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Expression of markers for both neuronal and glial cells in human amniotic epithelial cells
ELSEVIER
Neuroscience Letters 209 (1996) 9-12
NEUROSCIEHCf I[IIHS
Expression of markers for both neuronal and glial cells in human amniotic epithelial cells N o r i o S a k u r a g a w a a,*, R a m a s a m y T h a n g a v e l a, M a s a s h i M i z u g u c h i b, M o t o y u k i Hirasawa% I s a o K a m o ~ aDepartment ~f lnherited Metabolic Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan bDepartment of Mental Retardation and Birth De)%ctResearch, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan CDepartment of Ultrastructure Research, NationalInstitute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan Received 9 February 1996; revised version received 21 March 1996; accepted 22 March 1996
Abstract
Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. We attempted to detect various developmental antigens specific to neural cells by immunocytochemical methods. The cultured HAE cells displayed positive immunoreactivity to RCI, vimentin, A2B5, neurofilament proteins, microtubule-associated protein 2 (MAP2) and MAP2 kinase. In addition, the cells also demonstrated immunoreactivity to glial fibrillary acidic protein, CNPase, myelin basic protein and galactocerebroside. The appearance rate of positive cells was more than 50% in cells positive to RC1, A2B5, vimentin or neuronal markers, and 20--30% to glial cell markers. Double staining showed the heterogeneous appearance of oligodendrocyte lineage cells. These data indicate that HAE cells may have the putative multipotentiality of neurons, astrocytes and oligodendrocytes.
Keywords: Human amniotic epithelial cells; Neuronal markers; Glial markers; RC1; A2B5; Vimentin
From the embryological point of view, the amnion is composed of a single layer of epithelial cells which are in direct contact with the amniotic fluid. At about the 8th day after fertilization, a small cavity appears within the epiblast. This cavity enlarges to become the amniotic cavity. Human amniotic epithelial (HAE) cells are formed from amnioblasts which are adjacent to the cytotrophoblasts and line the amniotic cavity together with the rest of the epiblast. Accordingly, HAE cells may have the potentiality to differentia~te into various organs such as the brain, heart and liver. We report in this paper the immunocytochemical characteristics of HAE cells with a view to their putative multipotentiality of neural cells. The human amniotic epithelial tissue was mechanically peeled free from the chorion of a placenta obtained from an uncomplicated electiw~ cesarean section. The HAE cell layer was extensively scraped out to remove the underly-
* Corresponding author. Tel.: +81 423 412711, ext. 5151; fax: +81 423 461746.
ing tissues such as the spongy and fibroblast layers to eventually obtain a pure epithelial layer with basement membranes. The layer was then treated with 0.25% trypsin three times each for 20 min. HAE cells, obtained usually after the third treatment, were cultured in RPMI1640 medium supplemented with 10% fetal calf serum under a humidified atmosphere of 5% CO2 in air at 37°C. Cells grown on coverslips seated in a 24 well dish were immunocytochemically stained as follows. Unfixed cells were incubated with primary antibodies for 10 min. Then the cells were fixed with 4% paraformaldehyde in PBS(-) for 10 min at room temperature, followed by incubation with primary antibodies for 1 h. The coverslips were treated with secondary antibodies, biotinylated antimouse IgG (1:200; Vector) for 1 h, and then with peroxidase-conjugated streptavidin (Nichirei, Tokyo) for 1 h. The coverslips were washed again, followed by application of 0.2 mg/ml diaminobenzidine (Dojin, Osaka) in 0.05 M Tris-HCl (pH 7.4), to which H202 was added immediately before application to the slides. The slides
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 5 9 9 - 2
IV. Sakuragawa et al. / Neuroscience Letters 209 (1996) 9-12
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N. Sakuragawa et al. / Neuroscience Letters 209 (1996) 9-12
