The effect of taurine on human fetal brain cells proliferation in tissue culture

The effect of taurine on human fetal brain cells proliferation in tissue culture

NUTRITION RESEARCH, Vol. 12, pp. 179-185,1992 0271-5317/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved...

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NUTRITION RESEARCH, Vol. 12, pp. 179-185,1992 0271-5317/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

THE EFFECT OF TAURINE ON HUMAN FETAL BRAIN CELLS PROLIFERATION IN TISSUE CULTURE Xiaobin Han 1 MD PhD, Chen Xue-Cun MD, *Zhiling Pang MD Institute of Nutrition and Food Hygiene Chinese Academy of Preventive Medicine 29 Nan Wei Road, Beijing, China. *Tianjin Medical College, Tianjin, China.

ABSTRACT The purpose of this study is to investigate the effect of taurine on the human fetal brain cells proliferation with the method of pure cerebral neuronal culture free of glial cells, grown in a serum-free environment. We found that taurine was necessary for neuronal survival, neurite extension. Taurine, on the other hand, has the trophic effect on the human fetal brain cells proliferation determined by [3H]-thymidine incorporation. These results establish taurine as a putative human fetal brain neurontrophic factor in the process of human brain development. KEY WORDS: Neuronal cell culture, Taurine, Brain development

INTRODUCTION The possible role of taurine in the developing mammalian nervous system has been the subject of much recent research and conjecture (1,2). It has been known for some time that the concentration of taurine in newborn mammalian brain is several-fold greater than in mature brain of the same species, and that the decrease occurred approximately by the time of weaning (3). The levels of taurine in human milk are 258 mg/1, about 10 times higher than that of the cow's milk (4). Gaull demonstrate that taurine must be provided to the infant from the milk (5). Infants fed various formulas have urine and plasma taurine concentration lower than infants fed pooled human milk (6-8). Further studies demonstrated that the taurine-depleted kittens had persistence of cells in the external granule cell layer associated with a reduced concentration of taurine. The establishment of pure neuronal culture, and identification of new growth factors using these culture, have been the milestones of contemporary neuronal development studies in the peripheral nervous system. A pure neuronal population grown in a defined chemical medium is also necessary for molecular investigations of brain neuronal development because of the obscure and uncontrollable environment surrounding neurons in the intact central nervous system, and on the investigation of the growth factor essential for the development of cerebral neurons in a serum-free environment.

Correspondence: Dr. Xiaobin Han, Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine, 29 Nan Wei Road, 100050 Beijing, China. 179

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MATERIAI~ AND METHODS

Cell culture Fragments of human fetal brain between the 12-14th week of gestation (estimated by menstrual history and ultrasonic examination) were obtained from legal abortions. Tissue from the brain stem was pooled separate from that of the cerebral hemispheres. The cerebral hemispheres were removed to HBSS medium to prepare the cell suspensions with the method of mechanical cell dissociation. Subsequently the cells were seeded on plastic culture dishes (35 mm) precoated with poly-D-lysine (Sigma, MW 8000; 1 mg/ml in borate buffer pH 8.3) by an overnight incubation followed by 3 washes with triple-distilled water. The seeding density was 3 x 10~ cells/ml in tube and 1 x 105 cells/ml in 24-well plate. For the initial 24h, the cells were maintained in a medium containing 10% fetal cattle serum (FCS). Thereafter the cultures were kept in a chemically defined medium that was changed every 3rd day. Cultures were maintained in an humidified atmosphere of 95% air/ 5% CO 2 at 37~ All results reported here were abstained from cells taken from a total of 10 fetuses (12-14 weeks). The chemically defined medium consisted of Minimum Essential Medium (MEM)/F-12 (1:1)(Sigma) with 1.2g of NaHCO 3. It was supplemented with the following constituents to give the following final concentration: transferrin 10 mg/ml, putrescine 100 #M, insulin 5 mg/dl, sodium selenite 30 nM.

