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Differentiation of primary and secondary fibroblasts in cell culture systems
Mutation Research, 256 (1991) 233-242
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© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50
MUTAGI 00164
Differentiation of primary and secondary fibroblasts in cell culture systems Klaus Bayreuther, Pal I. Francz, Jochen Gogol, Constanze Hapke, Monika Maier and Hans-Georg Meinrath Institut fiir Genetik, Unicersith't Hohenheim, D-7000 Stuttgart 70 (Germany) (Accepted 17 June 1991)
Keywords: Stem cell system; Terminal differentiation; Ageing; Apoptosis; Transformation: Genetic programme
Summary As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an l 1-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems. In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-$2-$3, three mitotic fibroblasts (MF) differentiate along the sequence MF I - M F I I - M F III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment. The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo. Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence. Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations.
Correspondence: Dr. K. Bayreuther, Institut fiir Genetik, UniversitS.t Hohenheim, D-7000 Stuttgart 70 (Germany).
Nowadays methods of molecular cell biology are available that allow the characterization of the biological and biochemical properties of the
234
cells making up secondary cell populations in vitro. A comparison of the characteristics of the cell types in secondary populations in vitro with those of the primary cell types studied in ex viva-in vitro systems is destined to bring forth information about the relatedness of the cell types in the secondary and primary cell systems. In addition, the investigation of cells in primary cell systems established and analyzed before they start to divide ex viva-in vitro, will permit deductions about the biological and biochemical characteristics of the fibroblast equivalent cell types making up the corresponding cell system in viva. Comparative studies of the cellular composition of primary fibroblast populations and the biological and biochemical properties of the primary cells as a function of the age of the donor in an ex viva-in vitro system and of the cellular composition of secondary fibroblast populations and biological and biochemical properties of the secondary cells as a function of the in vitro age of the population studied, will give an answer to the frequently discussed question about the value of secondary in vitro cell systems for the study of general and special problems of cell biology. Higher eukaryotic animal and human organisms are made up of about 200 cell systems (Bloom and Fawcett, 1975). The majority are cell systems with specialized functions, e.g., keratinoblasts/ keratinocytes. In addition, there exist two cell systems with helper functions. These are the glia cell systems of the brain and the fibroblasts of the connective tissues. It is very likely that all cell systems are stem cell systems with a design resembling that of the hemopoietic stem cell system (Metcalf, 1977, 1988). The rapid progress in the understanding of the molecular mechanisms and the molecular control of the differentiation of cells in the distinct branches of the hemopoietic stem cell system is the result of the strict employment of molecular methods specific for the stem cell systems in the secondary in vitro, in the primary ex viva-in vitro, and in the in viva populations studied (Metcalf, 1977, 1988). We have collected evidence that primary fibroblast populations (ex viva-in vitro) isolated from prenatal or postnatal connective tissue of skin and lung of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Mollenhauer
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and Bayreuther, 1986), and man (Bayreuther et al., 1988b) develop as a function of the advancing in viva age of the donors along the progressive stages of a stem cell system. In addition, secondary fibroblast populations derived from the connective tissue of prenatal and postnatal skin and lung of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979), and man (Bayreuther et al., 1988a), including the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991), develop along a terminal differentiation sequence typical for stem cell systems, when mitotic stem cells and fibroblasts were passaged serially and when postmitotic fibroblasts were kept under appropriate conditions in stationary culture in vitro for longer periods of time (Figs. 1, 2). Results
Stem cell compartment Primary and secondary stem cells in citro Mesenchymal cells were isolated from the morphogenetic field of the wing of the Valo chicken at embryonic stage 20 and seeded into primary clonal culture. Primary clonal populations are serially subcloned for up to five times until heterogenous or homogenous clonal populations made up of cells with the characteristic morphology of fibroblasts, chondroblasts, and myoblasts could be distinguished. Primary and secondary clones studied showed the same [3~S]methionine polypeptide pattern of the nu-
235
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clear and cytoplasmic fraction, indicating that they are made up of identical cells, the stem cell S1. Tertiary clones exhibited three polypeptide pattern, one of the stem cell S1 and two new ones characterizing the stem cells S2a and S2b. In the fourth and fifth subcloning steps, clones with up to six different polypeptide marker combinations could be identified, three polypeptide patterns of S1, S2a, S2b, and three of the committed stem cells S3a, b, c for fibroblasts, or chondroblasts, or myoblasts. These findings make the following developmental pathway of the mesenchymal stem cells likely. S1 has developmental potential for fibroo blasts and chondroblasts plus fibroblasts and myoblasts. S1 proliferates and segregates two types of the stem cell $2, S2a with two potential functions for fibroblasts and chondroblasts, and S2b with developmental functions for fibroblasts and myoblasts. S2a and S2b proliferate again and
throw off three types of stem cells S3a, b, c. The $3 cells are committed stem cells with developmental quality for fibroblasts (S3a), chondroblasts (S3b), and myoblasts ($3c). The committed $3 cells move on to the progenitor compartments, in which the progenitor cells proliferate and develop along the fibroblast, the chondroblast, and the myoblast progenitor cell lineages. For these investigations, a new medium, serum, and factor composition had to be worked out, which supports the proliferation and differentiation of fibroblasts, chondroblasts, and myoblasts in a mixed population (Figs. 1, 2) (Hapke, 1990).
Fibroblast progenitor, maturing, degenerating, and transforming compartments Primary fibroblasts ex vivo-in vitro Primary human skin fibroblast populations were established ex vivo-in vitro from postnatal
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Fig. 3. Development o• apoptosis and transformation in the C3H mouse fibroblast stem cell system ( A - F 120 x ). (A) Postmitotic fibroblast PMF VI. (B) Nuclear fragmentation in early stages of PMF VIIb. (C) Budding of nuclear fragments and small amounts of cytoplasm through the cell wall. (D) Transformed mitotic fibroblasts TMF II with PMF VI in the background. (E) Postmitotic fibroblast PMF VIIa in apoptosis with pycnotic nucleus and contracted cytoplast. (F) PMF VIIa in late stages of apoptosis, expressing senescent cell antigen band 3. (G) Specific [35S]methionine polypeptide markers in PMF VI, TMF I and TMF II, NTMF I and NTMF II, as indicated by arrow (pl: 6.3, Mr: 32 kDa).
