Journal of Neuroscience Research 27:697-705 (1990)
Characterization of Adult Human Astrocytes Derived From Explant Culture M.L. Estes, R.M. Ransohoff, J.T. McMahon, B.S. Jacobs, and B.P. Barna Departments of Pathology (M.L.E., J.T.M.), Neurology (M.L.E., R.M.R.), Iinmunopathology (B.S.J., B.P.B.), and Molecular Biology (R.M.R.), and The Mellen Center for Multiple Sclerosis Treatment and Research (R.M.R.), Cleveland Clinic Foundation Four different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP( + ) cells also proliferated, bromo-deoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material. Key words: cell culture, glial fibrillary acidic protein, ultrastructure, proliferation
ing natural and pathologic phenomena such as persistent viral infection or growth regulation of glial cells (Ponten and MacIntyre, 1968; Gilden et al., 1976; Kim, 1985; Kennedy and Fok-Seang, 1986; Rutka et al., 1986). Cell cultures from adult human autopsy or biopsy brain have been initiated by a variety of techniques, and from a variety of pathologic substrates; not surprisingly, a wide range of cellular phenotypes have been described. To originate human brain cell cultures highly enriched for astrocytes, we have utilized a simplified explant method from surgical specimens obtained either during procedures for treatment of intractable epilepsy or disabling cerebellar tremor. Cultured cells have been used to study effects of inflammatory cytokines on glia, as well as astrocyte production of components of the CNS renini angiotensin system (Milsted ct al., 1990). In this report, we describe the morphology, ultrastructural attributes, growth characteristics, and immunocytochemical analysis of these cells.
INTRODUCTION A variety of functions have been attributed to astrocytes in vivo, including support of neuronal and oligodendroglial development, maintenance of ionic and metabolic homeostasis, and immunologic surveillance and effector function (Kimelberg and Norenberg, 1989). Nearly 20 years ago, it was observed that primary cultures of neonatal rodent brain could be enriched for astrocytes (Shein, 1965; Ponten and MacIntyre, 1968; Booher and Sensenbrenner, 1972). Such cultures have been studied intensively, in an attempt to correlate properties of cultured astrocytes with proposed functions in the intact CNS (Kimelberg, 1983). Techniques to enrich primary cultures for the growth of astrocytes have been well-suited to take advantage of the neonatal CNS as starting material; these have included selection of tissue for culture at times of maximal gliogenesis; or protocols for enzymatic dispersion of tissue followed by densitygradient centrifugation, differential adhesion, and antimetabolite treatment. More recently, similar cultures of human brain cells have been established, with the intention of study0 1990 Wiley-Liss, Inc.
MATERIALS AND METHODS Cell Cultures Three human glial cell lines (PlN, P2N, W3N) were established from temporal lobe tissue obtained during lobectomy for intractable epilepsy. One glial cell line (CAMS) was established from cerebral white matter obtained during thalamotomy for cerebellar tremor in a patient with severe, progressive multiple sclerosis. A human fibroblast cell line (WEF) was established from normal skin biopsy tissue taken for diagnostic purposes in a patient with a progressive myoclonic seizure disorder. Tissues were finely minced and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (PBS) (GIBCO, Grand Island, NY), l-glutamine and antibiotics, as previously described by Barna et a1 (1989a). Sera Received July 3. 1990; revised July 3 I , 1990; accepted July 3 1, 1990. Address Reprints to Melinda L. Estes, M.D., Cleveland Clinic Foundation. I Clinic Center (L25), Cleveland, OH 44195.
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Fig. 1. Crystal violet stained culture dish of P1N showing typical small astrocytic cells with central, oval nuclei, variable amounts of cytoplasm and short, thick cytoplasmic processes. Bar = 100 p. did not contain detectable levels of interferon activity and all reagents were endotoxin-free (less than 0.03 ng/ ml) . Monolayers of established cultures were dispersed with 0.05% trypsin in versene (GIBCO) and subcultured once to provide a stock of primary passage cells for cryogenic storage and characterization. Studies were carried out with second through fourth passage cells from cryogenic storage except where noted.
Immunofluorescence For immunofluorescence screening, coverslip cultures were acetone fixed (10 sec), blocked with 20% normal goat serum (30 min), and stained with polyclonal rabbit anti-bovine glial fibrillary acidic protein (GFAP) (Dako, Santa Barbara, CA; I :50 and 1 :100) followed by fluorescein isothiocyanate (FITC) conjugated goat antirabbit F(ab)'2 immunoglobulin (Accurate, Westbury, NY, 1:20). All cells were examined with fluorescence microscopy by a single obscrver (M.L.E.). Twenty high power fields ( X 200) were examined on each coverslip and the number of cells showing positive staining counted. This was expresed as a percentage of the total number of cells within the field; approximately 100 cells in 20 high power fields were examined. Additional immunofluorescence staining was performed using mouse monoclonal antihuman fibronectin (a gift from Dr. Robert H. Miller, Case Western Reserve University) (1 :50) and mouse monoclonal vimentin antibody (a gift from Dr. Robert H. Miller) (1:100). For each coversiip, control cells were examined using preimmune serum or without primary antibody. For double-label immunofluorescence analysis of cell-surface fibronectin and cytoplasmic GFAP, cells
Fig. 2 . Culture PIN was incubated with interferon gamma (10 Uiml) for 72 hrs. Cells were dispersed enzymatically and percentage HLA-DR expression was determined by flow cytometry as previously described (Barna et al., 1989a). Data represent means -C SEM for two to three experiments at each cell passage. were stained for 2.5 hr with mouse anti-human fibronectin monoclonal antisera (1:50), followed by tetramethylrhodamine isothiocyanate (TRITC) conjugated goat antimouse antisera (Sigma, 1:50), briefly washed, fixed in 70% ethanol, and stained with rabbit anti-bovine GFAP followed by goat anti-rabbit FITC conjugate. Experiments wcre done in parallel on coverslip cultures of fibroblasts and glial cells.
