Differentiation, Dedifferentiation, and Transdifferentiation of BALB/c 3T3 T Mesenchymal Stem Cells: Potential Significance in Metaplasia and Neoplasia

Rodney L. Sparks, Elizabeth I. Seibel-Ross, Marjorie L. Wier, et al. Cancer Res 1986;46:5312-5319.

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[CANCER RESEARCH 46, 5312-5319, October 1986]

Differentiation, Dedifferentiation, and Transdifferentiation of BALB/c 3T3 T Mesenchymal Stem Cells: Potential Significance in Metaplasia and Neoplasia1
Rodney L. Sparks, Elizabeth I. Seibel-Ross, Marjorie L. Wier, and Robert E. Scott2
Section of Experimental Pathology, Departments of Pathology and Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905

process by which normal cells develop variations in cell size, shape, proliferation, and differentiation. The expression of defects in the control of cellular differentiation is The underlying mechanisms by which the expression of de thought to be of etiological significance in the early stages of carcinogen- fects in the control of differentiation could be associated with esis. This possibility is supported by a variety of experimental studies the development of metaplastic, dysplastic, preneoplastic, and including those that have established that metaplastic changes in cells neoplastic states are not clear. The hypothesis we favor suggests can represent preneoplastic lesions in vivo. To evaluate this question in that in normal stem cells the integrated control of cellular greater detail, we have used 3T3 T mesenchymal stem cells as a model differentiation and proliferation is stringently regulated, system. These cells express certain characteristics of preneoplastic cells even though they can regulate their proliferation and even though they whereas in metaplastic, dysplastic, preneoplastic, and neoplas can undergo nonterminaland terminal differentiation into adipocytes. For tic cells, these stringent regulatory mechanisms are partially or example, they are immortal and aneuploid, and they show a proclivity to completely abrogated. As a result such cells would be expected undergo spontaneous or induced neoplastic transformation compared to to show aberrant differentiation and/or proliferation (4, 7, 9normal human cells. The question we sought to answer in the current 22). In this regard, the term "stringent integrated control" experiments concerns whether predifferentiation growth arrest and/or describes the ability of stem cells or progenitor cells to make nonterminal differentiation in such preneoplastic cells is completely specific "decisions" that result in the expression of a specific reversible or whether these processes induce the expression of the new set of genes that stably programs the cells' subsequent prolif ABSTRACT eration and differentiation potentialities. A major focus of our experimental studies on carcinogenesis has therefore been directed at attempts to establish the biolog ical and molecular mechanisms that control cellular differentia tion in normal, preneoplastic, and cancerous cells. One diffi results show that, when nonterminally differentiated 3T3 T adipocytes culty with such an approach is to identify a model cell system are induced to dedifferentiate, they can subsequently redifferentiate into with which to study mechanisms for the regulation of cellular macrophages. We therefore suggest that preneoplasia as expressed in differentiation and its integral relationship to the control of 3T3 T mesenchymal stem cells is associated with the expression of cellular proliferation. In this regard, many cell systems have defects in the ability to integrally control cellular differentiation and proliferation. As a result, the data suggest that such cells express an been used in the past, but none has proven to be ideal. The reason for this is that the control of growth and differentiation increased proclivity to undergo metaplastic change and complete neo in many model systems is regulated by highly variable or poorly plastic transformation. defined regulatory mechanisms. For example, in some differ entiation systems specific physiological polypeptides are re quired to induce differentiation (7, 10, 20, 23-30), whereas in INTRODUCTION others they are not (8, 11, 12, 21, 22, 31-43). In some cells Carcinogenesis is a multistep process that has been reported DNA synthesis is required before expression of the differen to develop in association with the expression of defects in the tiated phenotype (11, 22, 27, 33), whereas in other cells it is control of both cellular proliferation and differentiation (1-8). not (26, 29, 31, 38-40, 42). Furthermore, in some cell types Recent studies furthermore suggest that the expression of de part of the differentiation program is reversible (23, 25, 26, 29, fects in the control of differentiation may occur in the early 32, 33, 35, 40), whereas in others it appears to be irreversible stages of carcinogenesis. For example, it has recently been (8,31,36,42,43). shown that UV irradiation, at dosages that initiate carcinogen By contrast, normal stem cells and/or progenitor cells appear esis, induces stable and heritable defects in stem cell differen to possess stringent regulatory mechanisms that integrate the tiation (7), and it has been demonstrated that an initiating control of cellular proliferation and differentiation. This con dosage of carcinogen can induce specific defects in the ability clusion is primarily based on in vitro studies using cells such as of mouse epidermal cells to terminally differentiate (8). The normal diploid human epidermal cells and hemopoietic stem fact that many metaplastic and dysplastic disease states that cells (1, 43-46). The expression of such stringent mechanisms are associated with aberrant differentiation represent preneo to integrally regulate cellular differentiation and proliferation plastic lesions also supports this conclusion. In this regard, correlates very well with the fact that normal cells, especially metaplasia is the process in which a normal stem cell changes those derived from humans and hamsters, show an extremely its pathway of differentiation so that one adult cell type is low frequency of spontaneous or carcinogen-induced neoplastic replaced by a different adult cell type, and dysplasia is the transformation (4, 5, 19, 45, 46). The goal of our current studies was to determine whether or Received 11/19/85; revised 7/7/86; accepted 7/9/86. The costs of publication of this article were defrayed in part by the payment not preneoplastic BALB/c 3T3 T mesenchymal stem cells can of page charges. This article must therefore be hereby marked advertisement in be demonstrated to possess defects in their regulatory mecha accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grant CA 28240andby the Mayo Foundation to R. E. S. nisms that integrally control cellular differentiation and prolif R. L. S. was partially funded by National Cancer Institute Training Grant CA eration. That is, in these studies we specifically evaluated 09441. whether the induction of differentiation activates a program 2To whom requests for reprints should be addressed, at 51 IB Guggenheim that limits cell proliferation and also limits the differentiation Building, Mayo Clinic, Rochester, MN 55905. 5312
that which is thought to occur in normal stem cells. The results show that arrest at both the predifferentiation state and at the nonterminal differentiation state is a completely reversible phenomenon that does not limit the cells1 subsequent growth or differentiation potential. In fact, the stable program that limits the cells' proliferativi' potential and reduces the cells' subsequent differentiation potential in a manner comparable to