Cell counts Cultured cells were visualized and photographed using a Nikon Diaphot inverted microscope equipped with phase-contrast and bright-field optics. Cell viability in suspensions of dissociated cells was determined by the ability of viable neurons to exclude the dye Trypan blue. Aliquots(four 100-#1 aliquots) of the cell suspension were mixed with 100 #1 of saline containing 0.8% Trypan blue; 10 minutes later stained and non-stained cells were counted with the aid of the microscope. Viability of cultured neurons was determined by morphological criteria as previously described (9); determinations were made on at least 4 separate cultures.

Electron microscopy After 3, 6, 9, 15, 18, 21 DIV, the cultures were studied by electron microscope. They were washed with Millonig's phosphate buffer and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in the same phosphate buffer for 1 hour. After washing, they were post-fixed with 1% osmium tetroxide for 1 hour and dehydrated in a graded series with ethanol and embedded in Spurr's resin. One #m semi-thin sections for light microscopy were stained with Toluidine blue. Thin sections were double-stained with uranyl acetate and lead citrate and observed at 50 kV in Hitachi HU-12 electron microscope.

[3H]-Thymidine incorporation Human fetal brain neuron cells were cultured in 24-well plate at 1 x 105 cells per well for 5 days in media with various concentrations of taurine. Following the incubation, cultures were

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incubated with [3H]-thymidine for the last 4 hour of cultures. The means and standard errors of the mean [SEMI of incorporated [3H]-thymidine were determined by liquid scintillation counting after cells were collected on glass-fibre filter. Statistics Data were analyzed by one-way analysis of variance. Dunnett's multiple variable test was used for specific post hoccomparisons. Student's t-test was used to compare two groups.

RESULTS Morphological aspect All cultures (60 mm diameter dishes) were observed under phase-contrast microscopy after plating. Neurite outgrowth began 2 hours after plating, and a neurite network was formed faster than with the control (FIG. 1). Cultures of cerebral hemisphere cells were maintained in the absence or presence of taurine and neuronal survival was determined by morphological criteria in the cultures on days 5,10,15,20,25 and 35. In untreated cultures there was a progressive loss of cells over the course of the experiment. The cells cultured in chemically defined medium without taurine survival for 4-5 weeks only, but cells survival was significantly enhanced in the presence at taurine from 5 weeks to 7 weeks. The ultrastructure of the cultured cells from experimental and control groups were different. In the presence of taurine, the neuronal cells were characterized by a round of oval-shaped nucleus with some heterochromatin. The perinuclear cytoplasm contained a lot of Golgi's complex, many elongated mitochondria, numerous polyribosomes, and multiple parallel stacks of rough endoplasmic reticulum(rER) resembling Nissl bodies. Abundant neuronal processes with well-defined, linearly arranged microtubule were found between the cell bodies. Occasionally, terminal boutons with clear vesicles were observed without synaptic contacts (FIG. 2). Effect of taurine on primary cerebral neuron cell survival

When added at the time of plating, taurine increased the number of surviving neuron cells at day 5 in vitro (FIG. 2) Significantly increased neuronal survival was obtained with 12.5-200 /~g/ml taurine ( P < 0.01) Effects of taurine on [3H]-thymidine incorporation in human fetal brain cell cultures

The effect of taurine on neuronal cell proliferation was assessed by [3H]-thymidine incorporation determined by liquid scintillation counting on the day 3, 6, 9,and 12 are plating. The cpm value of the neuronal cells in the presence of taurine is significantly higher than that

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(a)

(b)

(e)

(d)

(e)

(0

FIG. I Phase-contrast micrographs of cultured human fetal brain cells treated with or without taurine. Two-day-old cultures: (a). control; (b). taurine Four-day-old cultures: (c). control; (d). taurine Six-day-old cultures: (e). control; (f). taurine

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(a)

(b) FIG. 2 Electron microscopic appearance of neutonal cells in culture with taurine (a) or without taurine (b). Bipolar neuronal cell surrounded by abundant process; inset, terminal bouton with clear vesicles, typical rER stacks within the cytoplasm can be seen in the cultures added taurine (a). X 8000 of the control. A time course of taurine effect on thymidine incorporation is shown in a dose dependent manner in TABLE 1.

