimeras are always recipient cells (Friedenstein e t al., 1976c) (Fig. 6). Thus hemopoietic cells and stromal mechanocytes behave as two histogenetically independent cell lines both in radiochimeras and in heterotopic transplants.

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FIG.6. Experimental scheme for demonstrating the persistence of recipient stromal cells in bone marrow of radiochimeras (Friedenstein and Kuralesova, 1971).

Cited results seem to solve the problem of the possibility of stromal mechanocyte recruitment from both hemopoietic cells and from macrophages negatively. These results confirm the heterogeneity of cells within the reticular endothelial system (Van Furth, 1972) or, at any rate, deny the existence of a single category of precursor cells for this system in postnatal life. As for other mechanocytes, the majority of the results obtained with cell markers show that they are also histogenetically independent of hemopoietic cells. It was shown by Hellstrom et al., (1970) that connective tissue fibroblasts in canine radiochimeras are of recipient origin. These results are in agreement with data on cell identification in sarcomata induced in radiochimeras by implantation of Cellophane (Moyzhess and Prigozhina, 1972) or by introduction of strontium-90 (Barnes, 1971). The same results were obtained in investigations in mice parabionts; in wound healing fibroblasts regenerate without participation of the partner cells (Ross et al., 1970), and sarcomata induced by implantation of polyvinyl chloride plates arise from local and not from circulating cells (Moyzhess, 1975).PH chromosomes found in hemopoietic cells of patients with chronic myeloleukosis may serve as another marker. Macrophages in the patients’ blood are also labeled with PH chromosomes, Yet, attempts to find labeled fibroblasts proved unsuccessful; in all cases in which precise identification of cell types was carried out fibroblasts had no PH chromosomes (Maniatis et al., 1969; de la Chapelle et al., 1973).

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However, it was reported (Barnes and Khrushchov, 1968) that in murine radiochimeras the dividing cells (fibroblasts?) attached to the glass on the fifth to seventh days after the onset of sterile inflammation were of donor origin. At this time mainly macrophages and lymphocytes are found in the region of aseptic inflammation. That they are of donor origin in radiochimeras is beyond doubt (Valkman and Gowans, 1965). In the article of Barnes and Khrushchov (1968)the difficulty lay in histological identification of the cells carrying a chromosome marker, in the determination of whether or not they were fibroblasts. According to Barnes (1971), these investigators failed to succeed, and thus the article cannot be considered convincing as regards the determination of precursor of fibroblasts. All this shows that in postnatal life precursors common to mechanocytes and to hemopoietic and lymphoid cells either do not exist at all or are not utilized in the renewal of mechanocytes of hemopoietic, connective, and osteogenic tissue. As for precursors of mechanocytes circulating in the blood, no direct data on their histogenetic relations have been obtained so far. It is more probable that they are also not connected with hemopoietic cells. Yet the problem can be solved only by direct experimental analysis.

VI. Diploid Strains of Fibroblasts Diploid strains (Hayflick and Moorhead, 1961) may serve as valuable subjects for the analysis of precursor cells, thanks to the longterm proliferation of fibroblasts in vitro and an enormous increase in their number. The size of the fraction of proliferating cells in cultures of diploid strains may vary considerably. This is evidenced b y the well-known phenomenon of density-dependent inhibition (Stoker and Rubin, 1967). The proliferative pool in confluent cultures grows smaller, and finally cell division stops completely. Fibroblast proliferation also stops in preconfluent cultures when specific factors necessary for growth are excluded from culture media. This state is, however, reversible. On passaging or removal of part of the cell layer (wound cultures) (Todaro et al., 1965), or on addition of serum, dormant fibroblasts of stationary cultures enter the S period of the cell cycle. On serum stimulation the percentage of cells entering the S period in stationary cultures may rise to 100%for 3T3 cells and 81%for embryonal fibroblasts (Rudland et al., 1974). Thus most of the fibroblasts even in stationary cultures, though arrested at a specific point (evidently in