237 abdominal skin of 45 female donors of four age groups: 1-20, 20-40, 40-60, and 60-90 years, with 19, 7, 8, and 11 donors respectively. Primary skin fibroblasts were studied in explant cultures, in mass cultures of low density, and in clonal populations ex vivo-in vitro. In these three systems, primary populations were found to be composed of eight fibroblast cell types, the mitotic fibroblasts (MF) MF I, MF II, MF III in the fibroblast progenitor compartment, the postmitotic fibroblasts (PMF) PMF IV, PMF V, PMF VI in the fibroblast maturing compartment, PMF VIIa in the fibroblast apoptosis compartment, and PMF VIIb in the transforming compartment (Figs. 1, 2). In primary populations, MF I is a small spindle-shaped cell, MF II is an epitheloid cell, and MF III is a larger epitheloid cell. The postmitotic fibroblast PMF IV is a large spindle-shaped cell with the morphology of a myofibroblast, PMF V is a large epitheloid cell, and PMF VI is the largest epitheloid cell. PMF VI is the terminally differentiated end cell. PMF V | I a is a cell with a heteropycnotic nucleus and a contracted, degenerating cytoplast. PMF VIIb is an epitheloid cell with 10-20 nuclear fragments that develop to become transformed mitotic fibroblasts T M F I by cellular budding (Figs. 1, 2, 3) (Bayreuther et al., 1988b). The composition of primary fibroblast populations that migrate from skin explants during the first 4-7 days is a function of the age of the donor. In this time period, the cells are established and have expressed their cell type-specific morphology and have not yet started to divide (ex vivo-in vitro). Such populations are always heterogenous with respect to their combination of cell types. Populations migrating out of the explant of a 7-year-old donor are predominantly made up of mitotic fibroblasts MF II plus 10-20% postmitotic fibroblasts (PMFs), those of a 37year-old donor are composed mainly of MF II and MF III plus 10-20% PMFs, those of an 57-year-old donor are made up of MF II and MF III plus 10-30% PMFs, and those of an 87-yearold donor are made up of 40% MF II and MF III plus 60% PMFs. 5 - 8 days after explantation, the MF II and MF III cells of the migrated populations begin to proliferate and to displace the postmitotic fibroblasts. As a result, all explants of
donors of different ages are surrounded by homogenous populations of MF II 14 days after explantation at the latest. These MF II populations are secondary fibroblast populations. The morphological and biochemical properties of these secondary MF II populations are identical, regardless of the age of the donor of the explant (Bayreuther et al., 1988b). Primary low density mass cultures of skin fibroblasts extracted from skin material of human donors of different ages by collagenase/trypsin treatment can be analyzed 4 - 7 days after seeding. At this time, the fibroblasts have expressed their morphological characteristics and have not yet started to divide (ex vivo-in vitro). Primary low density mass cultures were started from the same skin material from which the primary explant populations were set up. The age-dependent changes in the frequencies of primary mitotic and postmitotic fibroblasts in the low density mass cultures were similar or identical to those found in primary explant populations (Bayreuther et al., 1988b). As described for the primary explant populations, the primary low density mass cultures are made up of about 40% mitotic fibroblasts MF II and MF III, even when the skin material of the oldest donors is analyzed. From these mitotic cells, secondary fibroblast cell lines with a progenitor compartment with 60 CPDs and a maturing compartment of postmitotic fibroblasts in stationary culture for about 140 days have been established in vitro (Bayreuther, unpublished data). Five different clonetypes can be identified in primary clone cultures 14 days after seeding. These consist either of uniform populations of MF I in clone types of fibroblasts (CTF) CTF I, MF II in CTF II, and MF III in CTF III, or mixtures of MF I and MF II in CTF I / I I , and MF II plus MF III in clone type CTF I I / I I I . The age-dependent changes in primary clone type frequencies are similar to those found for the mitotic fibroblast cell types in primary explants and low density mass populations (in a strict sense, primary clones are made up of secondary fibroblasts) (Bayreuther et al., 1988b). Primary low density mass cultures of donors in the age group 20-40 years, which are predominantly made up of MF II and MF III, can be
238 experimentally induced by UV (Rodemann et al., 1989a) or MMC (Francz et al., 1989) to differentiate to the postmitotic populations of cell type PMF IV after 4 days, PMF V after 8 days, and PMF VI after 21 days. When the [35S]methionine polypeptide patterns of the pure primary subpopulations of MF I, MF II, MF III and experimentally induced pure primary populations of PMF IV, PMF V, and PMF VI are compared with those of secondary sub-populations of MF I, MF II, MF III and spontaneously arisen or experimentally induced secondary populations of PMF IV, PMF V, and PMF VI of the secondary human skin fibroblast cell line HH8, cell typespecific [35S]methionine polypeptide markers of the primary and secondary cell types are found to be identical (Bayreuther et al., 1988a,b; Bayreuther, unpublished data). Primary fibroblast populations from skin and lung of BN rats and C3H mice of different in vivo ages were studied in (ex vivo-in vitro) primary explant cultures, low density mass cultures and clone cultures. Again, three mitotic fibroblast (MF) cell types (MF I, MF II, MF III) and five postmitotic fibroblast (PMF) cell types (PMF IV, PMF V, PMF VI, PMF VIIa, and PMF VIIb) were found in the three primary cell systems studied. The age-dependent unidirectional changes in the frequencies of the fibroblast cell types in primary explant populations, in primary low density mass populations, and of mitotic fibroblasts in primary clone types are identical. In primary clone cultures, foci of transformed mitotic fibroblasts TMF I develop (Maier, 1991; Mollenhauer and Bayreuther, 1986). When dermis of mature male BN rats was sliced horizontally and fibroblasts were allowed to migrate from the sections in vitro, it was discovered that there exists a stratification of the fibroblasts MF I, MF II, and MF III in the reticular layer and of the postmitotic fibroblasts in the papillary layer with PMF VI located in the boundary zone underneath the basal lamina (Francz, unpublished data). When the [35S]methionine polypeptide patterns of pure primary subpopulations of MF I, MF II, MF III and of experimentally induced primary subpopulations of PMF IV, PMF V, and PMF VI are compared with those of secondary subpopulations of MF I,
MF II, MF III and spontaneously arisen or experimentally induced secondary subpopulations of PMF IV, PMF V, and PMF VI of secondary BN rat skin fibroblast cell lines, cell type-specific [-SSS]methionine polypeptide markers of the primary and secondary cell types are found to be identical (Mollenhauer and Bayreuther, 1986; Kontermann and Bayreuther, 1979; Francz, unpublished data).
Secondary fibroblasts in citro A secondary human dermal fibroblast population was established from abdominal skin of a female donor 8 years of age. Mitotic populations of this cell line, designated HH8, underwent 53.6 + 6.0 cumulative population doublings (CPD) in 302 _+ 27 days. When the growth capacity of the mitotic fibroblasts is exhausted and when appropriate methods are applied, mitotic fibroblasts do not degenerate in a Phase III phenomenon (Hayflick and Moorhead, 1961; Hayflick, 1981), but shift spontaneously to postmitotic fibroblasts. The maximal lifespan of postmitotic HH8 fibroblasts in stationary culture was 418_+ 35 additional days (Bayreuther et al., 1988a). Mitotic and postmitotic secondary fibroblast populations of HH8 are heterogenous with reproducible unidirectional changes in the proportions of cell stages of mitotic and postmitotic fibroblasts. Secondary fibroblasts have the same relative shapes and sizes as primary fibroblasts. The reiterated changes in the populations of mitotic and postmitotic fibroblasts in the secondary populations make it evident that secondary fibroblasts develop in one direction along the eightstage terminal cell lineage MF I - M F II-MF IIIPMF IV-PMF V-PMF VI-PMF V I I a / P M F VIIb in four compartments (Figs. 1, 2) (Bayreuther et aI., 1988a). As described for primary clonal populations, five different types of clones arise in the secondary clonal cultures of HH8. These are the pure clones CTF I, CTF II, and CTF III, and the heterogenous clones CTF I / I I and CTF II/III. Changes in the frequencies of the distinct clone types can be observed as a function of the CPD level of the heterogenous populations. Thus, the changes in cell type and clone type frequencies in secondary low density mass cultures and clonal