were counterstained with Harris hematoxylin for 30 s, then washed with water. The following primary antibodies served as neuronal markers: mouse monoclonal antibodies (mAb) to neurofilament proteins (NF) (1:20; Serotec), microtubule-associated protein 2 (MAP2) (1:20; Chemicon), and MAP2 kinase (MK) (1:500; PharMingen). To identify developing glial cells, antibodies to glial fibrillary acidic protein (GFAP) (1:2; Serotec) and 2':3'cyclic nucleotide 3'-phosphodiesterase (CNPase) (1:50; Sigma) were used. Mouse mAb, RC1 (no dilution; a gift from Dr. Yamamoto), 'was used to label cortical radial glia. Vimentin (1:50; DAKO) served as a marker of neuronal or glial precursor cells, O-2A progenitor cells or type 2 astrocytes [6]. The mouse monoclonal A2B5 hybridoma supernatant (1:5; a gift from Dr. Kikuchi) was used to detect A2B5(+) aeurons, O-2A progenitor cells or type 2 astrocytes. To :~tain oligodendrocytes, the cells were first incubated with rabbit polyclonal Ab to myelin basic protein (MBP) (1[:200; DAKO) and rabbit polyclonal Ab to galactocerebroside (GC) (1:50; Sigma), followed by treatment with fluorescein-conjugated goat antirabbit IgG (1:50; Zymed) for 1 h. Double immunofluorescence staining was performed using A2B5 (visualized with rhodamine (TRITC)) and GC (visualized with fluorescein). Immunoblotting was done on an Immobilon polyvinylidene difluoride membrane (Millipore) according to a previously described method [7]. HAE cells displayed immunoreactivity to NF, MAP2 (Fig. 1A,B) and M K (data not shown). Antigenicity to MAP2 and M K was also detected by immunoblotting, which indicated the presence of bands of 300 kDa and 42 kDa, respectively (Fig. 2). Immunoreactivity to vimentin was also found in the cells (Fig. IF). Vimentin is initially expressed in nearly all neuronal precursor cells in vivo, and is gradually replaced by neurofilamew~ subunits shortly before the immature neurons become postmitotic [3,11]. Microtubules arise early in neuronal development, usually long before neurofilaments [9,10]. HAE cells were shown to contain NF, MAP2 and vimentin, suggesting that these cells differentiated to acquire the expression of neuronal markers. The cytoskeleton of HAE cells labeled strongly with RC 1 (Fig. 1G), indicating the presence of cortical radial glia [4]. GFAP (Fig. 112) was positively stained in the HAE cells and a band at 51 kDa was detected on the blots of HAE cell extracts (Fitg. 2), indicating the presence of cells of astrocytic lineage. Also, HAE cells displayed strong immunoreactivity to the surface marker A2B5
11
04 0.
{2.
kD -4,----215-~ "~-'-137---~ 71----~ 51
43-..~
-4--42
31---~ 18--.~ Fig. 2. Immunoblotting for MAP2, MK and GFAP in cellular extracts of HAE cells. A single immunoreactive band was detected, estimated at 300 kDa for MAP2, 42 kDa for MK and 51 kDa for GFAP. (Fig. 1E). A2B5-positive cells are neurons and O-2A lineage cells such as type 2 astrocytes and immature oligodendrocytes. With regard to the markers for developing oligodendroglia cells, CNPase (Fig. 1D), GC (Fig. 3) and MBP (data not shown) were positively stained. Judging from a proposed flow chart on the differentiation of the O-2A lineage [6], HAE cells possessed properties of both oligodendrocytes and type 2 astrocytes. The population of immunoreactive cells was different between cells positive to neuronal or glial cell markers. It was higher in NF (76.4%), MAP2 (49.6%), RC1 (66.2%), A2B5 (52.6%) and vimentin (50.6%) but lower in GFAP (26.3%) and CNPase (20.2%). Double staining using A2B5 and GC showed that all O-2A lineage cells appeared even in the same field of a microscope (Fig. 3), indicating heterogeneous differentiation of oligodendrocytes. Regarding preparation of HAE cells, the original method involved gently peeling the cells free from the underlying chorion to yield an epithelial monolayer on a basement membrane with an underlying collagen matrix containing a few fibroblasts [1]. In order to get pure cultured HAE cells, we performed further treatment with
Fig. 1. HAE cells were stained with primary antibodies. The cells revealed strong immunoreactivities to NF (A) and MAP2 (B), which are neuronal markers, as well as A2B5 (E). Coexpression of GFAP (C) and A2B5 (E) is similar to that of type 2 astrocytes. Among oligodendroglial markers, CNPase (D) was immunologically detected in the cells. Immunoreactivity to vimentin (F) and RCI (G) was found in the cells. Mouse lg G (1/~g/ml) was stained as a control. Magnifications were 200x. Fig. 3. Double staining of HAE cells for A2B5 (A) (visualized with TRITC) and GC (B) (visualized with FITC). Arrows indicate the same point in the two photographs. (1) Type 1 oligodendrocyte, A2B5 (-)/GC (-). (2) Type 2 oligodendrocyte, A2B5 (+)/GC (-). (3) Type 3 oligodendrocyte, immature, A2B5 (+)/GC (+). (4) Type 3 e ligodendrocyte, mature, A2B5 (-)/GC (+). Magnifications were 200×.