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TD

Fig. 1. Illustration of the stages of adipocyte differentiation of 3T3 T mesenchymal stem cells that are regulated during the G, phase of the cell cycle. Each step is a prerequisite for the next step to occur, and once a cell has achieved the properties of a specific state, it is competent to respond to the signal(s) that induces progression to the next state. First, cells growth arrest at a specific state in the G, phase of the cell cycle, designated GD(Arrow 1). Such cells can remain at the GD state, or they can reinitiate proliferation (large Arrow 5), or differentiate. If differentiation is induced, two steps are involved. Cells first undergo nonter minal differentiation (Arrow 2) to the GD state. At this state they are fully morphologically differentiated. These cells can remain nonterminally differen tiated or they can terminally differentiate (Arrow 3). In addition, cells at the GD state can reinitiate proliferation with (Arrows 4 and 5) or without (small Arrow 5) first losing the differentiated phenotype. (See Fig. 2 which illustrates the mor phology of each state.) TD, terminally differentiated state.

ensure that cultures were free of Mycoplasma contamination, the cells were periodically assayed by the method of Chen (51). New stock cultures were thawed from liquid nitrogen every 2 to 3 mo. Preparation of Plasma and Plasma Fractions That Induce Adipocyte Differentiation. The preparation of human plasma and plasma fractions that were used to induce differentiation in these cells has been described in detail elsewhere (26, 29, 30). Briefly, citrate-anticoagulated plateletpoor human plasma was derived from venous blood of normal donors. This fluid was used in its native state following further processing. Fractionation of human plasma was carried out according to a modifi cation of the technique described by Mann (52). More specifically, liai .was added to citrate-anticoagulated human plasma to a final concentration of 0.1 M, and this solution was then adjusted to pH 8.6. It was stirred for 30 min and then sedimented by centrifugation at 3600 x g for 25 min. The supernatant was removed, and the barium citrate sediment, designated the cake, was then partially solubilized by the addition of 0.9% NaCl solution: 0.02 M sodium citrate to bring the volume to one-third of the original volume of plasma that was present when the BaCI2 was added. This suspension was stirred for 45 min and then subjected to centrifugation at 3600 x g for 25 min. The supernatant was saved and was designated CEPH (26, 29, 30). This plasma fraction was typically depleted of components that induce the terminal phase of differentiation but did contain those factors that induce GD arrest and nonterminal differentiation (see Fig. 2). In some preparations, however, CEPH had to be further processed by heparin agarose affinity chromatography or other procedures to derive a CEPH fraction that was nearly devoid of terminal differentiation-inducing factors. To all such plasma preparations, heparin at a final concentration of 30 units/ml was added as an anticoagulant. Biotin was also added at a final concen tration of 1 x IO"5 M.