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populations are a function of the CPD level of the mitotic mass populations, and the changes of the frequencies of the postmitotic fibroblast cell types are a function of the period of time of postmitotic mass populations in stationary culture, i.e., the so-called in vitro age of the populations (Bayreuther et al., 1988a,b). They are similar or identical to those found for the cell type frequencies in primary explant populations, low density mass populations and clonal populations, which are a function of the in vivo age of the donors (Figs. 1, 2)(Bayreuther et al., 1988b). The shifts in the frequencies of mitotic and postmitotic fibroblasts in secondary mass populations of HH8 are accompanied by reiterated alterations of biochemical parameters, for example in the [35S]methionine polypeptide pattern of the developing mass populations. The [ 35S]methionine polypeptide patterns of homogenous subpopulations of MF I, MF II, MF III, PMF IV, PMF V, and PMF VI, isolated from heterogenous mass populations by the clone-ring technique, revealed that the six fibroblast morphotypes studied express their cell type-specific polypeptide pattern in the heterogenous populations. Thus, the [3-SS]methionine polypeptide patterns of the mixed populations of the cell line HH8 are the sums of the polypeptide patterns of the distinct fibroblast cell types that make up the total population at different stages of development (Bayreuther et al., 1988a). For the analysis of the biological and biochemical changes accompanying the cyto-differentiation of the secondary fibroblasts of HH8 in the cell lineage, methods have been developed for the selective enrichment of the three mitotic and four postmitotic fibroblasts from the heterogenous mitotic populations (Francz et al., 1989; Rodemann et al., 1989a). Almost homogenous mass populations of MF II with 78-87% purity can routinely be obtained at CPD level 28-34, those of MF III with 73-86% purity at CPD level 48-53. Another cell system suitable for this purpose is the quantitative clonal system, with MF I in fibroblast clone type CTF I, MF II in CTF II, and MF III in CTF III, with mean sizes of 4303, 2352, and 302 cells per clone type (Rodemann et al., 1989a). In order to achieve a selective enrichment of four postmitotic fibroblast cell types,
methods have been worked out for the experimental induction of the differentiation from a lower state (MF II, MF III) to a higher state (PMF IV, PMF V, PMF VI, and PMF VII) in the lineage of HH8 by physical methods (UV (Rodemann et al., 1989a) and electromagnetic fields (Rodemann et al., 1989b)) and chemical agents (MMC (Francz et al., 1989)). The [35S]methionine polypeptide patterns of nearly homogenous sub- or total populations of the mitotic fibroblasts MF I, MF II, and MF III and their corresponding clonal populations CTF I, CTF II, and CTF III are identical (Bayreuther et al., 1988a, Francz et al., 1989; Rodemann et al., 1989a). Likewise postmitotic fibroblast cell types PMF IV, PMF V, PMF VI, and PMF VII, either spontaneously arisen or experimentally induced, show identical [35S]methionine polypeptide patterns for cell type-specific marker proteins (Bayreuther et al., 1988a; Rodemann et al., 1989a). In the lineage of MF II ~ PMF VI, 14 cell type-specific marker proteins have been identified in the nuclear and cytoplasmic fractions, 24 cell type-specific marker proteins in the membrane-bound fraction, and 11 cell type-specific marker proteins in the secreted protein fraction (Francz et al., 1989). Degenerating postmitotic fibroblasts PMF VIIa express a new set of cell type-specific [35S]methionine polypeptides, indicating that PMF VIIa degenerates under the control of an apoptosis program (Fig. 3E,F). Cells in late apoptosis express the modified band 3 (Kay, 1984; Francz, unpublished data). Macromolecule content and synthesis have been investigated in nearly homogenous mass populations of 75-88% purity of MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII of the cell line HH8 in an attempt to characterize the differentiating fibroblasts by other biochemical parameters. The DNA content, RNA synthesis, protein synthesis, collagen synthesis, and protein content were determined in fully contact regulated mitotic populations at CPD 30 (78-87% MF II) and at CPD 52 (73-86% MF liD, those of PMF IV, PMF V, PMF VI, and PMF VIIa (all at about 75-85% purity) in experimentally induced postmitotic fibroblasts in stationary cultures. The DNA content increases in the course of cyto-differentiation. MF II has 2C DNA, MF III and PMF IV
240 contain 4C DNA, PMF V 8C DNA, 64% of PMF VI have 8C DNA and the remaining 36% have 16C DNA. The degenerating PMF VIIa has DNA contents in the range of 8C-2C. In nearly homogenous mass populations of MF II, MF Ill, PMF IV, PMF V, and PMF VI, RNA synthesis increases by a factor 9 (MF II = factor 1), protein synthesis increases by a factor 4.8, collagen synthesis by 6.8, and total protein content by 11.5, when MF II and PMF VI are compared (Francz, unpublished data). Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and postmitotic states in vitro has been described for cell line HH8 (Rodemann, 1989). Secondary fibroblast populations of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979), and of the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991) also develop along the same cell biological and cell biochemical differentiation sequence in the progenitor, maturing, degenerating, and transforming compartments of the fibroblast stem cell system. Spontaneous transformation has been observed for primary and secondary populations of dermal fibroblasts of C3H mice (Maier, 1991) and BN rats (Kontermann and Bayreuther, 1979; Mollenhauer and Bayreuther, 1986) and in secondary fibroblasts of the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991). In all cell populations studied, transformation starts by the fragmentation of the nucleus of PMF VIIb into 10-20 micronuclei (Fig. 3B). The micronuclei surrounded by a small cytoplast bud through the cell membrane (Fig. 3C) and give rise to transformed cell lines (Fig. 3D) with three transformed mitotic fibroblasts (TMF) TMF I, T M F II, and TMF III and five transformed postmitotic fibroblasts (TPMF) T P M F IV, T P M F V, T P M F VI, T P M F VIIa, T P M F VIIb. In T P M F VIIb, nuclear fragmentation and cellular budding occur again. After the budding of the micronuclei plus cytoplast through the cell membrane, three neoplastically transformed mitotic fibroblasts (NTMF) N T M F I, N T M F II, and N T M F III and five neoplastically transformed postmitotic fibroblasts (NTPMF) N T P M F IV, N T P M F V, N T P M F VI, NTPMF VIIa, N T P M F VIIb are established (Fig. 2). In the transformed cell lines T M F II represents the
stem line with 87-91% of the cells, in neoplastically transformed cell lines, N T M F II makes up the stem line with 85-89% of the cells in the population. This concept of the developmental pathway of the transformed and neoplastically transformed fibroblasts is supported by the finding that a marker polypeptide, expressed for the first time in PMF VI, remains expressed in T M F I and T M F II and N T M F I and N T M F II (Fig. 3G). Discussion
Primary and secondary fibroblast cell systems of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979; Mollenhauer and Bayreuther, 1986), and man (Bayreuther et al., 1988a,b), including the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991), have been studied with stem cell system-specific concepts and methods. The data show that all fibroblast cell systems studied are stem cell systems with 11 cells developing in an ll-stage differentiation sequence in five compartments of a stem cell system. In the stem cell compartment, stem cells (S) develop along the sequence S 1 - S 2 a / S 2 b - S 3 , in the fibroblast progenitor compartment mitotic fibroblasts (MF) progress along the lineage MF I - M F I I - M F III, in the fibroblast maturing compartment postmitotic fibroblasts (PMF) proceed in a line PMF I V - PMF V - P M F VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. PMF VI dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment (Figs. 1, 2, 3). When stem cell system-specific methods are applied in comparative studies of the cellular composition of primary and secondary fibroblast populations and of the biological and biochemical properties of the primary and secondary cells as a function of the age of the donor in an ex vivo-in vitro system or as a function of the in vitro age of the populations, it becomes evident that primary and secondary fibroblast cell systems are stem cell systems with an identical design. The reiterated appearance of the 11 cell types