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N. Sakuragawa et al. /Neuroscience Letters 209 (1996) 9-12
vigorous scraping with a cell scraper. Fibroblasts were easily distinguished from radial glia in tissue culture on the basis of morphology and immunoreactivity to antifibronectin antibodies. Fibroblasts are RC1 (+)/fibronectin (+) while astrocytes and radial glias were G F A P (+)/ fibronectin ( - ) [4]. Morphologically, H A E cells showed round shapes and cluster formations. Double-labeled staining (data not shown) showed cultured H A E cells were RC1 (+)/fibronectin ( - ) , indicating less contamination with fibroblasts. Since H A E cells expressed phenotypes of both neuronal and neuroglial cells, it was very interesting to note that an inherited pathological condition in human, tuberous sclerosis, also demonstrated heterogeneous phenotypic expression of neuronal, oligodendroglial and astroglial cells in abnormal brain tissue [2]. It has been speculated that these giant cells represent hamartomatous pluripotential cells or premature neuroglia retaining multipotential phenotypic expressions. Judging from the different population of cells immunoreactive to neuron and glial cell markers and the heterogeneous appearance of O-2A lineage cells, H A E cells could have the putative multipotentiality of neural cells. In the mammalian CNS, a multipotential precursor cell gives rise to a variety of neurons and glial cells [12]. This neural stem cell is thought to be recognizable by the expression of nestin, vimentin and RCI antigen [8]. Since H A E cells are R C I (+)/vimentin (+) cells, HAE cells may have the putative potentiality of neural stem cells. In order to define the characteristics of stem cells, self-renewal [5] should be tested in addition to immunoreactivity against the nestin antibody. Additionally, we are now investigating immunoreactivity using human amniotic epithelial cells to determine the in vivo immunoreactivity. The authors thank Dr. M. Yamamoto (Shriver Center) for his gift o f monoclonal antibody, RC1 and Dr. A. Kikuchi for her gift of anti-A2B5 antibody. W e thank Prof. Pearlman, H. Asou and S. Nakamura for valuable
comments. This work was supported by the Research Grant for Highly Advanced Medical Treatment and the Research Grant (5A-6) for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan. [1] Akle, C.A., Adinolfi, M., Welsh, K.L., Leibowitz, S. and McColl, 1., immunogenicity of human amniotic epithelial cells after transplantation into volunteers, Lancet, II (1981) 1003-1005. [2] Chou, T.M. and Chou, S.M., Tuberous sclerosis in the premature infant: a report of a case with immunohistochemistry on the CNS, Clin. Neuropathol., 8 (1989)45-52. [3] Cochard, P. and Paulin, D., Initial expression of neurofilaments and vimentin in the central and peripheral nervous system of the mouse embryo in vivo, J. Neurosci., 4 (1984) 2080-2094. [4] Culican, SM., Baumrind, N.L., Yamamoto, M. and Pearlman, A.L., Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes, J. Neurosci., l0 (1990) 684-692. 15] Davis, A.A. and Temple, S., A self-renewing multipotential stem cell in embryonic rat cerebral cortex, Nature, 372 (1994) 263266. [6] Dubois-Dalcq, M., Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway, EMBO J., 6 (1987) 2587-2595. [7] Mizuguchi, M., lkeda, K., Asada, M., Mizutani, S. and Kamoshita, S., Expression of Bcl-2 protein in murine neural cells in culture, Brain Res., 649 (1994) 197-202. [8] Nakafuku, M. and Nakamura, S., Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, J. Neurosci. Res., 41 (1995) 153-168. [9] Pachter, J.S. and Liem, R.K.H., The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve, Dev. Biol., 103 (1984) 200-210. [10] Peters, A., and Vaughn, J.E., Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves, J. Cell Biol., 32 (1967) 113-119. [11] Tapscott, S.J., Bennett, G.S., Toyama, Y., Kleinbart, F. and Holtzer, H., Intermediate filament proteins in the developing chick spinal cord, Dev. Biol., 86 (1981) 40-54. [12] Zimmerman, L., Lendahl, L., Cunningham, M., McKay, R., Parr, B., Gavin, B., Man, J., Vassileva, G. and McMahon, A., Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors, Neuron, 12 (1994) 11-14.