Dibutyryl Cyclic Adenosine Monophosphate (dbcAMP) Astrocytes, as opposed to fibroblasts, have been shown to undergo typical morphologic changes such as process elongation after exposure to dbcAMP (Shapiro, 1973). Cultures were washed free of culture medium and
Fig. 3. A: Cell line PIN demonstrating immunofluorescent cytoplasmic and process staining for GFAP. B: Paired field of PIN visualized by Nomarski differential interference contrast optics showing polygonal cells with central nuclei and broad elongate processes. C: Immunofluorescence staining of cell line PIN using preimmune serum as a negative control for GFAP. D: Paired field of PIN visualized by Nomarski differential interference contrast optics. E: GFAP immunoreactivity of cell line P2N. F: Paired phase contrast photomicrograph of cell line P2N showing several cells with abundant cytoplasm, round central nuclei, and elongate intertwining processes. G: Large glial cell in culture CAMS with GFAP immunoreactivity. H: Paired field of CAMS visualized by phase contrast microscopy demonstrating a typical large astrocyte with multiple, complex branched processes. A-H bar = 100 p.
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Fig. 4. Fibroblast cell line, WFN, displaying negative GFAP irnmunoreactivity. Bar = 100 p. incubated for 2 hr in serum-free RPMI 1640 containing 1.5 mM dbcAMP. Cells were then stained with 0.1% crystal violet for 30 min, washed with distilled water, and air-dried for microscopic evaluation of morphologic changes.
Light Microscopy Coverslip cultures were stained with hematoxylineosin or with crystal violet for morphologic examination. Sclected cells were photographed using Nomarski diff'erential-interference contrast microscopy or phase microsCOPY. Detection of Proliferating Cells Bromo-deoxyuridine (BrdU) incorporation was used to detect proliferating cells as described by Yong and Kim (1987) with modification. Cultures established on glass covcrslips in petri dishes were incubated in medium for 18 hr with 50 p M BrdU. Coverslips were then fixed in 70% ethanol and permeabilized in 0.1% Triton X- 100. After washing in phosphate-buffered saline (PBS), cells were treated for 10 min with 1 M HCI to denature native DNA, then neutralized in 0.1 M sodium borate, pH 9.2, for an additional 10 min. Coverslips were then immunostained sequentially for l-hr per stain with: rabbit anti-bovine GFAP, 1: 100 (Dako); FITC-conjugated goat anti-rabbit serum; monoclonal anti-BrdU (Becton-Dickinson, Mountain View, CA); and conjugated rabbit anti-mouse immunoglobulin. Two 5-minute washes in PBS followed each staining step. Coverslips were then mounted in 90% glycerol for microscopic determination of the percentage of GFAP-positive cells containing nuclear BrdU in 10 high power fields. Transmission Electron Microscopy Cells for transmission electron microscopy (TEM) were cultured on Permanox culture dishes (Miles, Na-
Fig. 5 . Double stained GFAP and fibronectin irnmunofluorescence cells in W3N. A: Cytoplasmic staining for GFAP is present. B: Fibronectin stained cells of W3N shuwing no immunoreactivity to fibronectin. A,B: bar = 100 p. perville, IL). The culture medium was replaced with 3.75% buffered glutaraldehyde and the cells fixed in the dishes for 2 hr. The cells were postfixed in 1% osmium tetroxide for 2 hr and embedded enbloc in Spurr's epoxy resin. Semithin (1 b) plastic embedded sections from selected blocks were stained with a mixture of toluidine blue and basic fuchsin. Blocks for TEM were selected and thin sections cut and subsequently stained with uranyl acetate and lead citrate. Blocks were examined using a Philips 400 T electron microscope.
RESULTS General Characteristics All glial cultures were heterogenous and contained cells of variablc s i a . Culturcs P l N , P2N, and W3N were similar. Both PIN and P2N were composed of greater than 90% small cells (1.5-40 nm). These cells were characterized by variable amounts of cytoplasm, central nuclei, and short, thick, branched processes (Fig.
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Fig. 6. Irnmunofluorescent staining of cell line W3N showing cytoplasmic positivity for vimentin. Bar = 100 p. 1). Cell line CAMS displayed similar cells comprising approximately 50% of all cells visualized. A smaller percentage of cells, less than 10% in cell lines P l N , P2N, and W3N, were large, 50-150 nm, pyramidal-shaped with abundant cytoplasm and large central nuclei. An occasional binucleate cell was observed. These cells, in culture CAMS, had extensive, complex branching processes (see Fig. 3H). Both glial cell types had processes radially distributed from the surface membrane. The fibroblast culture line (WEF) was distinctly Fig. 7. A: Culture W3N, stained with crystal violet. The cells different from the glial lines. The cells were spindle- show elongation of cytoplasmic processes and narrowing of shaped, flat, and grew in parallel arrays. Any processes cell bodies after dbcAMP treatment. B: Fibroblast cell line, WEF treated with dbcAMP, demonstrates elongate bipolar visualized were seen only at the poles of the cell. The cells growing in fascicles. No change in growth pattern is seen four glial cell cultures as well as the skin fibroblast line after dbcAMP treatment. A,B: bar = 100 p. demonstrated density-dependent inhibition of growth compatible with a non-neoplastic phenotype. Seeding cfficiencies in microtiter tray cultures were performed in three of the cell lines: P l N , 50 2 5%; P2N, 40 & 14%; and W3N, 81 L 7% (N = 4).Population doubling time P l N , P2N, W3N, and CAMS all demonstrated cytoplaswas calculated to be approximately 4 days in T-25 flasks mic staining for GFAP (Fig. 3). Percentage of GFAPinitially seeded with 200,000 cells. Cell cultures were positive cells for each culture was: P l N , 90%; P2N, finite, with gradual slowing of growth and acquisition of 80%; W3N, 90%; and CAMS, 75%. In contrast the fibroblast cell line, WFN, showed no cytoplasmic staining senescence by passages 7-9. Additional functional studies, specifically induc- with GFAP (Fig. 4). To further characterize the cells, fibronectin immution of expression of HLA-DR, also demonstrated gradnostaining was performed. The glial cultures showed no ual decline with senescence. Treatment with interferon membrane-associated fibronectin positivity in contrast to gamma was previously shown in early passage (2-4) the fibroblast culture, WFN; which demonstrated strong cells to result in membrane exprcssion of HLA-DR anpositive intracellular staining. Double staining for GFAP tigen (Barna et al., 1989a). Studies of culture PIN indiand fibronectin of the glial cell line W3N at passage 6 cated that by passage 6, HLA-DR induction was less showed GFAP-positive cells but not dual-labelled cells than 60% of that in passage 3 (Fig. 2). (Fig. 5 ) . Immunofluorescence The glial cell line W3N was also analyzed with All cell lines were stained for GFAP, the recog- vimentin immunostaining. In W3N, 80% of cells in culnized standard marker for astrocytes. The glial lines: ture demonstrated vimentin positivity (Fig. 6).