DISCUSSION Primary cultures of dissociated nervous tissue have been taken as good model systems in experimental cell research of the central nervous system. These cell cultures are potentially important tools for investigating morphological and biochemical aspects of nerve and glial cell differentiation, intracellular regulatory mechanisms, cellular interactions, and drug effects. Taurine appears to be essential for growth and survival of mammalian cells. Thus, lymphoblastoid cells do not grow in the absence of taurine (10), myocytes have poor viability

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in the absence of taurine (11), and taurine depletion causes degenerative changes in retinal cells. However, next to nothing was known of taurine in human brain development. The present study is the first time using cell culture of human fetal cerebral hemisphere neurons in investigating the effect of taurine on neuronal development. We found that neuronal survival was supported by taurine in human fetal cell cultures of cerebral hemispheres. The trophic action of taurine in the nerve fibre outgrowth and neuronal proliferation were also highly significant. The ability to study living neurons from developing human brain provides a means towards understanding the cellular and molecular mechanisms involved in human brain development and function. Our initial findings indicated that taurine could promote DNA synthesis of human fetal cerebral neurons. This finding suggested that taurine may act as a growth factor in the proliferation of human fetal brain cells.

TABLE 1

Effects of taurine on [3H]-thymidine incorporation in human fetal brain cell cultures taurine (ug/ml) 0 2.5 200 400

[3H]-thymidine (cpm/weU) 516 • 916 • 1282 • 1688 •

45 192" 76** 187"*

Results shown are the mean + S.E.M. of 3 independent experiments done in triplicate wells. * P < 0.01 vs. control ** P < 0.001 vs. control.

ACKNOWLEDGEMENTS This study was supported by the National Natural Science foundation of China, No 39070719. The authors would like to thank Dr. Wenzi Chang, Dr. Lianying Li, Dr. Li Liu and Ms Jiamei Chen for their technical assistance in the project.

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Sturman JA, Gaull GE. Taurine in the brain and liver of the developing human and monkey. J Neurochem 1975; 25:831-835.

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Gaull GE. Taurine in the nutrition of human infant. Acta Paediatr Scand Suppl 1982; 296:38-47.

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Gaull GE, Rassin DK, Raiha NCR, Heinonen K. Milk protein quantity and quality in low-birth-weight infants. III. Effects on sulfur amino acids in plasma and urine. J Pediatr 1977; 90:348-355.

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Rassin DK, Gaull GE, Jarvenpaa AL, Raiha NCR. Feeding the low-birth-weight infant. II Effects of taurine and cholesterol supplementation on amino acids and cholesterol. Pediatrics 1983; 71:179-186.

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Martensson J, Finnstrom O. Metabolic effects of a human milk adapted formula on sulfur amino acid degradation in full-term infants. Early Hum Dev 1985; 11:333-339.

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Mattson MP, Dou P, Kater SB. Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. J Neurosci 1988; 8:2087-2100.

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Gaull GE, Wright CE, Tallan HH. Taurine in human lymphoblastoid cells: Uptake and role in proliferation. In: Sulfur Amino Acids: Biochemical and Clinical Aspects, Eds K Kuriyama, RJ Huxtahle, H Iwata. Alan R Liss, Inc: New York 1983; pp 297-304.

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Hunter EG. Adult ventricular myocytes isolated from CHF 146 and CHF 147 cardiomyopathic hamsters. Can J Physiol Pharm 1986; 64:1503-1506.

Accepted for publication October 18, 1991.

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