Neuroscience Letters 209 (1996) 9-12
NEUROSCIEHCf I[IIHS
Expression of markers for both neuronal and glial cells in human amniotic epithelial cells N o r i o S a k u r a g a w a a,*, R a m a s a m y T h a n g a v e l a, M a s a s h i M i z u g u c h i b, M o t o y u k i Hirasawa% I s a o K a m o ~ aDepartment ~f lnherited Metabolic Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan bDepartment of Mental Retardation and Birth De)%ctResearch, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan CDepartment of Ultrastructure Research, NationalInstitute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187, Japan Received 9 February 1996; revised version received 21 March 1996; accepted 22 March 1996
Abstract
Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. We attempted to detect various developmental antigens specific to neural cells by immunocytochemical methods. The cultured HAE cells displayed positive immunoreactivity to RCI, vimentin, A2B5, neurofilament proteins, microtubule-associated protein 2 (MAP2) and MAP2 kinase. In addition, the cells also demonstrated immunoreactivity to glial fibrillary acidic protein, CNPase, myelin basic protein and galactocerebroside. The appearance rate of positive cells was more than 50% in cells positive to RC1, A2B5, vimentin or neuronal markers, and 20--30% to glial cell markers. Double staining showed the heterogeneous appearance of oligodendrocyte lineage cells. These data indicate that HAE cells may have the putative multipotentiality of neurons, astrocytes and oligodendrocytes.
Keywords: Human amniotic epithelial cells; Neuronal markers; Glial markers; RC1; A2B5; Vimentin
From the embryological point of view, the amnion is composed of a single layer of epithelial cells which are in direct contact with the amniotic fluid. At about the 8th day after fertilization, a small cavity appears within the epiblast. This cavity enlarges to become the amniotic cavity. Human amniotic epithelial (HAE) cells are formed from amnioblasts which are adjacent to the cytotrophoblasts and line the amniotic cavity together with the rest of the epiblast. Accordingly, HAE cells may have the potentiality to differentia~te into various organs such as the brain, heart and liver. We report in this paper the immunocytochemical characteristics of HAE cells with a view to their putative multipotentiality of neural cells. The human amniotic epithelial tissue was mechanically peeled free from the chorion of a placenta obtained from an uncomplicated electiw~ cesarean section. The HAE cell layer was extensively scraped out to remove the underly-
* Corresponding author. Tel.: +81 423 412711, ext. 5151; fax: +81 423 461746.
ing tissues such as the spongy and fibroblast layers to eventually obtain a pure epithelial layer with basement membranes. The layer was then treated with 0.25% trypsin three times each for 20 min. HAE cells, obtained usually after the third treatment, were cultured in RPMI1640 medium supplemented with 10% fetal calf serum under a humidified atmosphere of 5% CO2 in air at 37°C. Cells grown on coverslips seated in a 24 well dish were immunocytochemically stained as follows. Unfixed cells were incubated with primary antibodies for 10 min. Then the cells were fixed with 4% paraformaldehyde in PBS(-) for 10 min at room temperature, followed by incubation with primary antibodies for 1 h. The coverslips were treated with secondary antibodies, biotinylated antimouse IgG (1:200; Vector) for 1 h, and then with peroxidase-conjugated streptavidin (Nichirei, Tokyo) for 1 h. The coverslips were washed again, followed by application of 0.2 mg/ml diaminobenzidine (Dojin, Osaka) in 0.05 M Tris-HCl (pH 7.4), to which H202 was added immediately before application to the slides. The slides
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 5 9 9 - 2
IV. Sakuragawa et al. / Neuroscience Letters 209 (1996) 9-12
10
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3
N. Sakuragawa et al. / Neuroscience Letters 209 (1996) 9-12