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in primary fibroblast populations of skin of 45 human donors, skin and lung of 26 BN rat donors, and the reiterated age-related changes in the cell type composition of the primary fibroblast populations (Bayreuther et al., 1988b; Mollenhauer and Bayreuther, 1986) make it very likely that mitotic fibroblast equivalents (MFE) and postmitotic fibroblast equivalents (PMFE) of the primary fibroblasts exist in vivo and that age-related changes in the frequencies of the fibroblast equivalents result from the differentiation along the cell lineage MFE I - M F E I I - M F E III and PMFE IV-PMFE V-PMFE VI-PMFE V I I a / P M F E VIIb in vivo. The development of the cells is under the control of a genetic program, which is expressed by the reproducible, unidirectional and cell typespecific changes of the [35S]methionine polypeptide pattern in the terminal differentiation cell lineage (Bayreuther et al., 1988a; Francz et al., 1989). In addition the mitotic and postmitotic fibroblasts have a cell type-specific DNA content, RNA synthesis, protein synthesis, collagen synthesis, and protein content (Francz, unpublished data). Normally the differentiation sequence proceeds spontaneously, but it can be speeded up by a number of physical and chemical agents (UV (Rodemann et al., 1989a), MMC (Francz et al., 1989)). Spontaneously arisen and experimentally induced fibroblast cell types have identical polypeptide patterns, macromolecule synthesis, and macromolecule content, indicating that normal terminal differentiation and experimentally speeded up terminal differentiation follow the same genetically programed pathway (Francz et al., 1989; Rodemann, 1989a). These data support the unifying concept of the molecular mechanisms of cellular aging and cellular neoplastic transformation (Kontermann and Bayreuther, 1979). According to this concept, normal physiological cellular aging is the result of the terminal differentiation of the fibroblasts in the fibroblast stem cell system, followed by either apoptosis or transformation. The course of the morphological and biochemical multistage differentiation sequence of fibroblasts can be speeded up by endogenous factors, e.g., Duchenne muscular dystrophy fibroblasts (Rodemann and Bayreuther, 1984, 1986), by exogenous factors such as UV
(Rodemann, 1989a) and mitomycin C (Francz et al., 1989), by compounds (sucrose) not degradable by lysosomes (Bayreuther, unpublished data), and by persistent infections with viruses (rubella virus) (Bayreuther, unpublished data). These conditions or treatments increase the probability of differentiation along the genetic program of the mitotic and postmitotic fibroblast cell lineage, and thus induce pathophysiological cellular aging. At higher in vitro age, the mitotic and postmitotic fibroblasts of the renewing progenitor and the maturing compartments in the dermal fibroblast stem cell systems of BN rats or man are completely depleted (Kontermann and Bayreuther, 1979; Bayreuther et al., 1988b). In contrast, the primary low density mass cultures of the oldest human donors are made up of about 40% mitotic fibroblasts MF II and MF III, which can give rise to secondary fibroblast cell lines in vitro with a proliferative capacity of 60 CPDs and postmitotic populations 140 days in stationary culture (Bayreuther, unpublished data). This finding is surprising, since the total number of the fibroblasts that can be extracted from a given volume of dermis decreases as a function of increasing donor age, by a factor 10 when dermis of a 7-year-old donor and an 87-year-old donor is compared. The decrease in the cell number of fibroblasts in situ in the presence of fibroblasts capable of proliferating in vitro makes it likely that the factors that normally regulate their proliferation in vivo have become abnormal in their qualitative a n d / o r quantitative composition as a function of the in vivo age of the dermis (Bayreuther, unpublished data). The terminally differentiated end cell PMF VI degenerates as PMF VIIa under the control of a genetic program by apoptosis. In spontaneously occurring or experimentally induced apoptosis, three new cell type PMF VIIa-specific [35S] methionine polypeptides are expressed that turn off the synthesis of all [35S]methionine polypeptides by a hitherto unknown mechanism. In later stages of apoptosis, the modified band 3 is expressed (Kay, 1984; Francz, unpublished data). The evolution of a molecular mechanism for the programed removal of terminally differentiated end cells is additional evidence for the genetically programed control of the balanced development
242 of the fibroblast in proliferating, m a t u r i n g , a n d degenerating compartments. In the stem cell systems of C 3 H mice (Maier, 1991), BN rats ( K o n t e r m a n n a n d Bayreuther, 1979; M o l l e n h a u e r a n d B a y r e u t h e r , 1986), a n d the h u m a n cell line WI38 (Gogol, 1991), transformed cells or neoplastically t r a n s f o r m e d cells arise by n u c l e a r f r a g m e n t a t i o n a n d cellular budding from P M F V I I b or T P M F VIIb. In transformed a n d neoplastically t r a n s f o r m e d cell lines, three mitotic ( T M F , N T M F ) a n d five postmitotic fibroblasts ( T P M F , N T P M F ) with cell biological a n d cell biochemical individuality can be identified (Fig. 2). In contrast to the n o r m a l fibroblasts, the progression along the d i f f e r e n t i a t i o n lineage of most of the t r a n s f o r m e d a n d neoplastically t r a n s f o r m e d fibroblasts is blocked at differentiation stages T M F II a n d N T M F II. Because of that, T M F II a n d N T M F II are the p r e d o m i n a n t cell types (stem line) with 84% a n d 79% cells of the p o p u l a t i o n s studied (Francz, u n p u b l i s h e d data). T h e s e data are in d i s a g r e e m e n t with the theory that assumes that carried on cells of the stem cell c o m p a r t m e n t give rise to sarcoma cells a n d stresses the i m p o r t a n c e of t e r m i n a l l y differe n t i a t e d or aged fibroblasts for the d e v e l o p m e n t of the t r a n s f o r m e d cells. F i b r o b l a s t p o p u l a t i o n s have so far always b e e n t r e a t e d as h o m o g e n o u s n o n - d i f f e r e n t i a t i n g cell p o p u l a t i o n s a n d most of the data o b t a i n e d are i r r e p r o d u c i b l e (Hayflick, 1981). W h e n stem cell system-specific concepts a n d m e t h o d s are applied, d e f i n e d fibroblast p o p u l a t i o n s can be m a d e available for qualitative a n d / o r q u a n t i t a t i v e studies of p r o b l e m s in g e n e r a l cell biology, b i o c h e m istry a n d virology, a n d more specialized issues like t e r m i n a l d i f f e r e n t i a t i o n , aging, apoptosis, a n d t r a n s f o r m a t i o n . Cell biological a n d biochemical studies of properly d e f i n e d fibroblast p o p u l a tions, i.e., pools of cell p o p u l a t i o n s with a verified c o n s t a n t c o m p o s i t i o n of fibroblast cell types a n d thus with identical biochemical p a r a m e t e r s , are d e s t i n e d to provide r e p r o d u c i b l e results in the field of fibroblast cell biology in future.
Aknowledgements S u p p o r t e d by grants of the D e u t s c h e Fors c h u n g s g e m e i n s c h a f t a n d the B u n d e s m i n i s t e r i u m ffir F o r s c h u n g u n d T e c h n o l o g i e .
References Bayreuther, K., H.P. Rodemann, R. Hommel, K. Dittmann, M. Albiez and P.I. Francz (1988a) Human skin fibroblasts in vitro differentiate along a terminal cell lineage, Proc. Natl. Acad. Sci. (U.S.A.), 85, 5112-5116. Bayreuther, K., H.P. Rodemann, P.1. Francz and K. Maier (1988b) Differentiation of fibroblast stem cells, in: B. Lord and T.M. Dexter (Eds.), Stem Cells, J. Ceil. Sci. Suppl. 10, 115-130. Bloom, W., and D.W. Fawcett (1975) A Textbook of Histology, 10th edn., Saunders, Philadelphia, PA. Francz, P.I., K. Bayreuther and H.P. Rodemann (1989) Cytoplasmic, nuclear, membrane-bound and secreted [35S]methionine-labelled polypeptide pattern in differentiating fibroblast stem cells in vitro, J. Cell. Sci., 92, 231-239. Gogol, J. (1991) Diplomarbeit, Universit~itHohenheim. Hapke, C. (1990) Diplomarbeit, Universit~itHohenheim. Hayflick, L. (1981) Cell death in vitro, in: I.D. Bowen and G. Lockshin (Eds.), Cell Death in Biology and Pathology, Chapman and Hall, London, pp. 243-285. Hayflick, L., and P.S. Moorhead (1961) The serial cultivation of human diploid cell strains, Exp. Cell. Res., 25,585-621. Kay, M.M.B. (1984) Localization of senescent cell antigen on band 3, Proc. Natl. Acad. Sci. (U.S.A.), 81, 5753-5757. Kontermann, K., and K. Bayreuther (1979) The cellular aging of rat fibroblasts in vitro is a differentiation process, Gerontology, 25, 261-274. Maier, M. (1991) Diplomarbeit, UniversitS_tHohenheim. Metcalf, D. (1977) Hemopoietic colonies, in: Recent Results in Cancer Research 61, Springer, Berlin. Met~alf, D. (1988) The Molecular Control of Blood Cells, Harvard University Press, Cambridge, MA. Mollenhauer, J.. and K. Bayreuther (1986) Donor age related changes in the morphology, growth potential, and collagen biosynthesis in rat fibroblast populations in vitro, Differentiation, 32, 165-172. Rodemann, H.P. (1989) Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin (MMC)-induced post-mitotic differentiation states in vitro, Differentiation, 42, 37-43. Rodemann, H.P., and K. Bayreuther (1984) Abnormal collagen metabolism in cultured human skin fibroblasts from patients with Duchenne muscular dystrophy, Proc. Natl. Acad. Sci. (U.S.A.), 81, 5130-5134. Rodemann, H.P., and K. Bayreuther (1986) Differential degradation of [35S]methioninepolypeptides in Duchenne muscular skin fibroblasts in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 83, 2086-2090. Rodemann, H.P., K. Bayreuther, P. Francz, K. Dittmann and M. Albiez (1989a) Selective enrichment and biochemical characterization of seven human skin fibroblast cell types in vitro, Exp. Cell Res., 180, 84-93. Rodemann, H.P., G. Pfleiderer and K. Bayreuther (1989b) The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields, Exp. Cell Res., 182, 610-621.