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4% of cells after 18 hr of culture demonstrated proliferation as measured by BrdU uptake and also showed GFAP positivity (Fig. 8). Proliferation of all four cell lines has been demonstrated by thymidine uptake and cell events (Barna et a!. , 1989b; Ransohoff et al., 1989).
TEM In order to evaluate ultrastructural features of astrocytes in tissue culture, all glial lines and the fibroblast cell line were examined by TEM. The cells in PIN, P2N, W3N, and CAMS all had similar ultrastructural features including: lobular nuclei with prominent chromatin, abundant cytoplasmic filaments (measuring approximately 10 nm in diameter), complex often Y-shaped mitochondria, glycogen pools, and dense bodies in varying numbers (Fig. 9). The cytoplasmic filaments often condensed in the perinuclcar region or near the surface membrane. No desmosomes or tonofilaments were seen. Scattered microtubules, measuring 20-2.5 nm in diameter, were present. The fibroblast cell line had elongate, bipolar cells with cytoplasmic filaments localized to the region immediately beneath the surface membrane. The nuclei were central, without lobulation, and contained a single nucleolus. No desmosomes were visualized.
DISCUSSION Fig. 8. Double-labelled imrnunocytochernistry of cell line P1N demonstrating: A: cytoplasmic GFAP positivity and B: nuclear BrdU positivity in the same astrocyte. A,B: bar = 100 P*..
dbcAMP Treatment Because astrocytes are known to exhibit characteristic morphologic changes after treatment with dbcAMP, glial lines were examined in this fashion. Examination of culture5 P l N , P2N, W3N, and CAMS after dbcAMP treatment indicated the presence of morphologic changes characteristic of astrocytes (Fig. 7A). Elongation of processes and narrowing of cell bodies were detectable in 25-5096 of cells after dbcAMP treatment. The fibroblast culture line (WEF) showed no morphologic changes in response to dbcAMP treatment (Fig. 7B). Indentity of Proliferating Cells Incorporation of BrdU uptake is a measure of cellular proliferation. Evaluation of coverslip cultures of P 1N glial cells by double-labelled immunocytochemistry demonstrated BrdU nuclear staining in GFAP-positive cells. We have previously reported that culture PIN expressed GFAP in >YO% of cells when stained with immunoperoxidase (Barna et al., 1989a). Approximately
Astrocyte-enriched cultures have proven to be valuable models for neurobiologists. However, correlation between in vitro and in vivo events, always tenuous, is rendered more difficult by the variable morphologic and functional phenotypes obtained in different laboratories by differing culture methods. In this regard, we have established astrocyte-enriched cultures of human brain from several pathologic substrates, and chardcterized features of these cultured cells to facilitate comparison with results described by other investigators. Additionally, our method is a simplified explant technique which allows establishment of cell lines from adult, human brain utilizing limited amounts of surgical material. The majority of cells in early passage in these cultures appeared to be astrocytic, as indicated by morphology, ultrastructure (lobular nuclei, branched mitochondria, and cytoplasmic filaments), and the presence of typical cytoplasmic GFAP immunoreactivity (Eng et a]. , 1971; Uyeda et al., 1972; Antanitus et al., 197.5; Bock et a]., 1977). The origin of proliferating astrocytes in cultures derived from mature brain has yet to be established (Norton et a]., 1988). In our cultures, the majority of cells stained positively for both GFAP (mean = 84%), and vimentin 80% indicating that the intermediate filaments were composed of both vimentin and GFAP, a situation which has been proposed to be characteristic of
Fig. 9. A-C: Electron micrographs of cell line P1N demonstrating a cell w i t h round nucleus and prominent chromatin (A), prominent cytoplasmic filaments of 10 p. in diameter (B), and complex, Y-shaped mitochondria intersparsed with abundant cytoplasmic dense bodies (C). A-C: bar = 1 F.