potential of these stem cells as it would in normal stem cells. In order to explain the experiments that were performed as part of this study, it is essential to understand the regulatory mechanisms that have been documented to control the differ entiation and proliferation of 3T3 T stem cells (15, 26, 29, 47, 48). A model of these cell cycle-dependent regulatory processes is illustrated and fully explained in Fig. 1, and the morpholog ical characteristics of cells at different states depicted in the model are illustrated in Fig. 2. With regard to this model, the following general questions were asked concerning whether the regulatory mechanisms for the control of differentiation and proliferation in 3T3 T stem cells are aberrant. Does the process of predifferentiation growth arrest with or without the subse quent expression of nonterminal differentiation restrict the cells' subsequent proliferation potential and/or the cells' differ

Cell Proliferation Assays. The ability of cells to undergo proliferative responses was measured by colony formation assays or by assaying changes in cell density. Cell density was determined by trypsinizing the cells and counting them with either a Coulter Counter or on a micro scopically calibrated grid at various times during their culture. Colony formation was assayed by plating the cells at clonal densities (50 cells/ dish) in DMEM containing 30% FCS plus insulin (50 /ig/ml). Dishes were maintained up to 3 wk, and the numbers and sizes of the colonies were determined at successive intervals. Adipocyte Differentiation Assays. Adipocyte differentiation was quantitated morphologically using phase-contrast microscopy. The ex tent of differentiation was determined by counting duplicate wells or flasks for each group. At least 200 cells/well were counted, and cells were designated to be adipocytes if they contained numerous lipid droplets in the cytoplasm. It has been previously established that this morphological criterion correlates well with the enzyme markers of entiation potential and multipotentiality? The results provide a differentiation, such as lipoprotein lipase and glycerol-3-phosphate negative answer to these questions and thereby argue in favor dehydrogenase (25, 29, 30). of the conclusion that 3T3 T stem cells are preneoplastic cells Induction and Isolation of Predifferentiation Growth-arrested Cells at that express defects in the stringency with which the integrated the GDState and Nonterminally Differentiated (<;) Adipocytes. li.\I li/ control of cellular differentiation and proliferation is regulated. e 3T3 T mesenchymal stem cells were first induced to undergo predif ferentiation growth arrest at the GD state by culture in DMEM con taining human plasma for 4 days. These cells were then refed DMEM MATERIALS AND METHODS containing 10% FCS to reverse the arrest process and to return the cells to the proliferative cycle. Thereafter, they were assayed to deter Cells and Tissue Culture. The BALB/c 3T3 T mesenchymal stem cell mine if the process of growth arrest at the GD state programmed the line (a gift from Dr. L. Diamond) was used in these studies. It was cells to express a different differentiation and/or proliferation poten originally subcloned from clone A31 3T3 cells because of its propensity tiality. to differentiate into adipocytes (49). It has, however, subsequently been Cells were also induced to undergo nonterminal differentiation by shown that 3T3 T cells are a mesenchymal stem cell that can be induced to differentiate into other cell types, including macrophages (25, 50). culture in DMEM containing 25% CEPH, and similar assays were then Stock cultures of these cells were grown at 37Cin a humidified performed. To prepare the latter type of cell populations, rapidly atmosphere of 10% CO2. The stock culture medium consisted of growing 3T3 T stem cells were rinsed twice with PBS and then disso antibiotic-free DMEM3 containing 4500 mg of glucose per liter and ciated from the tissue culture dishes nonenzymatically. This was accom 10% heat-inactivated fetal calf serum (KC Biological, Lenexa, KS). To plished by incubating the cells in 0.1% EDTA in PBS (pH 7.4) for 10 to 20 min. Dissociated cells were then plated onto Lab-Tek 100-mm 3 The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; bacteriological plates in DMEM containing 25% CEPH. The culture FCS, fetal calf serum; GD, a distinct state in the (., phase of the cell cycle in of cells on bacteriological plates in this medium markedly inhibits which cells undergo predifferentiation growth arrest; GD, a distinct state in the G i phase of the cell cycle in which there is nonterminal differentiation of cellular proliferation and increases the kinetics of differentiation so adipocytes; PBS, phosphate-buffered saline; MF, macrophage foci. that maximal adipocyte differentiation is obtained within 5 days (47, 5313