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© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50
MUTAGI 00164
Differentiation of primary and secondary fibroblasts in cell culture systems Klaus Bayreuther, Pal I. Francz, Jochen Gogol, Constanze Hapke, Monika Maier and Hans-Georg Meinrath Institut fiir Genetik, Unicersith't Hohenheim, D-7000 Stuttgart 70 (Germany) (Accepted 17 June 1991)
Keywords: Stem cell system; Terminal differentiation; Ageing; Apoptosis; Transformation: Genetic programme
Summary As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an l 1-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems. In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-$2-$3, three mitotic fibroblasts (MF) differentiate along the sequence MF I - M F I I - M F III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment. The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo. Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence. Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations.
Correspondence: Dr. K. Bayreuther, Institut fiir Genetik, UniversitS.t Hohenheim, D-7000 Stuttgart 70 (Germany).
Nowadays methods of molecular cell biology are available that allow the characterization of the biological and biochemical properties of the
234
cells making up secondary cell populations in vitro. A comparison of the characteristics of the cell types in secondary populations in vitro with those of the primary cell types studied in ex viva-in vitro systems is destined to bring forth information about the relatedness of the cell types in the secondary and primary cell systems. In addition, the investigation of cells in primary cell systems established and analyzed before they start to divide ex viva-in vitro, will permit deductions about the biological and biochemical characteristics of the fibroblast equivalent cell types making up the corresponding cell system in viva. Comparative studies of the cellular composition of primary fibroblast populations and the biological and biochemical properties of the primary cells as a function of the age of the donor in an ex viva-in vitro system and of the cellular composition of secondary fibroblast populations and biological and biochemical properties of the secondary cells as a function of the in vitro age of the population studied, will give an answer to the frequently discussed question about the value of secondary in vitro cell systems for the study of general and special problems of cell biology. Higher eukaryotic animal and human organisms are made up of about 200 cell systems (Bloom and Fawcett, 1975). The majority are cell systems with specialized functions, e.g., keratinoblasts/ keratinocytes. In addition, there exist two cell systems with helper functions. These are the glia cell systems of the brain and the fibroblasts of the connective tissues. It is very likely that all cell systems are stem cell systems with a design resembling that of the hemopoietic stem cell system (Metcalf, 1977, 1988). The rapid progress in the understanding of the molecular mechanisms and the molecular control of the differentiation of cells in the distinct branches of the hemopoietic stem cell system is the result of the strict employment of molecular methods specific for the stem cell systems in the secondary in vitro, in the primary ex viva-in vitro, and in the in viva populations studied (Metcalf, 1977, 1988). We have collected evidence that primary fibroblast populations (ex viva-in vitro) isolated from prenatal or postnatal connective tissue of skin and lung of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Mollenhauer
FibroblosLtronsformin9 com~rtment . ~ ~PMFVllb
CPO Celt number
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Fig. 1. Diagrammatic representation of the organization of the normal primary and secondary fibroblast stem cell system.
and Bayreuther, 1986), and man (Bayreuther et al., 1988b) develop as a function of the advancing in viva age of the donors along the progressive stages of a stem cell system. In addition, secondary fibroblast populations derived from the connective tissue of prenatal and postnatal skin and lung of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979), and man (Bayreuther et al., 1988a), including the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991), develop along a terminal differentiation sequence typical for stem cell systems, when mitotic stem cells and fibroblasts were passaged serially and when postmitotic fibroblasts were kept under appropriate conditions in stationary culture in vitro for longer periods of time (Figs. 1, 2). Results
Stem cell compartment Primary and secondary stem cells in citro Mesenchymal cells were isolated from the morphogenetic field of the wing of the Valo chicken at embryonic stage 20 and seeded into primary clonal culture. Primary clonal populations are serially subcloned for up to five times until heterogenous or homogenous clonal populations made up of cells with the characteristic morphology of fibroblasts, chondroblasts, and myoblasts could be distinguished. Primary and secondary clones studied showed the same [3~S]methionine polypeptide pattern of the nu-
235
Stem cell comportment
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b Fig. 2. Diagrammatic representation of the organization of the normal fibroblast stem cell system and the emerging transformed and neoplastically transformed cell system.
clear and cytoplasmic fraction, indicating that they are made up of identical cells, the stem cell S1. Tertiary clones exhibited three polypeptide pattern, one of the stem cell S1 and two new ones characterizing the stem cells S2a and S2b. In the fourth and fifth subcloning steps, clones with up to six different polypeptide marker combinations could be identified, three polypeptide patterns of S1, S2a, S2b, and three of the committed stem cells S3a, b, c for fibroblasts, or chondroblasts, or myoblasts. These findings make the following developmental pathway of the mesenchymal stem cells likely. S1 has developmental potential for fibroo blasts and chondroblasts plus fibroblasts and myoblasts. S1 proliferates and segregates two types of the stem cell $2, S2a with two potential functions for fibroblasts and chondroblasts, and S2b with developmental functions for fibroblasts and myoblasts. S2a and S2b proliferate again and
throw off three types of stem cells S3a, b, c. The $3 cells are committed stem cells with developmental quality for fibroblasts (S3a), chondroblasts (S3b), and myoblasts ($3c). The committed $3 cells move on to the progenitor compartments, in which the progenitor cells proliferate and develop along the fibroblast, the chondroblast, and the myoblast progenitor cell lineages. For these investigations, a new medium, serum, and factor composition had to be worked out, which supports the proliferation and differentiation of fibroblasts, chondroblasts, and myoblasts in a mixed population (Figs. 1, 2) (Hapke, 1990).
Fibroblast progenitor, maturing, degenerating, and transforming compartments Primary fibroblasts ex vivo-in vitro Primary human skin fibroblast populations were established ex vivo-in vitro from postnatal
236
A
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NTMFI NTMFII
Fig. 3. Development o• apoptosis and transformation in the C3H mouse fibroblast stem cell system ( A - F 120 x ). (A) Postmitotic fibroblast PMF VI. (B) Nuclear fragmentation in early stages of PMF VIIb. (C) Budding of nuclear fragments and small amounts of cytoplasm through the cell wall. (D) Transformed mitotic fibroblasts TMF II with PMF VI in the background. (E) Postmitotic fibroblast PMF VIIa in apoptosis with pycnotic nucleus and contracted cytoplast. (F) PMF VIIa in late stages of apoptosis, expressing senescent cell antigen band 3. (G) Specific [35S]methionine polypeptide markers in PMF VI, TMF I and TMF II, NTMF I and NTMF II, as indicated by arrow (pl: 6.3, Mr: 32 kDa).