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immature astrocytcs in situ (Dahl, 1981; Bighami et al., 1982; Bovolenta et al., 1984). Norton et al. (1988) suggested that the occurrence of GFAP( + )himentin( ) cells in cultures of adult rodent brain could be accounted for by the proliferation in these cultures of vimentin( )/ GFAP( -) glial progenitors, followed by differentiation into a vimentin( +)/GFAP( + ) astrocytic phenotype. By analogy, it is possible that glial progenitors give rise to the proliferating GFAP( ) population in these cultures. It is interesting to speculate about the lineage of the GFAP( -) cells which comprise a minority population within our cultures initially, but which constitute an increasing fraction with higher passage number. The GFAP( -) cells appear identical morphologically and ultrastructurally to the GFAP( ) cells; furthermore, they are immunoreactive with monospecific antisera to vimentin to the same extent as the GFAP( l)cells. It appears unlikely that they are fibroblastic in origin, as indicated by their morphology, growth characteristics, and lack of cell-surface immunoreactivity with antisera to fibronectin. It is possible that they represent GFAP-nonexpressing glial cells; however, this question cannot be addressed without further criteria for identifying cells of glial lineage. Some characteristics of cells in the present study are consistent with the results reported previously by Rorke et a1 (1975), Gilden et a]. (1975, 1976) in their description of explant cultures of human adult brain cells. In particular, our cultures contained variable numbers of cells of widely-differing morphology by invertcdoptic phase microscopy, and the forms observed corresponded roughly to those described by Rorke et al. (1975). Additionally, like Rorke et al. (1975), we did not find that cell morphology or growth could be reliably predicted from the pathologic material from which the cultures were initiated. In the present study, the four glial cell lines had a mean immunoreactivity with GFAP antisera of 84% in early passages, similar to results described by Gilden et al. (1976). However, we noted a distinct diminution of GFAP( + ) cells with increasing passage, unlike Gilden et al. (1976). Furthermore, cultures initiated by our methods could not be maintained beyond the eighth or ninth passage, in contrast to the routine attainment of 10-20 passages by these investigators. Results of preliminary autoradiographic studies had indicated that 56% of GFAP-positive cells incorporated tritiated thymidine after 48 hr of culture. Shorter term BrdU studies also demonstrated that 4% of GFAP-positive cells had nuclear BrdU positivity. This degree of DNA synthesis is comparable to that reported by Yong and Kim (1987) for cultures of BrdU and GFAP-labelled human fetal astrocytes. Such results indicate that our culture conditions, although differing greatly from those
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of Yong and Kim, are nevertheless supportive of astrocyte proliferation. In additional functional studies. we previously demonstrated by double-labelling, that GFAP-positive human astrocytes expressed HLA-DR after exposure to interferon gamma (Barna et al., 1989a). This responsiveness to interferon gamma diminished with passage and appearcd to parallel a loss of GFAPpositive cells as discussed above. Our culture system differs markedly from that described by Rutka et al. (1986). The immunocytochemical and ultrastructural characteristics of cells described here [early predominance of GFAP( ) cells, complex mitochondria, abundant intermediate filaments] are not compatible with the leptomeningeal features [uniform predominance of fibronectin( ) cells, desmosomes, tonofilaments] reported in their investigation. Modulation of glial phenotype by manipulation of culture conditions is a well recognized phenomenon. Raff et al. (1983) demonstrated the serum dependence of glial progenitor cell differentiation into either astrocytes or oligodendrocytes. Similarly, several investigative groups have shown that the C-6 rat glioma cell line is capable of a range of astrocytic or oligodendrocytic phenotypes depending upon culture conditions (Liao et al., 1978; Maltese and Volpe, 1979). The effect of isolation technique on glial cell phenotype is less well understood and it is not clear, e.g., whether the spontaneous expression of HLA-DR found in some human astrocyte cultures is related to the source of material or isolation technique (Kim, 1985; Pulver et al., 1987). Because of the limitations in quantity of human material for culture, it remains critical to carefully analyze and preserve astrocyte populations derived from such sources. It will be important to determine whether functionally distinct subpopulations of astrocytes can be identified and characterized by such comparative studies of cell cultures obtained by differing culture methods.
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ACKNOWLEDGMENTS This work is supported in part by NIH 1454-HL (M.L.E.) and NCI-ROI CA 49950 (B .P.B.). R.M.R. is a Harry M . Weaver Neuroscience Scholar of the National Multiple Sclerosis Society. We are grateful to Dr. Robert H. Miller, Center for Neurosciences, Case Western Reserve University School of Medicine, for helpful discussion and for the gift of antisera; to Dr. Christine H. Block for her thoughtful review of the manuscript; and to Ms. Denise Egleton for secrelarial support.
REFERENCES Antanitus DS, Choi RH, Lapham LW (1975): lrnrnunofluorescence staining of astrocytes in-vitro using antiserum to glial fibrillary acidic protein. Brain Res 89:363-367.
Adult Human Astrocytes Barna BP, Chou SM, Jacobs B. Yen-Lieberman B. Ransohoff RM (1989a): Interferon-B impairs induction of HLA-DR antigen expression in cultured human astrocytes. J Neuroimmunol 23: 45-53. Barna B, Chou S, Bona S, Jacobs B, Estes ML, Ransohoff R (1989b): Tumor necrosis factor alpha (TNFa): mitogen cultured nonneoplastic human astrocytes. Ann Neurol 26: 148. Bignami A, Raju T, Dahl D (1982): Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons. Dev Biol 9 I :286-295. Bock E, Moller M, Nissen C, Sensenbrenner M (1977): Glial fibrillary acidic protein in primary astroglial cell cultures derived from newborn rat brain. FEBS Lett 83:207-211. Booher J, Sensenbrenner M (1972): Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2:97-105. Bovolenta P, Liem RKH, Mason CA (1984): Development of cerebellar astroglia: transitions in form and cytoskeletal content. Dev Biol 102:248-259. Dahl D (198 I): The vimentin-GFA protein transition in rat neuroglia cytoskeleton occurs at the time of myelination. J Neurosci Res 61741-748. Eng LF, Vanderhaegen JJ, Bignami A, Gerstl B (1971): .4n acidic protein isolated from fibrous astrocytes. Brain Res 28:35 1-354. Gilden DH, Devlin M , Wroblewska 2. Friedman H, Rorke LB, Santoli D, Koprowski H (1975): Human brain in tissue culture. 1. Acquisition, initial processing and establishment of brain cell cultures. J Comp Neurol 161:295-306. Gilden DH, Wroblewskia Z, Eng LF, Rorke LB (1976): Human brain in tissue culture. V. Identification of glial cells by immunofluorescence. J Neurol Sci 29: 177-184. Kennedy PG, Fok-Seang J (1986): Studies on the development, antigenic phenotype and function of human glial cells in tissue culture. Brain 109: 1261-1277. Kim S U (1985): Antigen expression by glia cells grown in culture. J Neuroimmunol 8:255-282. Kimelberg HE ( 1983): Primary astrocyte cultures: a key to astrocyte function. Cell Mol Neurobiol 3:l-16. Kimelberg HK, Norenberg MD (Apr 1989): Astrocytes. Scientific American. pp. 66-76. Liao CL. Eng LF, Hermann MM, Bensh KG (1978): Clial fibrillary acidic protein: solubility characteristics, relation to cell growth