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Fig. 2. Phase-contrast micrographs which illustrate the morphology of control 3T3 T mcscnchymal stem cells and their appearance during various stages of adipocyte differentia tion, a, rapidly growing 3T3 T cells in 10% FCS; b. cells arrested at the prcdifferentiation arrest state (Go) after 4 days in 25rhuman plasma; <. which have progressed to the cells nonterminal differentiation (G0 ) state after 6 to 8 days in 25"t human plasma: d, adipocytes which have reached the terminal differentia tion state, x 286.

achieved in 30% FCS and insulin (50 ig/ml). nce clones were estab O lished they were maintained in DMEM containing 10% FCS. (b) The second method used to isolate clones derived from single nonterminally differentiated adipocytes was to plate gradient-purified adipocytes in 96-well microtiter plates. Briefly, the cell density of the gradient-purified adipocytes was adjusted in an attempt to plate the adipocytes in the microtiter plates at approximately one cell/well. After attachment (4 to 24 h), the wells were screened by phase-contrast microscopy, and wells with more than one cell were eliminated. All of the remaining wells were then immediately rescreened to ensure that there was only one cell/well. Clones which had been derived from single nonterminally differentiated adipocytes were obtained using both of the procedures described above. Induction of Macrophage Differentiation. BALB/c 3T3 mesenchymal stem cell clones which had been derived from nonterminally differen cells were induced to undergo adipocyte differentiation and arrest at tiated (Go-) adipocytes as described above were induced to differentiate the GD state as described above. They were then purified and plated by into macrophages by the plasma clot technique as described previously two different techniques for cloning, (a) Cells for cloning were plated (25). In this system macrophage differentiation appears to be induced at low density in 100 nini dishes (Falcon) which had glass cloning chips as a result of the clotting process and subsequent clot lysis. Briefly, covering the bottom of the dish. After cell attachment, the dishes were cells grown in monolayer cultures were fed DMEM with 25% human scanned microscopically to find cloning chips which had single adipo plasma without anticoagulants. Soon after mixing DMEM (which cytes that had attached. The cloning chips which had single adipocytes contains calcium) and plasma, the clotting system was activated so that were then transferred to 12-well (one chip/well) cluster plates (Costar). a plasma clot formed on the cell monolayer. Within 2 to 4 days the Each well with a cloning chip was screened immediately to verify that initial clot underwent lysis as a result of fibrinolysis. This medium, there was only a single adipocyle on the cloning chip. This single cell which was once again liquid, was aspirated, and the cells were refed cloning procedure ensured that any clones which arose were derived fresh medium with plasma. Typically, the appearance of MF occurred from a single cell. Restimulation of growth and clonal expansion was concomitantly with the lysis of the second clot. 5314 53). On Day 5, there was typically greater than 90% adipocyte differ entiation; most of these cells are at the GD- state (see Fig. 1). These cells were then washed free of the medium, rinsed twice with PBS, and incubated in 0.1% EDTA in PBS to dissociate the cells. These cells were layered on a gradient of 10% bovine serum albumin in PBS and subjected to centrifugation as previously reported (47). This technique causes nonfat cells to pellet at the bottom of the tube, whereas adipo cytes remain suspended in the upper layer or at the interface between the two layers of the gradient (47, 53). The purified adipocytes (>95%) were resuspended and plated in DMEM containing 30% FCS and insulin (50 jtg/ml). This high concentration of FCS and insulin has been shown previously to stimulate the proliferation of adipocytes which are arrested at GD' (53) and to initiate their return to the proliferative cell cycle and their loss of the adipocyte phenotype. Cloning of Nonterminal!)' Differentiated Adipocytes. BALB/c 3T3 T