237 abdominal skin of 45 female donors of four age groups: 1-20, 20-40, 40-60, and 60-90 years, with 19, 7, 8, and 11 donors respectively. Primary skin fibroblasts were studied in explant cultures, in mass cultures of low density, and in clonal populations ex vivo-in vitro. In these three systems, primary populations were found to be composed of eight fibroblast cell types, the mitotic fibroblasts (MF) MF I, MF II, MF III in the fibroblast progenitor compartment, the postmitotic fibroblasts (PMF) PMF IV, PMF V, PMF VI in the fibroblast maturing compartment, PMF VIIa in the fibroblast apoptosis compartment, and PMF VIIb in the transforming compartment (Figs. 1, 2). In primary populations, MF I is a small spindle-shaped cell, MF II is an epitheloid cell, and MF III is a larger epitheloid cell. The postmitotic fibroblast PMF IV is a large spindle-shaped cell with the morphology of a myofibroblast, PMF V is a large epitheloid cell, and PMF VI is the largest epitheloid cell. PMF VI is the terminally differentiated end cell. PMF V | I a is a cell with a heteropycnotic nucleus and a contracted, degenerating cytoplast. PMF VIIb is an epitheloid cell with 10-20 nuclear fragments that develop to become transformed mitotic fibroblasts T M F I by cellular budding (Figs. 1, 2, 3) (Bayreuther et al., 1988b). The composition of primary fibroblast populations that migrate from skin explants during the first 4-7 days is a function of the age of the donor. In this time period, the cells are established and have expressed their cell type-specific morphology and have not yet started to divide (ex vivo-in vitro). Such populations are always heterogenous with respect to their combination of cell types. Populations migrating out of the explant of a 7-year-old donor are predominantly made up of mitotic fibroblasts MF II plus 10-20% postmitotic fibroblasts (PMFs), those of a 37year-old donor are composed mainly of MF II and MF III plus 10-20% PMFs, those of an 57-year-old donor are made up of MF II and MF III plus 10-30% PMFs, and those of an 87-yearold donor are made up of 40% MF II and MF III plus 60% PMFs. 5 - 8 days after explantation, the MF II and MF III cells of the migrated populations begin to proliferate and to displace the postmitotic fibroblasts. As a result, all explants of
donors of different ages are surrounded by homogenous populations of MF II 14 days after explantation at the latest. These MF II populations are secondary fibroblast populations. The morphological and biochemical properties of these secondary MF II populations are identical, regardless of the age of the donor of the explant (Bayreuther et al., 1988b). Primary low density mass cultures of skin fibroblasts extracted from skin material of human donors of different ages by collagenase/trypsin treatment can be analyzed 4 - 7 days after seeding. At this time, the fibroblasts have expressed their morphological characteristics and have not yet started to divide (ex vivo-in vitro). Primary low density mass cultures were started from the same skin material from which the primary explant populations were set up. The age-dependent changes in the frequencies of primary mitotic and postmitotic fibroblasts in the low density mass cultures were similar or identical to those found in primary explant populations (Bayreuther et al., 1988b). As described for the primary explant populations, the primary low density mass cultures are made up of about 40% mitotic fibroblasts MF II and MF III, even when the skin material of the oldest donors is analyzed. From these mitotic cells, secondary fibroblast cell lines with a progenitor compartment with 60 CPDs and a maturing compartment of postmitotic fibroblasts in stationary culture for about 140 days have been established in vitro (Bayreuther, unpublished data). Five different clonetypes can be identified in primary clone cultures 14 days after seeding. These consist either of uniform populations of MF I in clone types of fibroblasts (CTF) CTF I, MF II in CTF II, and MF III in CTF III, or mixtures of MF I and MF II in CTF I / I I , and MF II plus MF III in clone type CTF I I / I I I . The age-dependent changes in primary clone type frequencies are similar to those found for the mitotic fibroblast cell types in primary explants and low density mass populations (in a strict sense, primary clones are made up of secondary fibroblasts) (Bayreuther et al., 1988b). Primary low density mass cultures of donors in the age group 20-40 years, which are predominantly made up of MF II and MF III, can be
238 experimentally induced by UV (Rodemann et al., 1989a) or MMC (Francz et al., 1989) to differentiate to the postmitotic populations of cell type PMF IV after 4 days, PMF V after 8 days, and PMF VI after 21 days. When the [35S]methionine polypeptide patterns of the pure primary subpopulations of MF I, MF II, MF III and experimentally induced pure primary populations of PMF IV, PMF V, and PMF VI are compared with those of secondary sub-populations of MF I, MF II, MF III and spontaneously arisen or experimentally induced secondary populations of PMF IV, PMF V, and PMF VI of the secondary human skin fibroblast cell line HH8, cell typespecific [35S]methionine polypeptide markers of the primary and secondary cell types are found to be identical (Bayreuther et al., 1988a,b; Bayreuther, unpublished data). Primary fibroblast populations from skin and lung of BN rats and C3H mice of different in vivo ages were studied in (ex vivo-in vitro) primary explant cultures, low density mass cultures and clone cultures. Again, three mitotic fibroblast (MF) cell types (MF I, MF II, MF III) and five postmitotic fibroblast (PMF) cell types (PMF IV, PMF V, PMF VI, PMF VIIa, and PMF VIIb) were found in the three primary cell systems studied. The age-dependent unidirectional changes in the frequencies of the fibroblast cell types in primary explant populations, in primary low density mass populations, and of mitotic fibroblasts in primary clone types are identical. In primary clone cultures, foci of transformed mitotic fibroblasts TMF I develop (Maier, 1991; Mollenhauer and Bayreuther, 1986). When dermis of mature male BN rats was sliced horizontally and fibroblasts were allowed to migrate from the sections in vitro, it was discovered that there exists a stratification of the fibroblasts MF I, MF II, and MF III in the reticular layer and of the postmitotic fibroblasts in the papillary layer with PMF VI located in the boundary zone underneath the basal lamina (Francz, unpublished data). When the [35S]methionine polypeptide patterns of pure primary subpopulations of MF I, MF II, MF III and of experimentally induced primary subpopulations of PMF IV, PMF V, and PMF VI are compared with those of secondary subpopulations of MF I,
MF II, MF III and spontaneously arisen or experimentally induced secondary subpopulations of PMF IV, PMF V, and PMF VI of secondary BN rat skin fibroblast cell lines, cell type-specific [-SSS]methionine polypeptide markers of the primary and secondary cell types are found to be identical (Mollenhauer and Bayreuther, 1986; Kontermann and Bayreuther, 1979; Francz, unpublished data).
Secondary fibroblasts in citro A secondary human dermal fibroblast population was established from abdominal skin of a female donor 8 years of age. Mitotic populations of this cell line, designated HH8, underwent 53.6 + 6.0 cumulative population doublings (CPD) in 302 _+ 27 days. When the growth capacity of the mitotic fibroblasts is exhausted and when appropriate methods are applied, mitotic fibroblasts do not degenerate in a Phase III phenomenon (Hayflick and Moorhead, 1961; Hayflick, 1981), but shift spontaneously to postmitotic fibroblasts. The maximal lifespan of postmitotic HH8 fibroblasts in stationary culture was 418_+ 35 additional days (Bayreuther et al., 1988a). Mitotic and postmitotic secondary fibroblast populations of HH8 are heterogenous with reproducible unidirectional changes in the proportions of cell stages of mitotic and postmitotic fibroblasts. Secondary fibroblasts have the same relative shapes and sizes as primary fibroblasts. The reiterated changes in the populations of mitotic and postmitotic fibroblasts in the secondary populations make it evident that secondary fibroblasts develop in one direction along the eightstage terminal cell lineage MF I - M F II-MF IIIPMF IV-PMF V-PMF VI-PMF V I I a / P M F VIIb in four compartments (Figs. 1, 2) (Bayreuther et aI., 1988a). As described for primary clonal populations, five different types of clones arise in the secondary clonal cultures of HH8. These are the pure clones CTF I, CTF II, and CTF III, and the heterogenous clones CTF I / I I and CTF II/III. Changes in the frequencies of the distinct clone types can be observed as a function of the CPD level of the heterogenous populations. Thus, the changes in cell type and clone type frequencies in secondary low density mass cultures and clonal
239