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phases and cellular localization in rat C-6 glioma cells: an immunoradiometric and immunohistologic study. J Neurochem 3011 181-1186. Maltcse WA, Volpc JJ (1979): Induction of an oligodcndroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium. J Cell Physiol 101:459-469. Milsted A, Barna BP, Ransohoff RM, Brosnihan KB, Ferrario CM (1990): Astrocyte cultures derived from human brain tissue express angiotensinogen mKNA. Proc Natl Acad Sci USA (in press). Norton WT, Farooq M, Chiu FC, Bottenstein JE (1988): Pure astrocyte cultures derived from cells isolated from mature brain. Glia 1:403-414. Ponten J , Maclntyre EM (1968): Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:465486. Pulver M, Carrel S, Mach JP, de Tribolet N (1987): Cultured human fetal astrocytes can be induced by interferon-y to express HLADR. J Neuroimmunol 14: 123-133. Raff MC, Miller RH, Noble M (1983): A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 305:390-396. Ransohoff RM. Chou SM, Jacobs B, Rudick RA, Estes ML, Barna BP (1989): Cytokine effects on multiple sclerosis astrocytes. Presented at the Workshop on Genes and Susceptibility to Multiple Sclerosis. Rorke LB, Gilden DH, Wroblewska S , Santoli D (1975): Human brain in tissue culture. IV. Morphological characteristics. J Comp Neurol 161:329-340. Rutka JT, Kleppe-Hoifodt H , Emma DA, Giblin JR, Dougherty DV, McCulloch JR, deArmond SJ, Rosenblum ML (1986): Characterization of normal human brain cultures: evidence for the outgrowth of leptomeningeal cells. Lab Invest 55:71-85. Shapiro DL (1973): Morphological and biochemical alterations in foetal rat brain cells cultured in the presence of monobutyryl cyclic AMP. Nature 241:203-204. Shein HM (1965): Propagation of human fetal spongioblasts and astrocytes in dispersed cell cultures. Exp Cell Res 40554-569. Uyeda CT, Eng LF, Bignami AT (1972): Immunological study of the glial fibrillary acidic protein. Brain Res 37:81-89. Yong VW, Kim SU (1987): A new double labelling immunofluoresccnce technique for the determination of proliferation of human astrocytes in culture. J Neurosci Methods 21:9-16.
Characterization of Adult Human Astrocytes Derived From Explant Culture M.L. Estes, R.M. Ransohoff, J.T. McMahon, B.S. Jacobs, and B.P. Barna Departments of Pathology (M.L.E., J.T.M.), Neurology (M.L.E., R.M.R.), Iinmunopathology (B.S.J., B.P.B.), and Molecular Biology (R.M.R.), and The Mellen Center for Multiple Sclerosis Treatment and Research (R.M.R.), Cleveland Clinic Foundation Four different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP( + ) cells also proliferated, bromo-deoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material. Key words: cell culture, glial fibrillary acidic protein, ultrastructure, proliferation
ing natural and pathologic phenomena such as persistent viral infection or growth regulation of glial cells (Ponten and MacIntyre, 1968; Gilden et al., 1976; Kim, 1985; Kennedy and Fok-Seang, 1986; Rutka et al., 1986). Cell cultures from adult human autopsy or biopsy brain have been initiated by a variety of techniques, and from a variety of pathologic substrates; not surprisingly, a wide range of cellular phenotypes have been described. To originate human brain cell cultures highly enriched for astrocytes, we have utilized a simplified explant method from surgical specimens obtained either during procedures for treatment of intractable epilepsy or disabling cerebellar tremor. Cultured cells have been used to study effects of inflammatory cytokines on glia, as well as astrocyte production of components of the CNS renini angiotensin system (Milsted ct al., 1990). In this report, we describe the morphology, ultrastructural attributes, growth characteristics, and immunocytochemical analysis of these cells.
INTRODUCTION A variety of functions have been attributed to astrocytes in vivo, including support of neuronal and oligodendroglial development, maintenance of ionic and metabolic homeostasis, and immunologic surveillance and effector function (Kimelberg and Norenberg, 1989). Nearly 20 years ago, it was observed that primary cultures of neonatal rodent brain could be enriched for astrocytes (Shein, 1965; Ponten and MacIntyre, 1968; Booher and Sensenbrenner, 1972). Such cultures have been studied intensively, in an attempt to correlate properties of cultured astrocytes with proposed functions in the intact CNS (Kimelberg, 1983). Techniques to enrich primary cultures for the growth of astrocytes have been well-suited to take advantage of the neonatal CNS as starting material; these have included selection of tissue for culture at times of maximal gliogenesis; or protocols for enzymatic dispersion of tissue followed by densitygradient centrifugation, differential adhesion, and antimetabolite treatment. More recently, similar cultures of human brain cells have been established, with the intention of study0 1990 Wiley-Liss, Inc.
MATERIALS AND METHODS Cell Cultures Three human glial cell lines (PlN, P2N, W3N) were established from temporal lobe tissue obtained during lobectomy for intractable epilepsy. One glial cell line (CAMS) was established from cerebral white matter obtained during thalamotomy for cerebellar tremor in a patient with severe, progressive multiple sclerosis. A human fibroblast cell line (WEF) was established from normal skin biopsy tissue taken for diagnostic purposes in a patient with a progressive myoclonic seizure disorder. Tissues were finely minced and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (PBS) (GIBCO, Grand Island, NY), l-glutamine and antibiotics, as previously described by Barna et a1 (1989a). Sera Received July 3. 1990; revised July 3 I , 1990; accepted July 3 1, 1990. Address Reprints to Melinda L. Estes, M.D., Cleveland Clinic Foundation. I Clinic Center (L25), Cleveland, OH 44195.