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Macrophage Differentiation Assays. We previously showed that 3T3 T-derived differentiated macrophages contained numerous lysosomes and phagosomes, were positive for cell surface la antigens, were capable of phagocytosis of iron particles, formed MF, and were positive for acid phosphatase and nonspecific esterase activity. In the present study the ability of BALB/c 3T3 T cells, and the various adipocytes clones and other cell lines, to differentiate into macrophages was determined morphologically and by enzyme cytochemistry as previously described (25). Phase-contrast microscopy was used to demonstrate that MF had formed. In this type of macrophage differentiation induction system, foci of tightly aggregated cells form (approximately 0.1 mm in diame ter), and only cells within the foci express all of the characteristics of macrophages. To verify that these foci were indeed macrophages, the cells were subjected to two different enzyme cytochemical stains for enzymes characteristic of macrophages, i.e., acid phosphatase and nonspecific esterase. Stains were done in situ with the cells still adherent to the tissue culture dishes. This allowed direct comparison microscop ically between the differentiated MF and adjacent undifferentiated cells. Acid phosphatase activity was determined by a modification of the method described by Li et al. (54). Briefly, the medium was aspirated from the tissue culture flasks, and the cells were allowed to air dry. They were then fixed in situ with a methanol-acetone fixative for 30 s, washed with distilled water, and air dried again. The cells were then stained and counterstained according to Li et al. (54). Acid phosphatase activity was detected as dark red to purple granules in the cytoplasm. WEHI 265.1 cells (a BALB/c monocytic leukemia cell line; kindly provided by Dr. D. J. McKean, Mayo Clinic) were used as positive controls for acid phosphatase activity (55). Nonspecific esterase activity was determined by a modification of the procedure described by Li et al. (56) and Yam et al. (57). Briefly, the medium was aspirated from the tissue culture flasks, and the cells were allowed to air dry. They were then fixed with cold buffered formalin/acetone for 30 s, washed with distilled water, and air dried again. The cells were then stained and counterstained according to Li et al. (56) and Yam et al. (57). Nonspecific esterase activity was determined as dark red precipitates in the cytoplasm. P388D, (a murine macrophage tumor cell line; kindly provided by Dr. D. J. McKean, Mayo Clinic) and WEHI 265.1 cells were used as positive controls for nonspecific esterase activity (55, 58).

IO*
345012345

TIME (Days)

Fig. 4. Illustration of the ability of 3T3 T stem cells to resume proliferation with normal kinetics after having been in differentiation-promoting medium for 4 days and reaching the predifferentiation arrest state GD. In the first panel (passage 1), Go-arrested cells (O) have been replated in 10% PCS and their growth kinetics compared to control (C) 3T3 T cells which have never seen differentia tion-promoting medium (). anels 2 to 4 represent subsequent serial passages. P In the lower right-hand corner of each panel is the approximate number of population doublings (/'/)hat have occurred during each passage. The ordinate t represents cell density/cm2, and the abscissa represents the time (days) for each passage.

RESULTS The general objective of this study was to determine if strin gent regulation of the integrated control of differentiation and proliferation exists at the GD and GD states. More specifically, our studies asked if arrest at either of these states programmed the cells to subsequently differentiate more efficiently or to restrict their proliferative capacity. Studies were also performed to determine if the process of nonterminal differentiation into

cells with the adipocyte phenotype programs such cells to lose their prior differentiation multipotentiality. Predifferentiation Arrest at the (,, tate Is Reversible and S Does Not Program a Change in Subsequent Differentiation or Proliferation Potentialities. The ability of BALB/c 3T3 T mesenchymal stem cells to growth arrest at the GD state and then differentiate when cultured in heparinized medium containing human plasma is documented in Fig. 3. The data show that within 3 to 4 days after addition of this medium the cells arrest their growth at the GD state, and within 5 to 6 days thereafter differentiation is evident. These results are similar to previously reported work (26, 29, 30) and were derived from studies using tissue culture flasks. The results presented in Fig. 4 illustrate the data which show that exposure of the 3T3 T stem cells to differentiation-promoting medium to induce growth arrest at the GD state for 4 days does not alter the proliferative potential of these cells once the differentiation signal is removed and the cells are restimulated to proliferate. More specifically, if the cells were first arrested at the predifferentiation arrest state, then trypsinized and replated at low density (approximately 5 x IO3cells/cm2) in DMEM containing 10% PCS, the first panel (passage 1) shows that the cells which had previously been arrested at the GD state proliferate well, although at a somewhat slower initial rate than native BALB/c 3T3 T cells that had not been exposed to differentiation-promoting medium. Fig. 4, furthermore, shows that, in subsequent passages, the cells that had previously been arrested at the GD state showed comparable proliferation kinetics to native 3T3 T stem cells and maintained a comparable growth rate for at least 4 passages or 18 popula tion doublings. The fact that the cells regained their previous proliferative potential and maintained it for at least 18 popu lation doublings indicates that arrest at the Gn state has no detectable effect (either negative or positive) on proliferative potential once the differentiation stimulus is removed and the cells reenter the cell cycle. Experiments were next performed to determine if previously Go-arrested cells, which had been stimulated with 30% FCS and insulin to reenter the cell cycle and resume proliferation, will differentiate more efficiently upon reintroduction of the differentiation stimulus. This question was evaluated by using