populations are a function of the CPD level of the mitotic mass populations, and the changes of the frequencies of the postmitotic fibroblast cell types are a function of the period of time of postmitotic mass populations in stationary culture, i.e., the so-called in vitro age of the populations (Bayreuther et al., 1988a,b). They are similar or identical to those found for the cell type frequencies in primary explant populations, low density mass populations and clonal populations, which are a function of the in vivo age of the donors (Figs. 1, 2)(Bayreuther et al., 1988b). The shifts in the frequencies of mitotic and postmitotic fibroblasts in secondary mass populations of HH8 are accompanied by reiterated alterations of biochemical parameters, for example in the [35S]methionine polypeptide pattern of the developing mass populations. The [ 35S]methionine polypeptide patterns of homogenous subpopulations of MF I, MF II, MF III, PMF IV, PMF V, and PMF VI, isolated from heterogenous mass populations by the clone-ring technique, revealed that the six fibroblast morphotypes studied express their cell type-specific polypeptide pattern in the heterogenous populations. Thus, the [3-SS]methionine polypeptide patterns of the mixed populations of the cell line HH8 are the sums of the polypeptide patterns of the distinct fibroblast cell types that make up the total population at different stages of development (Bayreuther et al., 1988a). For the analysis of the biological and biochemical changes accompanying the cyto-differentiation of the secondary fibroblasts of HH8 in the cell lineage, methods have been developed for the selective enrichment of the three mitotic and four postmitotic fibroblasts from the heterogenous mitotic populations (Francz et al., 1989; Rodemann et al., 1989a). Almost homogenous mass populations of MF II with 78-87% purity can routinely be obtained at CPD level 28-34, those of MF III with 73-86% purity at CPD level 48-53. Another cell system suitable for this purpose is the quantitative clonal system, with MF I in fibroblast clone type CTF I, MF II in CTF II, and MF III in CTF III, with mean sizes of 4303, 2352, and 302 cells per clone type (Rodemann et al., 1989a). In order to achieve a selective enrichment of four postmitotic fibroblast cell types,
methods have been worked out for the experimental induction of the differentiation from a lower state (MF II, MF III) to a higher state (PMF IV, PMF V, PMF VI, and PMF VII) in the lineage of HH8 by physical methods (UV (Rodemann et al., 1989a) and electromagnetic fields (Rodemann et al., 1989b)) and chemical agents (MMC (Francz et al., 1989)). The [35S]methionine polypeptide patterns of nearly homogenous sub- or total populations of the mitotic fibroblasts MF I, MF II, and MF III and their corresponding clonal populations CTF I, CTF II, and CTF III are identical (Bayreuther et al., 1988a, Francz et al., 1989; Rodemann et al., 1989a). Likewise postmitotic fibroblast cell types PMF IV, PMF V, PMF VI, and PMF VII, either spontaneously arisen or experimentally induced, show identical [35S]methionine polypeptide patterns for cell type-specific marker proteins (Bayreuther et al., 1988a; Rodemann et al., 1989a). In the lineage of MF II ~ PMF VI, 14 cell type-specific marker proteins have been identified in the nuclear and cytoplasmic fractions, 24 cell type-specific marker proteins in the membrane-bound fraction, and 11 cell type-specific marker proteins in the secreted protein fraction (Francz et al., 1989). Degenerating postmitotic fibroblasts PMF VIIa express a new set of cell type-specific [35S]methionine polypeptides, indicating that PMF VIIa degenerates under the control of an apoptosis program (Fig. 3E,F). Cells in late apoptosis express the modified band 3 (Kay, 1984; Francz, unpublished data). Macromolecule content and synthesis have been investigated in nearly homogenous mass populations of 75-88% purity of MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII of the cell line HH8 in an attempt to characterize the differentiating fibroblasts by other biochemical parameters. The DNA content, RNA synthesis, protein synthesis, collagen synthesis, and protein content were determined in fully contact regulated mitotic populations at CPD 30 (78-87% MF II) and at CPD 52 (73-86% MF liD, those of PMF IV, PMF V, PMF VI, and PMF VIIa (all at about 75-85% purity) in experimentally induced postmitotic fibroblasts in stationary cultures. The DNA content increases in the course of cyto-differentiation. MF II has 2C DNA, MF III and PMF IV
240 contain 4C DNA, PMF V 8C DNA, 64% of PMF VI have 8C DNA and the remaining 36% have 16C DNA. The degenerating PMF VIIa has DNA contents in the range of 8C-2C. In nearly homogenous mass populations of MF II, MF Ill, PMF IV, PMF V, and PMF VI, RNA synthesis increases by a factor 9 (MF II = factor 1), protein synthesis increases by a factor 4.8, collagen synthesis by 6.8, and total protein content by 11.5, when MF II and PMF VI are compared (Francz, unpublished data). Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and postmitotic states in vitro has been described for cell line HH8 (Rodemann, 1989). Secondary fibroblast populations of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979), and of the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991) also develop along the same cell biological and cell biochemical differentiation sequence in the progenitor, maturing, degenerating, and transforming compartments of the fibroblast stem cell system. Spontaneous transformation has been observed for primary and secondary populations of dermal fibroblasts of C3H mice (Maier, 1991) and BN rats (Kontermann and Bayreuther, 1979; Mollenhauer and Bayreuther, 1986) and in secondary fibroblasts of the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991). In all cell populations studied, transformation starts by the fragmentation of the nucleus of PMF VIIb into 10-20 micronuclei (Fig. 3B). The micronuclei surrounded by a small cytoplast bud through the cell membrane (Fig. 3C) and give rise to transformed cell lines (Fig. 3D) with three transformed mitotic fibroblasts (TMF) TMF I, T M F II, and TMF III and five transformed postmitotic fibroblasts (TPMF) T P M F IV, T P M F V, T P M F VI, T P M F VIIa, T P M F VIIb. In T P M F VIIb, nuclear fragmentation and cellular budding occur again. After the budding of the micronuclei plus cytoplast through the cell membrane, three neoplastically transformed mitotic fibroblasts (NTMF) N T M F I, N T M F II, and N T M F III and five neoplastically transformed postmitotic fibroblasts (NTPMF) N T P M F IV, N T P M F V, N T P M F VI, NTPMF VIIa, N T P M F VIIb are established (Fig. 2). In the transformed cell lines T M F II represents the
stem line with 87-91% of the cells, in neoplastically transformed cell lines, N T M F II makes up the stem line with 85-89% of the cells in the population. This concept of the developmental pathway of the transformed and neoplastically transformed fibroblasts is supported by the finding that a marker polypeptide, expressed for the first time in PMF VI, remains expressed in T M F I and T M F II and N T M F I and N T M F II (Fig. 3G). Discussion
Primary and secondary fibroblast cell systems of Valo chicken (Hapke, 1990), C3H mice (Maier, 1991), BN rats (Kontermann and Bayreuther, 1979; Mollenhauer and Bayreuther, 1986), and man (Bayreuther et al., 1988a,b), including the normal human embryonic lung fibroblast cell line WI38 (Gogol, 1991), have been studied with stem cell system-specific concepts and methods. The data show that all fibroblast cell systems studied are stem cell systems with 11 cells developing in an ll-stage differentiation sequence in five compartments of a stem cell system. In the stem cell compartment, stem cells (S) develop along the sequence S 1 - S 2 a / S 2 b - S 3 , in the fibroblast progenitor compartment mitotic fibroblasts (MF) progress along the lineage MF I - M F I I - M F III, in the fibroblast maturing compartment postmitotic fibroblasts (PMF) proceed in a line PMF I V - PMF V - P M F VI. PMF VI is the terminally differentiated end cell of the fibroblast stem cell system. PMF VI dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment (Figs. 1, 2, 3). When stem cell system-specific methods are applied in comparative studies of the cellular composition of primary and secondary fibroblast populations and of the biological and biochemical properties of the primary and secondary cells as a function of the age of the donor in an ex vivo-in vitro system or as a function of the in vitro age of the populations, it becomes evident that primary and secondary fibroblast cell systems are stem cell systems with an identical design. The reiterated appearance of the 11 cell types
241