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Fig. 1. Crystal violet stained culture dish of P1N showing typical small astrocytic cells with central, oval nuclei, variable amounts of cytoplasm and short, thick cytoplasmic processes. Bar = 100 p. did not contain detectable levels of interferon activity and all reagents were endotoxin-free (less than 0.03 ng/ ml) . Monolayers of established cultures were dispersed with 0.05% trypsin in versene (GIBCO) and subcultured once to provide a stock of primary passage cells for cryogenic storage and characterization. Studies were carried out with second through fourth passage cells from cryogenic storage except where noted.
Immunofluorescence For immunofluorescence screening, coverslip cultures were acetone fixed (10 sec), blocked with 20% normal goat serum (30 min), and stained with polyclonal rabbit anti-bovine glial fibrillary acidic protein (GFAP) (Dako, Santa Barbara, CA; I :50 and 1 :100) followed by fluorescein isothiocyanate (FITC) conjugated goat antirabbit F(ab)'2 immunoglobulin (Accurate, Westbury, NY, 1:20). All cells were examined with fluorescence microscopy by a single obscrver (M.L.E.). Twenty high power fields ( X 200) were examined on each coverslip and the number of cells showing positive staining counted. This was expresed as a percentage of the total number of cells within the field; approximately 100 cells in 20 high power fields were examined. Additional immunofluorescence staining was performed using mouse monoclonal antihuman fibronectin (a gift from Dr. Robert H. Miller, Case Western Reserve University) (1 :50) and mouse monoclonal vimentin antibody (a gift from Dr. Robert H. Miller) (1:100). For each coversiip, control cells were examined using preimmune serum or without primary antibody. For double-label immunofluorescence analysis of cell-surface fibronectin and cytoplasmic GFAP, cells
Fig. 2 . Culture PIN was incubated with interferon gamma (10 Uiml) for 72 hrs. Cells were dispersed enzymatically and percentage HLA-DR expression was determined by flow cytometry as previously described (Barna et al., 1989a). Data represent means -C SEM for two to three experiments at each cell passage. were stained for 2.5 hr with mouse anti-human fibronectin monoclonal antisera (1:50), followed by tetramethylrhodamine isothiocyanate (TRITC) conjugated goat antimouse antisera (Sigma, 1:50), briefly washed, fixed in 70% ethanol, and stained with rabbit anti-bovine GFAP followed by goat anti-rabbit FITC conjugate. Experiments wcre done in parallel on coverslip cultures of fibroblasts and glial cells.
Dibutyryl Cyclic Adenosine Monophosphate (dbcAMP) Astrocytes, as opposed to fibroblasts, have been shown to undergo typical morphologic changes such as process elongation after exposure to dbcAMP (Shapiro, 1973). Cultures were washed free of culture medium and
Fig. 3. A: Cell line PIN demonstrating immunofluorescent cytoplasmic and process staining for GFAP. B: Paired field of PIN visualized by Nomarski differential interference contrast optics showing polygonal cells with central nuclei and broad elongate processes. C: Immunofluorescence staining of cell line PIN using preimmune serum as a negative control for GFAP. D: Paired field of PIN visualized by Nomarski differential interference contrast optics. E: GFAP immunoreactivity of cell line P2N. F: Paired phase contrast photomicrograph of cell line P2N showing several cells with abundant cytoplasm, round central nuclei, and elongate intertwining processes. G: Large glial cell in culture CAMS with GFAP immunoreactivity. H: Paired field of CAMS visualized by phase contrast microscopy demonstrating a typical large astrocyte with multiple, complex branched processes. A-H bar = 100 p.
Fig. 3.
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Fig. 4. Fibroblast cell line, WFN, displaying negative GFAP irnmunoreactivity. Bar = 100 p. incubated for 2 hr in serum-free RPMI 1640 containing 1.5 mM dbcAMP. Cells were then stained with 0.1% crystal violet for 30 min, washed with distilled water, and air-dried for microscopic evaluation of morphologic changes.
Light Microscopy Coverslip cultures were stained with hematoxylineosin or with crystal violet for morphologic examination. Sclected cells were photographed using Nomarski diff'erential-interference contrast microscopy or phase microsCOPY. Detection of Proliferating Cells Bromo-deoxyuridine (BrdU) incorporation was used to detect proliferating cells as described by Yong and Kim (1987) with modification. Cultures established on glass covcrslips in petri dishes were incubated in medium for 18 hr with 50 p M BrdU. Coverslips were then fixed in 70% ethanol and permeabilized in 0.1% Triton X- 100. After washing in phosphate-buffered saline (PBS), cells were treated for 10 min with 1 M HCI to denature native DNA, then neutralized in 0.1 M sodium borate, pH 9.2, for an additional 10 min. Coverslips were then immunostained sequentially for l-hr per stain with: rabbit anti-bovine GFAP, 1: 100 (Dako); FITC-conjugated goat anti-rabbit serum; monoclonal anti-BrdU (Becton-Dickinson, Mountain View, CA); and conjugated rabbit anti-mouse immunoglobulin. Two 5-minute washes in PBS followed each staining step. Coverslips were then mounted in 90% glycerol for microscopic determination of the percentage of GFAP-positive cells containing nuclear BrdU in 10 high power fields. Transmission Electron Microscopy Cells for transmission electron microscopy (TEM) were cultured on Permanox culture dishes (Miles, Na-
Fig. 5 . Double stained GFAP and fibronectin irnmunofluorescence cells in W3N. A: Cytoplasmic staining for GFAP is present. B: Fibronectin stained cells of W3N shuwing no immunoreactivity to fibronectin. A,B: bar = 100 p. perville, IL). The culture medium was replaced with 3.75% buffered glutaraldehyde and the cells fixed in the dishes for 2 hr. The cells were postfixed in 1% osmium tetroxide for 2 hr and embedded enbloc in Spurr's epoxy resin. Semithin (1 b) plastic embedded sections from selected blocks were stained with a mixture of toluidine blue and basic fuchsin. Blocks for TEM were selected and thin sections cut and subsequently stained with uranyl acetate and lead citrate. Blocks were examined using a Philips 400 T electron microscope.