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the Go phase of the cell cycle) (Seifert and Rudland, 1974), are nevertheless ready to enter the proliferation stage. During the phase of logarithmic growth fibroblasts have been shown to synthesize procollagen, which is secreted into the medium (Malek et aZ., 1967). Collagen fibers are formed in vitro at postconfluence cell densities when several layers of cells are present in cultures (Goldberg and Green, 1964). However, complete differentiation of mechanocytes in diploid fibroblast cultures does not occur. Cells of bone marrow fibroblast strains, for example, have been shown (Friedenstein et d., 1970) to differentiate into osteoblasts and osteocytes after reverse transplantation in vivo, but when in culture they behave as proliferating fibroblasts. Fibroblast diploid strains may be thus considered mechanocyte precursor cell populations, since they are composed of proliferating cells and since new mechanocytes are formed as a result of their cell division. Final differentiation of fibroblasts in these in vitro cell populations is, however, inhibited. The main factor limiting fibroblast multiplication in dense cultures is the availability of medium ingredients rather than limited surface to which cells may attach (fibroblasts are capable of forming several cell layers in cultures) (Dulbecco and Elkington, 1973). Because of physicochemical conditions at the interface of a liquid phase and the cell surface, a diffusion boundary layer is created. In this layer the concentration of substances actively utilized or secreted by cells may differ manyfold from that of the remaining medium. Experiments with recirculation of the medium in cultures (Stoker, 1973), as well as with changes in the medium (Wiebel and Baserga, 1968), showed that at excessive cell density some substances stimulating the entry of fibroblasts into the S period become inaccessible to them. The list of agents stimulating proliferation of fibroblasts in vitro is fairly extensive. It includes macromolecular serum factors (Todaro et al., 1965; Eagle and Levine, 1967; Vasiliev et al., 1969), aphetoprotein (Vaheri et d., 1973), a series of low-molecular-weight substances in the culture medium (Holley and Kiernan, 1974), enzymes (Selton and Rubin, 1970) and, in particular, thrombin of plasma (Chen and Buchman, 1975), insulin and polypeptide hormone purified from bovine brain and pituitary glands, glucocorticoids (Gospodarovicz and Moran, 1974), substances inflicting damage on microtubular cell structures (Vasiliev et al., 1971), and such metal ions as Zn2+,Cd2+,Hg2+ (Rubin, 1975), and Ca2+(Dulbecco and Elkington 1975). These factors differ in intensity, fresh serum being the most active stimulus. Some factors, such as hormone of pituitary gland and insulin, evidently affect the cellular surface; others, such as low-

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molecular-weight components in the medium, act inside the cells. Stimulation of fibroblast proliferation is accompanied by changes in the ratio of 3’,5’-cyclic AMP to 3’,5’-cyclic GMP, which may serve as an intracellular signal for a transition from the Go or GI period to the S period (Siefert and Rudland, 1974; Moens et al., 1975). Which of the above factors participates in regulating mechanocyte proliferation in vivo is not yet clear. Although almost all the fibroblasts in diploid strains retain the ability to synthesize DNA, they evidently differ in proliferative potency. Clonogenic properties of separate cells obtained from the culture are far from identical; on passaging not all cells form colonies, and the latter differ in size. On the cloning of separate cells from Wi-38 and Wi-26 diploid strain cultures (Smith and Hayflick, 1974), about 85%of the cells proved capable of one or more division cycles. Fifty percent of them exhibit a level of population doubling no higher than 8, that is, they form colonies of 100 or fewer cells, and only about 20% possess a level of population doubling higher than 10 (up to 40 to 60, depending on the age of the culture). The distribution curve of the number of clonogenic cells with different levels of population doubling shows that cells with a population doubling level higher than 8 to 10 (high) on the one hand, and from 1 to 8 (low) on the other, represent two different subpopulations. It is between these two categories of clonogenic cells that a sharp break is observed on the distribution curve. With aging of the strain the number of cells with a high level of population doubling goes down. At the same time the number of cells incapable of division increases (Cristafalo and Sharf, 1973). The plating efficiency of cells from fibroblast colonies in 12-day-old bone marrow cultures is 20%on the average, the value varying over a wide range for individual colonies. The remaining cells are able to form clusters of less than 50 cells (the conventional value of a cell cluster considered a colony) (Friedenstein et al., 1974b). Thus both clonogenic and nonclonogenic cells are recruited from among the progeny of initially explanted clonogenic cells (FCFC). It has been shown that the first nonclonogenic cells appear in colonies only after several cell doublings. There is no direct evidence, however, that cells that lose clonogenic properties cannot acquire them anew, that is, FCFC + nonclonogenic cell transformation is irreversible. This does not allow one at present to assert the existence of a special line of clonogenic stem cells from which nonclonogenic cells with limited proliferative potency are recruited in vitro, although this interpretation seems most attractive. It would be interesting to determine the