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Fig. 3. Illustration of the kinetics of 3T3 T growth arrest and differentiation into adipocytes. This figure represents the typical kinetics seen in tissue culture flasks when cells are induced to differentiate with heparinized DMEM containing 25% human plasma. ,percentage of adipocytes; O, cell density (cells/cm2 X 10-').

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Table 1 Nonlerminally differentiated adipocytes induced to dedifferentiate and then redifferentiate into macrophages Each of clones FC-1, FC-4, FC-5, and FC-6 was isolated from single adipocytes as described in "Materials and Methods." Differentiation assays Macrophage foci formation Mesenchymal stem cells cultured in human plasma clots to induce macro phage differentiation" 3T3T Clone Clone Clone Clone Rapidly 3T3T Clone Clone Clone FC-1 FC-4 FC-5 FC-6 growing mesenchymal stem cells FC-1 FC-4 FC-5 Nonspecific esterase activity Acid phosphatase activity

Rapidly growing control cell types Human fibroblasts NR Human monocytic leukemia line (WEHI 265.1) Human macrophage tumor cell line (P388D1) MR " The plasma clot technique for inducing macrophage formation utilized 25% human plasma in nonheparinized DMEM. All other cells (transformed; undifferentiated) were tested during logarithmic growth. * NR, not relevant because these cell lines grow in a poorly adherent manner.