were counterstained with Harris hematoxylin for 30 s, then washed with water. The following primary antibodies served as neuronal markers: mouse monoclonal antibodies (mAb) to neurofilament proteins (NF) (1:20; Serotec), microtubule-associated protein 2 (MAP2) (1:20; Chemicon), and MAP2 kinase (MK) (1:500; PharMingen). To identify developing glial cells, antibodies to glial fibrillary acidic protein (GFAP) (1:2; Serotec) and 2':3'cyclic nucleotide 3'-phosphodiesterase (CNPase) (1:50; Sigma) were used. Mouse mAb, RC1 (no dilution; a gift from Dr. Yamamoto), 'was used to label cortical radial glia. Vimentin (1:50; DAKO) served as a marker of neuronal or glial precursor cells, O-2A progenitor cells or type 2 astrocytes [6]. The mouse monoclonal A2B5 hybridoma supernatant (1:5; a gift from Dr. Kikuchi) was used to detect A2B5(+) aeurons, O-2A progenitor cells or type 2 astrocytes. To :~tain oligodendrocytes, the cells were first incubated with rabbit polyclonal Ab to myelin basic protein (MBP) (1[:200; DAKO) and rabbit polyclonal Ab to galactocerebroside (GC) (1:50; Sigma), followed by treatment with fluorescein-conjugated goat antirabbit IgG (1:50; Zymed) for 1 h. Double immunofluorescence staining was performed using A2B5 (visualized with rhodamine (TRITC)) and GC (visualized with fluorescein). Immunoblotting was done on an Immobilon polyvinylidene difluoride membrane (Millipore) according to a previously described method [7]. HAE cells displayed immunoreactivity to NF, MAP2 (Fig. 1A,B) and M K (data not shown). Antigenicity to MAP2 and M K was also detected by immunoblotting, which indicated the presence of bands of 300 kDa and 42 kDa, respectively (Fig. 2). Immunoreactivity to vimentin was also found in the cells (Fig. IF). Vimentin is initially expressed in nearly all neuronal precursor cells in vivo, and is gradually replaced by neurofilamew~ subunits shortly before the immature neurons become postmitotic [3,11]. Microtubules arise early in neuronal development, usually long before neurofilaments [9,10]. HAE cells were shown to contain NF, MAP2 and vimentin, suggesting that these cells differentiated to acquire the expression of neuronal markers. The cytoskeleton of HAE cells labeled strongly with RC 1 (Fig. 1G), indicating the presence of cortical radial glia [4]. GFAP (Fig. 112) was positively stained in the HAE cells and a band at 51 kDa was detected on the blots of HAE cell extracts (Fitg. 2), indicating the presence of cells of astrocytic lineage. Also, HAE cells displayed strong immunoreactivity to the surface marker A2B5
11
04 0.
{2.
kD -4,----215-~ "~-'-137---~ 71----~ 51
43-..~
-4--42
31---~ 18--.~ Fig. 2. Immunoblotting for MAP2, MK and GFAP in cellular extracts of HAE cells. A single immunoreactive band was detected, estimated at 300 kDa for MAP2, 42 kDa for MK and 51 kDa for GFAP. (Fig. 1E). A2B5-positive cells are neurons and O-2A lineage cells such as type 2 astrocytes and immature oligodendrocytes. With regard to the markers for developing oligodendroglia cells, CNPase (Fig. 1D), GC (Fig. 3) and MBP (data not shown) were positively stained. Judging from a proposed flow chart on the differentiation of the O-2A lineage [6], HAE cells possessed properties of both oligodendrocytes and type 2 astrocytes. The population of immunoreactive cells was different between cells positive to neuronal or glial cell markers. It was higher in NF (76.4%), MAP2 (49.6%), RC1 (66.2%), A2B5 (52.6%) and vimentin (50.6%) but lower in GFAP (26.3%) and CNPase (20.2%). Double staining using A2B5 and GC showed that all O-2A lineage cells appeared even in the same field of a microscope (Fig. 3), indicating heterogeneous differentiation of oligodendrocytes. Regarding preparation of HAE cells, the original method involved gently peeling the cells free from the underlying chorion to yield an epithelial monolayer on a basement membrane with an underlying collagen matrix containing a few fibroblasts [1]. In order to get pure cultured HAE cells, we performed further treatment with
Fig. 1. HAE cells were stained with primary antibodies. The cells revealed strong immunoreactivities to NF (A) and MAP2 (B), which are neuronal markers, as well as A2B5 (E). Coexpression of GFAP (C) and A2B5 (E) is similar to that of type 2 astrocytes. Among oligodendroglial markers, CNPase (D) was immunologically detected in the cells. Immunoreactivity to vimentin (F) and RCI (G) was found in the cells. Mouse lg G (1/~g/ml) was stained as a control. Magnifications were 200x. Fig. 3. Double staining of HAE cells for A2B5 (A) (visualized with TRITC) and GC (B) (visualized with FITC). Arrows indicate the same point in the two photographs. (1) Type 1 oligodendrocyte, A2B5 (-)/GC (-). (2) Type 2 oligodendrocyte, A2B5 (+)/GC (-). (3) Type 3 oligodendrocyte, immature, A2B5 (+)/GC (+). (4) Type 3 e ligodendrocyte, mature, A2B5 (-)/GC (+). Magnifications were 200×.