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factors affecting the ratio of clonogenic to nonclonogenic cells in diploid cell strains. There are data (Smith and Hayflick, 1974) indicating that the biological age of a culture is one of them. It may be suggested that it is associated with the gradual accumulation of oxidative damage occurring during proliferation in culture, since tocopherol confers on Wi-38 cells a long-term ability to proliferate in vitro (Pecker and Smith, 1974). Questions concerning the few nondividing cells found in diploid strains even after serum stimulation remain unanswered. The extent to which they represent mature, completely differentiated fibroblasts has not been determined. VII. Concluding Remarks Among the cells of connective tissue, precursors of mechanocytes capable of protracted self-maintenance can be identified by the in vitro colony assay method. They behave as clonogenic cells and give rise to diploid strains of fibroblasts capable of sustained passaging. In cases in which reverse grafting in vivo was carried out, it was found that the differentiation features characteristic of mechanocytes of the source tissue are consistently retained by in vitro descendants of clonogenic cells. Thus self-maintained precursors determined to differentiate into definite types of mechanocytes are present in postnatal life. The question arises as to the degree of heterogeneity of clonogenic mechanocyte precursors within the tissue limits, as far as differentiation ability goes. It concerns, in particular, the problem of collagen types synthesized by separate clones. Collagen contains three polypeptide chains per molecule. During collagen synthesis the triple-helical precursor of collagen, procollagen, is produced. Procollagen is composed of p r o 4 1 and p r o 4 2 chains, which are ribosomal products (Kerwar et al., 1972) containing an additional peptide at the amino terminal of their respective chains (Dehm et aZ., 1972). The amino terminal extensions of the precursor chains are excised by the specific enzyme procollagen peptidase to yield mature collagen chains (Lapiere et al., 1971). Recent studies have established the existence of several chemically distinct collagens. Type I collagen has the chain composition [ a I ( I ) l ~ 2and seems to be the only collagen to occur in mature bone (Miller, 1973). In cartilage type I1 collagen, [a1(11)I3,predominates (Miller and Matukas, 1969). In the skin two different types of collagen are synthesized: type I (predominantly) and type I11 with the chain composition [aI(III)], (Church et al., 1973; Penttinen et al., 1975). In addition, type

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IV collagen, [cYI(IV)]~, has been described in basement membrane from renal glomerulus and lens capsule (Kefalides, 1972). It is not clear whether the type of collagen may be considered a clonal property when several collagen types are produced in a given tissue, yet data on collagen synthesis in lung tissue cultures (Bradley et al., 1974) show that this is quite possible. One may expect that clonal differences may also be found for peptide extensions of procollagen molecules. For clones of mechanocytes from hemopoietic organs a distinguishing property may be the type of inductive microenvironment they create. Beside interorganic differences in the microenvironment, intraorganic ones are obviously present. This is evidenced by the persistence of thymus-dependent and thymus independent zones in lymphoid organs and of regions in the spleen where erythroid colonies occur more frequently than myeloid ones (Metcalf and Moore, 1971). One may expect that intraorganic differences in the microenvironment are due to mosaicism of stromal mechanocytes. However, no data are available indicating whether differences among separate clones of stromal mechanocytes exist, to say nothing of the character of these differences. In addition to determined precursors inducible mechanocyte precursors are also found in adult mammals. For example, there are two kinds of precursors of osteogenic cells: determined and inducible. In order to realize their osteogenic potency determined precursors do not require additional inductive stimuli. Inducible osteogenic precursors do not exhibit osteogenic potency unless they are subjected to the action of local inducers of osteogenesis. No new determined selfmaintained osteogenic precursors are formed on induction; the inducer provides only for recruitment (or for storage) of immediate osteoprogenitor cells with a limited ability to proliferate. Therefore ectopic bone represents a temporary structure. It is sustained only as long as the action of the inducer is in effect. Clonogenic precursors from different organs, the spleen being among them, may serve as the cells in which osteogenesis is induced. However, clonogenic cells of spleen origin include committed precursors of spleen stromal mechanocytes; their in uitro descendants form spleen stromal tissue on regrafting in uiuo. Whether every clone of spleen mechanocytes is susceptible to induced osteogenesis is not known. Thus either the committed precursors of spleen stromal mechanocytes are simultaneously inducible osteogenic precursors, or the determined and the inducible precursors comprise different categories of cells localized in one organ.