in primary fibroblast populations of skin of 45 human donors, skin and lung of 26 BN rat donors, and the reiterated age-related changes in the cell type composition of the primary fibroblast populations (Bayreuther et al., 1988b; Mollenhauer and Bayreuther, 1986) make it very likely that mitotic fibroblast equivalents (MFE) and postmitotic fibroblast equivalents (PMFE) of the primary fibroblasts exist in vivo and that age-related changes in the frequencies of the fibroblast equivalents result from the differentiation along the cell lineage MFE I - M F E I I - M F E III and PMFE IV-PMFE V-PMFE VI-PMFE V I I a / P M F E VIIb in vivo. The development of the cells is under the control of a genetic program, which is expressed by the reproducible, unidirectional and cell typespecific changes of the [35S]methionine polypeptide pattern in the terminal differentiation cell lineage (Bayreuther et al., 1988a; Francz et al., 1989). In addition the mitotic and postmitotic fibroblasts have a cell type-specific DNA content, RNA synthesis, protein synthesis, collagen synthesis, and protein content (Francz, unpublished data). Normally the differentiation sequence proceeds spontaneously, but it can be speeded up by a number of physical and chemical agents (UV (Rodemann et al., 1989a), MMC (Francz et al., 1989)). Spontaneously arisen and experimentally induced fibroblast cell types have identical polypeptide patterns, macromolecule synthesis, and macromolecule content, indicating that normal terminal differentiation and experimentally speeded up terminal differentiation follow the same genetically programed pathway (Francz et al., 1989; Rodemann, 1989a). These data support the unifying concept of the molecular mechanisms of cellular aging and cellular neoplastic transformation (Kontermann and Bayreuther, 1979). According to this concept, normal physiological cellular aging is the result of the terminal differentiation of the fibroblasts in the fibroblast stem cell system, followed by either apoptosis or transformation. The course of the morphological and biochemical multistage differentiation sequence of fibroblasts can be speeded up by endogenous factors, e.g., Duchenne muscular dystrophy fibroblasts (Rodemann and Bayreuther, 1984, 1986), by exogenous factors such as UV
(Rodemann, 1989a) and mitomycin C (Francz et al., 1989), by compounds (sucrose) not degradable by lysosomes (Bayreuther, unpublished data), and by persistent infections with viruses (rubella virus) (Bayreuther, unpublished data). These conditions or treatments increase the probability of differentiation along the genetic program of the mitotic and postmitotic fibroblast cell lineage, and thus induce pathophysiological cellular aging. At higher in vitro age, the mitotic and postmitotic fibroblasts of the renewing progenitor and the maturing compartments in the dermal fibroblast stem cell systems of BN rats or man are completely depleted (Kontermann and Bayreuther, 1979; Bayreuther et al., 1988b). In contrast, the primary low density mass cultures of the oldest human donors are made up of about 40% mitotic fibroblasts MF II and MF III, which can give rise to secondary fibroblast cell lines in vitro with a proliferative capacity of 60 CPDs and postmitotic populations 140 days in stationary culture (Bayreuther, unpublished data). This finding is surprising, since the total number of the fibroblasts that can be extracted from a given volume of dermis decreases as a function of increasing donor age, by a factor 10 when dermis of a 7-year-old donor and an 87-year-old donor is compared. The decrease in the cell number of fibroblasts in situ in the presence of fibroblasts capable of proliferating in vitro makes it likely that the factors that normally regulate their proliferation in vivo have become abnormal in their qualitative a n d / o r quantitative composition as a function of the in vivo age of the dermis (Bayreuther, unpublished data). The terminally differentiated end cell PMF VI degenerates as PMF VIIa under the control of a genetic program by apoptosis. In spontaneously occurring or experimentally induced apoptosis, three new cell type PMF VIIa-specific [35S] methionine polypeptides are expressed that turn off the synthesis of all [35S]methionine polypeptides by a hitherto unknown mechanism. In later stages of apoptosis, the modified band 3 is expressed (Kay, 1984; Francz, unpublished data). The evolution of a molecular mechanism for the programed removal of terminally differentiated end cells is additional evidence for the genetically programed control of the balanced development
242 of the fibroblast in proliferating, m a t u r i n g , a n d degenerating compartments. In the stem cell systems of C 3 H mice (Maier, 1991), BN rats ( K o n t e r m a n n a n d Bayreuther, 1979; M o l l e n h a u e r a n d B a y r e u t h e r , 1986), a n d the h u m a n cell line WI38 (Gogol, 1991), transformed cells or neoplastically t r a n s f o r m e d cells arise by n u c l e a r f r a g m e n t a t i o n a n d cellular budding from P M F V I I b or T P M F VIIb. In transformed a n d neoplastically t r a n s f o r m e d cell lines, three mitotic ( T M F , N T M F ) a n d five postmitotic fibroblasts ( T P M F , N T P M F ) with cell biological a n d cell biochemical individuality can be identified (Fig. 2). In contrast to the n o r m a l fibroblasts, the progression along the d i f f e r e n t i a t i o n lineage of most of the t r a n s f o r m e d a n d neoplastically t r a n s f o r m e d fibroblasts is blocked at differentiation stages T M F II a n d N T M F II. Because of that, T M F II a n d N T M F II are the p r e d o m i n a n t cell types (stem line) with 84% a n d 79% cells of the p o p u l a t i o n s studied (Francz, u n p u b l i s h e d data). T h e s e data are in d i s a g r e e m e n t with the theory that assumes that carried on cells of the stem cell c o m p a r t m e n t give rise to sarcoma cells a n d stresses the i m p o r t a n c e of t e r m i n a l l y differe n t i a t e d or aged fibroblasts for the d e v e l o p m e n t of the t r a n s f o r m e d cells. F i b r o b l a s t p o p u l a t i o n s have so far always b e e n t r e a t e d as h o m o g e n o u s n o n - d i f f e r e n t i a t i n g cell p o p u l a t i o n s a n d most of the data o b t a i n e d are i r r e p r o d u c i b l e (Hayflick, 1981). W h e n stem cell system-specific concepts a n d m e t h o d s are applied, d e f i n e d fibroblast p o p u l a t i o n s can be m a d e available for qualitative a n d / o r q u a n t i t a t i v e studies of p r o b l e m s in g e n e r a l cell biology, b i o c h e m istry a n d virology, a n d more specialized issues like t e r m i n a l d i f f e r e n t i a t i o n , aging, apoptosis, a n d t r a n s f o r m a t i o n . Cell biological a n d biochemical studies of properly d e f i n e d fibroblast p o p u l a tions, i.e., pools of cell p o p u l a t i o n s with a verified c o n s t a n t c o m p o s i t i o n of fibroblast cell types a n d thus with identical biochemical p a r a m e t e r s , are d e s t i n e d to provide r e p r o d u c i b l e results in the field of fibroblast cell biology in future.
Aknowledgements S u p p o r t e d by grants of the D e u t s c h e Fors c h u n g s g e m e i n s c h a f t a n d the B u n d e s m i n i s t e r i u m ffir F o r s c h u n g u n d T e c h n o l o g i e .
References Bayreuther, K., H.P. Rodemann, R. Hommel, K. Dittmann, M. Albiez and P.I. Francz (1988a) Human skin fibroblasts in vitro differentiate along a terminal cell lineage, Proc. Natl. Acad. Sci. (U.S.A.), 85, 5112-5116. Bayreuther, K., H.P. Rodemann, P.1. Francz and K. Maier (1988b) Differentiation of fibroblast stem cells, in: B. Lord and T.M. Dexter (Eds.), Stem Cells, J. Ceil. Sci. Suppl. 10, 115-130. Bloom, W., and D.W. Fawcett (1975) A Textbook of Histology, 10th edn., Saunders, Philadelphia, PA. Francz, P.I., K. Bayreuther and H.P. Rodemann (1989) Cytoplasmic, nuclear, membrane-bound and secreted [35S]methionine-labelled polypeptide pattern in differentiating fibroblast stem cells in vitro, J. Cell. Sci., 92, 231-239. Gogol, J. (1991) Diplomarbeit, Universit~itHohenheim. Hapke, C. (1990) Diplomarbeit, Universit~itHohenheim. Hayflick, L. (1981) Cell death in vitro, in: I.D. Bowen and G. Lockshin (Eds.), Cell Death in Biology and Pathology, Chapman and Hall, London, pp. 243-285. Hayflick, L., and P.S. Moorhead (1961) The serial cultivation of human diploid cell strains, Exp. Cell. Res., 25,585-621. Kay, M.M.B. (1984) Localization of senescent cell antigen on band 3, Proc. Natl. Acad. Sci. (U.S.A.), 81, 5753-5757. Kontermann, K., and K. Bayreuther (1979) The cellular aging of rat fibroblasts in vitro is a differentiation process, Gerontology, 25, 261-274. Maier, M. (1991) Diplomarbeit, UniversitS_tHohenheim. Metcalf, D. (1977) Hemopoietic colonies, in: Recent Results in Cancer Research 61, Springer, Berlin. Met~alf, D. (1988) The Molecular Control of Blood Cells, Harvard University Press, Cambridge, MA. Mollenhauer, J.. and K. Bayreuther (1986) Donor age related changes in the morphology, growth potential, and collagen biosynthesis in rat fibroblast populations in vitro, Differentiation, 32, 165-172. Rodemann, H.P. (1989) Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin (MMC)-induced post-mitotic differentiation states in vitro, Differentiation, 42, 37-43. Rodemann, H.P., and K. Bayreuther (1984) Abnormal collagen metabolism in cultured human skin fibroblasts from patients with Duchenne muscular dystrophy, Proc. Natl. Acad. Sci. (U.S.A.), 81, 5130-5134. Rodemann, H.P., and K. Bayreuther (1986) Differential degradation of [35S]methioninepolypeptides in Duchenne muscular skin fibroblasts in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 83, 2086-2090. Rodemann, H.P., K. Bayreuther, P. Francz, K. Dittmann and M. Albiez (1989a) Selective enrichment and biochemical characterization of seven human skin fibroblast cell types in vitro, Exp. Cell Res., 180, 84-93. Rodemann, H.P., G. Pfleiderer and K. Bayreuther (1989b) The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields, Exp. Cell Res., 182, 610-621.