potentialities. The results show that growth arrest at the GD and GD' states has no effect on the subsequent growth and differentiation characteristics of 3T3 T stem cells. This suggests that even though 3T3 T stem cells express a well-characterized sequence of biological events that control their differentiation (see Fig. 1), these cells show specific abnormalities in their ability to be programmed by these events to influence their subsequent growth and differentiation potentialities. In con trast, in most if not all normal stem cells or progenitor cells that have been carefully studied in vivo or in vitro, the induction of cellular differentiation is associated with a decreased proliferative potential with or without modulation in the regulation of the kinetics of differentiation. More specifically, in vivo the induction of differentiation of multipotential hematopoietic stem cells to the erythroid lineage limits their proliferative potential to approximately five population doublings (64). Sim Fig. 7. Cytochemical demonstration of nonspecific esterase activity in clone FC-5 cells which have been induced to differentiate into macrophages. These cells ilarly, in vitro the induction of differentiation of human epider have formed a tightly aggregated macrophage focus (MF, arrow), and the cells in mal cells is associated with the essentially complete loss of the focus are stained with dark reddish-brown precipitates in the cytoplasm. The proliferative potential even before overt expression of differen cells surrounding the MF are undifferentiated and are negative for nonspecific esterase activity. X 350. tiation markers is detectable (41, 43). Another characteristic of normal stem cells is that they express highly stringent mechanisms to control differentiation mesenchymal stem cells, express defects in their mechanisms to control differentiation and proliferation. This hypothesis is pathways or lineages. That is, if a normal multipotential stem cell is induced to differentiate into a specific cell lineage, it based on evidence that normal stem cells express highly strin typically cannot reverse this program (1,6). Only in patholog gent mechanisms to integrate the control of cellular differentia tion and proliferation (6-8, 20, 21, 36) and that induction of ical states can mammalian stem cells or progenitor cells change such highly stringent regulatory mechanisms can serve to sup their pathway of differentiation. The classical pathological state press the process of carcinogenesis (9, 13, 18). We used 3T3 T wherein this process is evident is designated metaplasia. For mesenchymal stem cells as a model preneoplastic cell line example, chronic irritation of the trachea! epithelium can in because they are immortal, they are aneuploid, and they show duce a metaplastic change wherein the columnar ciliated res piratory epithelial cells are replaced by stratified squamous an increased proclivity to undergo neoplastic transformation, whereas truly normal cell populations studied in vitro, such as epithelium. Metaplastic changes in mesenchymally derived tis human epithelial cells or early passage hamster embryo cells, sues can also occur. For example, metaplasia of fibroblasts into do not (4, 16, 19, 21, 43, 45, 59-63). osteoblasts and chondroblasts can occur following injury, and myeloid metaplasia is also a well-defined clinical entity in In the current studies a series of experimental approaches was used to determine if 3T3 T mesenchymal stem cells show humans. In this regard it has also been established that meta defects in the stringency by which they integrally control their plastic changes can in specific circumstances represent a pre neoplastic process even though in most situations metaplasia is differentiation and proliferation. The first group of experiments assayed whether predifferentiation growth arrest at the GD state benign and reversible. The classical example of preneoplastic or nonterminal differentiation and arrest at the GD' state act as metaplasia is that which occurs in the stomach. Metaplastic a stringent regulatory process in 3T3 T cells to permanently changes in transitional epithelium of the bladder can also program the cells' subsequent proliferation and differentiation represent a preneoplastic lesion.
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DEFECTS. METAPLASIA AND NEOPLASIA leukaemic cells induced by a differentiation-inducing protein. Nat. New Biol., 257:276-278, 1972. 11. Fibach, E., Peled, T., and Rachmilewitz, E. A. Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60). J. Cell. Physiol.,//J: 152-158, 1982. 12. Marks, P. A., Chen, Z., Banks, J., and Rifkind, R. A. Erythroleukemia cells: variants inducible for hemoglobin synthesis without commitment to terminal cell division. Proc. Nati. Acad. Sci. USA. SO: 2281-2284. 1983. 13. Peehl, D. M., and Stanbridge, E. J. The role of differentiation in the suppression of tumorigenicity in human cell hybrids. Int. J. Cancer, 30:113120. 1982. 14. Sachs, L. Constitutive uncoupling of pathways of gene expression that control growth and differentiation in myeloid leukemia: a model for the origin and progression of malignancy. Proc. Nati. Acad. Sci. USA, 77: 6152-6156, 1980. 15. Scott, R. E., and Florine, D. L. Cell cycle models for the aberrant coupling of growth arrest and differentiation in hyperplasia, metaplasia, and neoplasia. Am. J. Pathol., 07:342-348. 1982. 16. Scott, R. E., Wille, J. J., Jr., Pittelkow, M. R.. and Sparks, R.L. Biological mechanisms of stem cell carcinogenesis: a concept for multiple phases in the initiation of carcinogenesis and the role of differentiation control defects. In: J. C. Barrett and R. W. Tennant (eds.). Mammalian Cell Transformation, Mechanisms of Carcinogenesis, and Assays for Carcinogens, pp. 67-80. New York: Raven Press, 1985. 