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The existence of both determined and inducible precursors is hardly confined to only one type of tissue composed of mechanocytes, namely, bone. Ectopic formation is also known for several other tissues, and thus the presence of both determined and inducible precursors may also be suggested. In particular, stromal tissue of lymphoid and hemopoietic organs are involved. There are various types of heterotopic development of hemopoietic tissue, such as myeloid transformation of the lymph nodes and additional lymph node formation. Since the sites of proliferation and differentiation of hemopoietic and lymphoid cells are assigned by strbmal mechanocytes responsible for the necessary microenvironment, some of these types may be a result of the heterotopic development of stromal tissue mechanocytes, connected either with the influx of determined stromal precursors into unaccustomed sites or with the formation of new stromal tissue at the expense of inducible precursors, as is the case with ectopic osteogenesis. The complete spectrum of self-maintained precursors of mechanocytes operating in postnatal life is thus far from being established. It remains to be learned how many separate categories of determined precursors are present in the body; whether inducible precursors are specific cells or whether precursors committed to a definite differentiation pathway can operate as inducible precursors of another; whether common progenitors of all types of mechanocytes are present in postnatal life, where they are localized and whether they are capable of recruiting not only immediate committed precursors (as is the case with bone induction) but new determined self-maintained precursors as well. The actual exchange of mechanocyte precursors between organs and tissues has not been shown so far. On the contrary, there are certain data indicating its absence. In fact, in t h ~ m i d i n e - ~cell H labeling in a mice parabiont (Ross et al., 1970), it was shown that in the healing wound of a second partner all fibroblasts remained unlabeled. Thus, in fibroblast formation, the participation of migrating cells in their intensive regeneration has not been established. The same is true of stromal reticular cells of hemopoietic organs and of 0s teogenic cells. On heterotopic transplantation of tissue fragments of hemopoietic organs, stromal and osteogenic mechanocytes are formed only from precursors transferred from donor cells without par1968; Frieticipation of any of the recipient cells (Friedenstein et d., denstein and Kuralesova, 1971). Meanwhile, the circulation of precursors of mechanocytes in blood has received direct confirmation (Luria et al., 1971). It is not the result of contamination of blood by fibroblasts of the blood vessel wall

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during blood sampling as was sometimes assumed, but evidence of the presence of clonogenic fibroblast precursors among nucleated blood cells. Where they enter the blood and where they migrate to in the blood remains unknown. It is not excluded that natural contamination, that is, chance penetration through capillaries of several organs of cells which then simply die, is involved. However, this is not the only possibility. Contemporary methods fail to demonstrate the histogenically common nature of mechanocytes and hemopoietic cells. On the contrary, the results obtained rather indicate its absence. Precursors of mechanocytes are self-maintained independently of hemopoietic cells throughout the life-span of an organism and are not replenished at their expense. Despite an assumption made more than once in descriptive histology, neither lymphocytes nor macrophages, which are both descendants of hemopoietic stem cells, are transformed into mechanocytes. The presence of special stem cells among mechanocyte precursors remains disputable, in spite of the existence of clonogenic cells. These cells with high self-maintenance ability seem to be the first to be suspected of being stem cells. Yet there must be some certainty that clonogenic cells really differ from other proliferative mechanocytes and that they serve as the necessary starters in mechanocyte renewal. Thus far stem cell lines with such properties seem to b e proved only for ectopic bone tissue. Clonogenic precursors are the cells responsible for self-maintenance of ectopic bone and are different from immediate osteogenic precursors; the generation of ectopic bone cells occurs as the result of constant recruitment of osteoprogenitor cells from clonogenic precursors. But whether mechanocytes in other tissues are also supplied with stem cells remains unproved although quite possible. Stem cell existence has been established unambiguously for tissues with extensive cell renewal. Mechanocytes, on the contrary, have a very slow renewal rate. During the life-span the total number of cell divisions mechanocyte precursors undergo is considerably smaller than that of hemopoietic or lymphoid precursor cells. Therefore it is not excluded that structurally the population of progenitors of mechanocytes will appear fundamentally different as compared to the population of hemopoietic cells. REFERENCES Barnes, D. W. (1971).Nature (London) 233,267. Barnes, D. W. H., and Khrushchov, N. G . (1968). Nature (London) 218,599.

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