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N. Sakuragawa et al. /Neuroscience Letters 209 (1996) 9-12
vigorous scraping with a cell scraper. Fibroblasts were easily distinguished from radial glia in tissue culture on the basis of morphology and immunoreactivity to antifibronectin antibodies. Fibroblasts are RC1 (+)/fibronectin (+) while astrocytes and radial glias were G F A P (+)/ fibronectin ( - ) [4]. Morphologically, H A E cells showed round shapes and cluster formations. Double-labeled staining (data not shown) showed cultured H A E cells were RC1 (+)/fibronectin ( - ) , indicating less contamination with fibroblasts. Since H A E cells expressed phenotypes of both neuronal and neuroglial cells, it was very interesting to note that an inherited pathological condition in human, tuberous sclerosis, also demonstrated heterogeneous phenotypic expression of neuronal, oligodendroglial and astroglial cells in abnormal brain tissue [2]. It has been speculated that these giant cells represent hamartomatous pluripotential cells or premature neuroglia retaining multipotential phenotypic expressions. Judging from the different population of cells immunoreactive to neuron and glial cell markers and the heterogeneous appearance of O-2A lineage cells, H A E cells could have the putative multipotentiality of neural cells. In the mammalian CNS, a multipotential precursor cell gives rise to a variety of neurons and glial cells [12]. This neural stem cell is thought to be recognizable by the expression of nestin, vimentin and RCI antigen [8]. Since H A E cells are R C I (+)/vimentin (+) cells, HAE cells may have the putative potentiality of neural stem cells. In order to define the characteristics of stem cells, self-renewal [5] should be tested in addition to immunoreactivity against the nestin antibody. Additionally, we are now investigating immunoreactivity using human amniotic epithelial cells to determine the in vivo immunoreactivity. The authors thank Dr. M. Yamamoto (Shriver Center) for his gift o f monoclonal antibody, RC1 and Dr. A. Kikuchi for her gift of anti-A2B5 antibody. W e thank Prof. Pearlman, H. Asou and S. Nakamura for valuable
comments. This work was supported by the Research Grant for Highly Advanced Medical Treatment and the Research Grant (5A-6) for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan. [1] Akle, C.A., Adinolfi, M., Welsh, K.L., Leibowitz, S. and McColl, 1., immunogenicity of human amniotic epithelial cells after transplantation into volunteers, Lancet, II (1981) 1003-1005. [2] Chou, T.M. and Chou, S.M., Tuberous sclerosis in the premature infant: a report of a case with immunohistochemistry on the CNS, Clin. Neuropathol., 8 (1989)45-52. [3] Cochard, P. and Paulin, D., Initial expression of neurofilaments and vimentin in the central and peripheral nervous system of the mouse embryo in vivo, J. Neurosci., 4 (1984) 2080-2094. [4] Culican, SM., Baumrind, N.L., Yamamoto, M. and Pearlman, A.L., Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes, J. Neurosci., l0 (1990) 684-692. 15] Davis, A.A. and Temple, S., A self-renewing multipotential stem cell in embryonic rat cerebral cortex, Nature, 372 (1994) 263266. [6] Dubois-Dalcq, M., Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway, EMBO J., 6 (1987) 2587-2595. [7] Mizuguchi, M., lkeda, K., Asada, M., Mizutani, S. and Kamoshita, S., Expression of Bcl-2 protein in murine neural cells in culture, Brain Res., 649 (1994) 197-202. [8] Nakafuku, M. and Nakamura, S., Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, J. Neurosci. Res., 41 (1995) 153-168. [9] Pachter, J.S. and Liem, R.K.H., The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve, Dev. Biol., 103 (1984) 200-210. [10] Peters, A., and Vaughn, J.E., Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves, J. Cell Biol., 32 (1967) 113-119. [11] Tapscott, S.J., Bennett, G.S., Toyama, Y., Kleinbart, F. and Holtzer, H., Intermediate filament proteins in the developing chick spinal cord, Dev. Biol., 86 (1981) 40-54. [12] Zimmerman, L., Lendahl, L., Cunningham, M., McKay, R., Parr, B., Gavin, B., Man, J., Vassileva, G. and McMahon, A., Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors, Neuron, 12 (1994) 11-14.