RESULTS General Characteristics All glial cultures were heterogenous and contained cells of variablc s i a . Culturcs P l N , P2N, and W3N were similar. Both PIN and P2N were composed of greater than 90% small cells (1.5-40 nm). These cells were characterized by variable amounts of cytoplasm, central nuclei, and short, thick, branched processes (Fig.
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Fig. 6. Irnmunofluorescent staining of cell line W3N showing cytoplasmic positivity for vimentin. Bar = 100 p. 1). Cell line CAMS displayed similar cells comprising approximately 50% of all cells visualized. A smaller percentage of cells, less than 10% in cell lines P l N , P2N, and W3N, were large, 50-150 nm, pyramidal-shaped with abundant cytoplasm and large central nuclei. An occasional binucleate cell was observed. These cells, in culture CAMS, had extensive, complex branching processes (see Fig. 3H). Both glial cell types had processes radially distributed from the surface membrane. The fibroblast culture line (WEF) was distinctly Fig. 7. A: Culture W3N, stained with crystal violet. The cells different from the glial lines. The cells were spindle- show elongation of cytoplasmic processes and narrowing of shaped, flat, and grew in parallel arrays. Any processes cell bodies after dbcAMP treatment. B: Fibroblast cell line, WEF treated with dbcAMP, demonstrates elongate bipolar visualized were seen only at the poles of the cell. The cells growing in fascicles. No change in growth pattern is seen four glial cell cultures as well as the skin fibroblast line after dbcAMP treatment. A,B: bar = 100 p. demonstrated density-dependent inhibition of growth compatible with a non-neoplastic phenotype. Seeding cfficiencies in microtiter tray cultures were performed in three of the cell lines: P l N , 50 2 5%; P2N, 40 & 14%; and W3N, 81 L 7% (N = 4).Population doubling time P l N , P2N, W3N, and CAMS all demonstrated cytoplaswas calculated to be approximately 4 days in T-25 flasks mic staining for GFAP (Fig. 3). Percentage of GFAPinitially seeded with 200,000 cells. Cell cultures were positive cells for each culture was: P l N , 90%; P2N, finite, with gradual slowing of growth and acquisition of 80%; W3N, 90%; and CAMS, 75%. In contrast the fibroblast cell line, WFN, showed no cytoplasmic staining senescence by passages 7-9. Additional functional studies, specifically induc- with GFAP (Fig. 4). To further characterize the cells, fibronectin immution of expression of HLA-DR, also demonstrated gradnostaining was performed. The glial cultures showed no ual decline with senescence. Treatment with interferon membrane-associated fibronectin positivity in contrast to gamma was previously shown in early passage (2-4) the fibroblast culture, WFN; which demonstrated strong cells to result in membrane exprcssion of HLA-DR anpositive intracellular staining. Double staining for GFAP tigen (Barna et al., 1989a). Studies of culture PIN indiand fibronectin of the glial cell line W3N at passage 6 cated that by passage 6, HLA-DR induction was less showed GFAP-positive cells but not dual-labelled cells than 60% of that in passage 3 (Fig. 2). (Fig. 5 ) . Immunofluorescence The glial cell line W3N was also analyzed with All cell lines were stained for GFAP, the recog- vimentin immunostaining. In W3N, 80% of cells in culnized standard marker for astrocytes. The glial lines: ture demonstrated vimentin positivity (Fig. 6).
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4% of cells after 18 hr of culture demonstrated proliferation as measured by BrdU uptake and also showed GFAP positivity (Fig. 8). Proliferation of all four cell lines has been demonstrated by thymidine uptake and cell events (Barna et a!. , 1989b; Ransohoff et al., 1989).
TEM In order to evaluate ultrastructural features of astrocytes in tissue culture, all glial lines and the fibroblast cell line were examined by TEM. The cells in PIN, P2N, W3N, and CAMS all had similar ultrastructural features including: lobular nuclei with prominent chromatin, abundant cytoplasmic filaments (measuring approximately 10 nm in diameter), complex often Y-shaped mitochondria, glycogen pools, and dense bodies in varying numbers (Fig. 9). The cytoplasmic filaments often condensed in the perinuclcar region or near the surface membrane. No desmosomes or tonofilaments were seen. Scattered microtubules, measuring 20-2.5 nm in diameter, were present. The fibroblast cell line had elongate, bipolar cells with cytoplasmic filaments localized to the region immediately beneath the surface membrane. The nuclei were central, without lobulation, and contained a single nucleolus. No desmosomes were visualized.
DISCUSSION Fig. 8. Double-labelled imrnunocytochernistry of cell line P1N demonstrating: A: cytoplasmic GFAP positivity and B: nuclear BrdU positivity in the same astrocyte. A,B: bar = 100 P*..