17. Scott, R. E., Wille, J. J., Jr., and Wier, M. L. Mechanisms for the initiation and promotion of carcinogenesis: a review and a new concept. Mayo Clin. Proc., 59: 107-117, 1984. Stanbridge, E. J., Der, C. J., Doersen, C-J., Nishimi, R. Y., Peehl, D. M., 18. Weissman, B. E., and Wilkinson, S. E. Human cell hybrids: analysis of transformation and tumorigenicity. Science (Wash. DC), 2/5: 252-259, 1982. 19. Ts'o, P. O. P., Bruce, S. A., Brown, A. R., and Miller, P. S. New view of carcinogenesis: implications for chemotherapy and human risk assessment. In: J. C. Barrett and R. W. Tennant (eds.). Mammalian Cell Transformation: Mechanisms of Carcinogenesis and Assays for Carcinogens, pp. 105-121. New York: Raven Press, 1985. 20. Wille, J. J., Jr., Maercklein, P. B., and Scott, R. E. Neoplastic transformation and defective control of cell proliferation and differentiation. Cancer Res., .-5139-5146, 1982. 21. Wille, J. J., Jr., Pittelkow, M. R., and Scott, R. E. Normal and transformed human prokeratinocytes express divergent effects of a tumor promoter on cell cycle-mediated control of proliferation and differentiation. Carcinogen esis (Lond.), 6: 1181-1187, 1985. 22. Yen, A. Control of HL-60 myeloid differentiation. Evidence of uncoupled growth and differentiation control, S-phase specificity, and two-step regula tion. Exp. Cell Res., 156:198-212, 1985. 23. Green, H., and Kehinde, O. Sublines of mouse 3T3 cells that accumulate lipid. Cell, ;.- 113-116, 1974. 24. Harrison, J. J., Soudry, E., and Sager, R. Adipocyte conversion of CHEF cells in serum-free medium. J. Cell Biol., 100: 429-434, 1985. 25. Krawisz, B. R., Florine, D. L., and Scott, R. E. Differentiation of fibroblastlike cells into macrophages. Cancer Res., 41: 2891-2899, 1981. 26. Krawisz, B. R., and Scott, R. E. Coupling of proadipocyte growth arrest and differentiation. I. Induction by heparinized medium containing human plasma. J. Cell Biol., 94: 394-399, 1982. 27. Kuri-Harcuch, W., and Marsch-Moreno, M. DNA synthesis and cell division related to adipose differentiation of 3T3 cells. J. Cell. Physiol., 14:39-44, 1983. 28. Nixon, B. T., and Green, H. Growth hormone promotes the differentiation of myoblasts and preadipocytes generated by azacytidine treatment of 1OT'/i cells. Proc. Nati. Acad. Sci. USA, 81: 3429-3432, 1984. 29. Scott, R. E., Florine, D. L., Wille, J. J., Jr., and Yun, K. Coupling of growth arrest and differentiation at a distinct state in the (., phase of the cell cycle: GD. Proc. Nati. Acad. Sci. USA, 79: 845-849. 1982. 30. Scott, R. E., Hoerl, B. J., Wille, J. J., Jr., Florine, D. L., Krawisz, B. R., and Yun, K. Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation. J. Cell Biol., 94:400-405, 1982. 31. Campione-Piccardo, J., Craig, J., Sun, J-J., and McBurney, M. W. Commit ment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid. Exp. Cell Res., 156: 544-552, 1985. 32. Dean, A., Erard, F., Schneider, A. B., and Schechter, A. N. Induction of hemoglobin accumulation in human K562 cells by hemin is reversible. Science (Wash. DC), 2/2:459-461, 1981. 33. Devlin, B. H., and Knigsberg,I. R. Reentry into the cell cycle of differen tiated skeletal myocytes. Dev. Biol., 95: 175-192, 1983. 34. Ebert, P. S., Wars, I., and Buell, D. N. 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Another group of experiments was therefore performed to determine if 3T3 T mesenchymal stem cells express the ability to undergo transfifferentiation and thereby express additional defects in the stringency with which they integrally regulate the control of their differentiation and proliferation. In these ex periments, 3T3 T stem cells were induced to undergo nonter minal differentiation into adipocytes. The adipocytes were then cloned and induced to lose the differentiated phenotype and reenter the cell cycle. Thereafter, we assayed whether prior adipocyte differentiation restricted the m nil potential t> of 3T3 i T mesenchymal stem cells as one would predict should occur in normal stem cells or whether 3T3 T stem cells could differ entiate into adipocytes, dedifferentiate, and redifferentiate into another cell type as occurs in some preneoplastic and metaplastic cells. The results clearly show that 3T3 T stem cells can undergo adipocyte differentiation, dedifferentiation, and redifferentiation (transdifferentiation) into macrophages that ex press specific marker enzymes including nonspecific esterase activity and acid phosphatase activity as well as expressing morphological characteristics of macrophages. To our knowl edge, this is the first clear demonstration of the transdifferen tiation of mammalian cells in vitro. We interpret these results to support the conclusion that 3T3 T mesenchymal stem cells represent a preneoplastic cell popu lation that expresses defects in the stringency with which the integrated control of cellular differentiation and proliferation is regulated. In addition, these and additional studies serve as the basis for our hypothesis which suggests that the expression of defects in the mechanisms that control the stringency by which the integrated control of cellular differentiation and proliferation is regulated may be a general characteristic of preneoplastic lesions and therefore may represent a very early lesion in the process of carcinogenesis. ACKNOWLEDGMENTS
The authors thank Dr. J. J. Wille, Jr., Dr. D. N. Estervig, and Dr. E. B. Leof and W. J. Hanson, B. J. Hoerl, and P. B. Maercklein for their advice and technical assistance.

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