dbcAMP Treatment Because astrocytes are known to exhibit characteristic morphologic changes after treatment with dbcAMP, glial lines were examined in this fashion. Examination of culture5 P l N , P2N, W3N, and CAMS after dbcAMP treatment indicated the presence of morphologic changes characteristic of astrocytes (Fig. 7A). Elongation of processes and narrowing of cell bodies were detectable in 25-5096 of cells after dbcAMP treatment. The fibroblast culture line (WEF) showed no morphologic changes in response to dbcAMP treatment (Fig. 7B). Indentity of Proliferating Cells Incorporation of BrdU uptake is a measure of cellular proliferation. Evaluation of coverslip cultures of P 1N glial cells by double-labelled immunocytochemistry demonstrated BrdU nuclear staining in GFAP-positive cells. We have previously reported that culture PIN expressed GFAP in >YO% of cells when stained with immunoperoxidase (Barna et al., 1989a). Approximately
Astrocyte-enriched cultures have proven to be valuable models for neurobiologists. However, correlation between in vitro and in vivo events, always tenuous, is rendered more difficult by the variable morphologic and functional phenotypes obtained in different laboratories by differing culture methods. In this regard, we have established astrocyte-enriched cultures of human brain from several pathologic substrates, and chardcterized features of these cultured cells to facilitate comparison with results described by other investigators. Additionally, our method is a simplified explant technique which allows establishment of cell lines from adult, human brain utilizing limited amounts of surgical material. The majority of cells in early passage in these cultures appeared to be astrocytic, as indicated by morphology, ultrastructure (lobular nuclei, branched mitochondria, and cytoplasmic filaments), and the presence of typical cytoplasmic GFAP immunoreactivity (Eng et a]. , 1971; Uyeda et al., 1972; Antanitus et al., 197.5; Bock et a]., 1977). The origin of proliferating astrocytes in cultures derived from mature brain has yet to be established (Norton et a]., 1988). In our cultures, the majority of cells stained positively for both GFAP (mean = 84%), and vimentin 80% indicating that the intermediate filaments were composed of both vimentin and GFAP, a situation which has been proposed to be characteristic of
Fig. 9. A-C: Electron micrographs of cell line P1N demonstrating a cell w i t h round nucleus and prominent chromatin (A), prominent cytoplasmic filaments of 10 p. in diameter (B), and complex, Y-shaped mitochondria intersparsed with abundant cytoplasmic dense bodies (C). A-C: bar = 1 F.
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immature astrocytcs in situ (Dahl, 1981; Bighami et al., 1982; Bovolenta et al., 1984). Norton et al. (1988) suggested that the occurrence of GFAP( + )himentin( ) cells in cultures of adult rodent brain could be accounted for by the proliferation in these cultures of vimentin( )/ GFAP( -) glial progenitors, followed by differentiation into a vimentin( +)/GFAP( + ) astrocytic phenotype. By analogy, it is possible that glial progenitors give rise to the proliferating GFAP( ) population in these cultures. It is interesting to speculate about the lineage of the GFAP( -) cells which comprise a minority population within our cultures initially, but which constitute an increasing fraction with higher passage number. The GFAP( -) cells appear identical morphologically and ultrastructurally to the GFAP( ) cells; furthermore, they are immunoreactive with monospecific antisera to vimentin to the same extent as the GFAP( l)cells. It appears unlikely that they are fibroblastic in origin, as indicated by their morphology, growth characteristics, and lack of cell-surface immunoreactivity with antisera to fibronectin. It is possible that they represent GFAP-nonexpressing glial cells; however, this question cannot be addressed without further criteria for identifying cells of glial lineage. Some characteristics of cells in the present study are consistent with the results reported previously by Rorke et a1 (1975), Gilden et a]. (1975, 1976) in their description of explant cultures of human adult brain cells. In particular, our cultures contained variable numbers of cells of widely-differing morphology by invertcdoptic phase microscopy, and the forms observed corresponded roughly to those described by Rorke et al. (1975). Additionally, like Rorke et al. (1975), we did not find that cell morphology or growth could be reliably predicted from the pathologic material from which the cultures were initiated. In the present study, the four glial cell lines had a mean immunoreactivity with GFAP antisera of 84% in early passages, similar to results described by Gilden et al. (1976). However, we noted a distinct diminution of GFAP( + ) cells with increasing passage, unlike Gilden et al. (1976). Furthermore, cultures initiated by our methods could not be maintained beyond the eighth or ninth passage, in contrast to the routine attainment of 10-20 passages by these investigators. Results of preliminary autoradiographic studies had indicated that 56% of GFAP-positive cells incorporated tritiated thymidine after 48 hr of culture. Shorter term BrdU studies also demonstrated that 4% of GFAP-positive cells had nuclear BrdU positivity. This degree of DNA synthesis is comparable to that reported by Yong and Kim (1987) for cultures of BrdU and GFAP-labelled human fetal astrocytes. Such results indicate that our culture conditions, although differing greatly from those
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of Yong and Kim, are nevertheless supportive of astrocyte proliferation. In additional functional studies. we previously demonstrated by double-labelling, that GFAP-positive human astrocytes expressed HLA-DR after exposure to interferon gamma (Barna et al., 1989a). This responsiveness to interferon gamma diminished with passage and appearcd to parallel a loss of GFAPpositive cells as discussed above. Our culture system differs markedly from that described by Rutka et al. (1986). The immunocytochemical and ultrastructural characteristics of cells described here [early predominance of GFAP( ) cells, complex mitochondria, abundant intermediate filaments] are not compatible with the leptomeningeal features [uniform predominance of fibronectin( ) cells, desmosomes, tonofilaments] reported in their investigation. Modulation of glial phenotype by manipulation of culture conditions is a well recognized phenomenon. Raff et al. (1983) demonstrated the serum dependence of glial progenitor cell differentiation into either astrocytes or oligodendrocytes. Similarly, several investigative groups have shown that the C-6 rat glioma cell line is capable of a range of astrocytic or oligodendrocytic phenotypes depending upon culture conditions (Liao et al., 1978; Maltese and Volpe, 1979). The effect of isolation technique on glial cell phenotype is less well understood and it is not clear, e.g., whether the spontaneous expression of HLA-DR found in some human astrocyte cultures is related to the source of material or isolation technique (Kim, 1985; Pulver et al., 1987). Because of the limitations in quantity of human material for culture, it remains critical to carefully analyze and preserve astrocyte populations derived from such sources. It will be important to determine whether functionally distinct subpopulations of astrocytes can be identified and characterized by such comparative studies of cell cultures obtained by differing culture methods.
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ACKNOWLEDGMENTS This work is supported in part by NIH 1454-HL (M.L.E.) and NCI-ROI CA 49950 (B .P.B.). R.M.R. is a Harry M . Weaver Neuroscience Scholar of the National Multiple Sclerosis Society. We are grateful to Dr. Robert H. Miller, Center for Neurosciences, Case Western Reserve University School of Medicine, for helpful discussion and for the gift of antisera; to Dr. Christine H. Block for her thoughtful review of the manuscript; and to Ms. Denise Egleton for secrelarial support.
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