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Human Amniotic Epithelial Cells Express Specific Markers of Nerve Cells and Migrate Along the Nerve Fibers in the Corpus Callosum***☆

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Human Amniotic Epithelial Cells Express Specific Markers of Nerve Cells and Migrate Along the Nerve Fibers in the Corpus Callosum***☆ NEURAL REGENERATION RESEARCH Volume 7, Issue 1, January 2012 www.nrronline.org Cite this article as: Neural Regen Res. 2012;7(1):41-45. Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum***☆ Zhiyuan Wu1, 2, Guozhen Hui2, Yi Lu2, Tianjin Liu3, Qin Huang3, Lihe Guo3 1Department of Neurosurgery, Changzhou Second People’s Hospital, Nanjing Medical University, Nanjing 213000, Jiangsu Province, China 2Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China 3Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Abstract Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using Zhiyuan Wu☆, Doctor, Associate chief physician, immunocytochemical methods, we investigated the expression of neuronal phenotypes Department of Neurosurgery, (microtubule-associated protein-2, glial fibrillary acidic protein and nestin) in human amniotic Changzhou Second People’s epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and Hospital, Nanjing Medical proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic University, Nanjing 213000, Jiangsu Province, China; epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the Department of Neurosurgery, grafted cells could integrate with the host and migrate 1-2 mm along the nerve fibers in corpus the First Affiliated Hospital of callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind Soochow University, Suzhou of seed cells for use in neurograft. 215006, Jiangsu Province, China Key Words: human amniotic epithelial cells; cell transplantation; microtubule-associated protein-2; glial fibrillary acidic protein; nestin; neurotrophin; central nervous system injury; neural regeneration Corresponding author: Guozhen Hui, Professor, Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou INTRODUCTION RESULTS 215006, Jiangsu Province, China; Lihe Guo, Researcher, Institute of Human amniotic epithelial cells (HAECs) Morphology of the isolated and cultured Biochemistry and Cell have the potential to differentiate into HAECs and the expression of neuronal Biology, Shanghai Institutes [1] for Biological Sciences, nervous system cells , and can synthesize specific markers Chinese Academy of and release brain-derived neurotrophic The HAECs became adherent within 2 days Sciences, Shanghai 200031, 6 China factor, neurotrophin-3 and nerve growth after the isolation and 1 × 10 HAECs could [email protected]; factor[2]. Therefore, HAECs are potential be harvested from an amniotic membrane at [email protected] seed cells for cell transplantation. a size of 15 cm × 15 cm. Under light Received: 2011-09-25 Importantly HAECs do not express microscopy, the adherent HAECs were Accepted: 2011-11-12 (N20110802001/WLM) HLA-associated antigen, which avoids the growing in clusters, in round or polygonal immune rejection present in cell shapes, and contained large nuclei and Wu ZY, Hui GZ, Lu Y, Liu TJ, [3] Huang Q, Guo LH. Human transplantation . characteristic vesicular fat droplets in the amniotic epithelial cells A number of existing studies demonstrate cytoplasm (Figure 1). The cells proliferated express specific markers of nerve cells and migrate along that HAEC transplantation may be an slowly and were passaged every 2-3 weeks. the nerve fibers in the corpus effective treatment for ischemic callosum. Neural Regen Res. 2012;7(1):41-45. neurological impairment, Parkinson’s disease and spinal cord injury[4-5], with no www.crter.cn www.nrronline.org limitations such as scarcity of seed cell sources, difficulty in sample harvesting, or doi:10.3969/j.issn.1673-5374. 2012.01.007 legal and ethical issues. However, a systematic study of these cells has not been undertaken and their biological characteristics still remain unclear. This study sought to establish stable HAEC culture conditions, elucidate the biological Figure 1 Morphology of the human amniotic characteristics, and observe the migration of epithelial cells in primary culture (hematoxylin- HAECs after transplantation, in a broader eosin staining, light microscopy, × 200). attempt to provide novel seed cells for stem Adherent cells were round or polygonal, with large nuclei and characteristic vesicular fat cell transplantation in the treatment of droplets in cytoplasm (arrow). neurological diseases. 41 Wu ZY, et al. / Neural Regeneration Research. 2012;7(1):41-45. Immunohistochemical and cytochemical staining demonstrated that microtubule-associated protein-2 (MAP-2), glial fibrillary acidic protein (GFAP) and nestin were expressed in both the amniotic membrane and the cultured HAECs. Nestin expression was markedly reduced in the cultured HAECs (Figure 2). AD Figure 3 Morphology of the adherent rat glial cells. A spindle shape, small cell body and long processes were present (hematoxylin-eosin staining, light microscopy, × 200). A BE CF B Figure 2 Expression of glial fibrillary acidic protein (A, D), microtubule-associated protein-2 (B, E), and nestin (C, F) C in amnion tissue and the cultured human amniotic epithelial cells (light microscopy, × 200). (A-C) Amniotic membrane (immunohistochemistry). (D-F) Primary cultures of human amniotic epithelial cells (immunocytochemistry). Glial fibrillary acidic protein (arrows), microtubule-associated protein-2 (arrows), and nestin (arrows) were expressed in both the amnion membrane and the cultured human amniotic epithelial cells, and the number of nestin-positive cells was reduced in the cultured Figure 4 Morphology of rat glial cells in different cells. culture mediums (hematoxylin-eosin staining, light microscopy, × 100). (A) In 10 % fetal bovine serum + RPMI1640 medium, the Morphology of the cultured rat glial cells cultured cells grew well with extending cell bodies and Under light microscopy, the adherent rat glial cells were long processes. irregular spindle-shaped cells, with small cell bodies and (B) In human amniotic epithelial cell conditioned medium, long protrusions. Cells were vigorously proliferating and the cultured cells showed similar changes and the cell body was wide. were passaged every 4-5 days (Figure 3). Effect of HAECs conditioned medium on the (C) In serum-free RPMI1640 medium, most of the cultured cells died and a small amount of cells adhered with thin morphology of rat glial cells shape and poor refraction. Hematoxylin-eosin staining showed that rat glial cells could not survive in serum-free RPMI1640 and the Effect of HAEC conditioned medium on proliferation majority of cells died. A small number of residual cells of rat glial cells in vitro could adhere but showed thin shape and poor refraction. The proliferation of rat glial cells was determined with Rat glial cells grew well in the HAEC conditioned trypan blue staining and 3-(4, 5-Dimethylthiazol-2-yl)-2, medium, with extended bodies and long processes 5-diphenyltetrazolium bromide (MTT) assay. Results (Figure 4). found that HAEC conditioned medium promoted the 42 Wu ZY, et al. / Neural Regeneration Research. 2012;7(1):41-45. proliferation of rat glial cells (Figures 5, 6). can differentiate into nerve cells, suggesting that HAECs may exhibit nerve cell-associated biological 3.0 characteristics. In this study, both amniotic membrane ) 4 tissue and the cultured HAECs expressed the nerve 2.5 tissue markers GFAP, MAP-2 and nestin, as detected by 2.0 HAEC conditioned medium immunohistochemical/cytochemical staining. In addition, 1.5 (×10 count Serum-free RPMI1640 the nestin-positive cells were present in HAECs before 1.0 cell and after culture, but the number was significantly 0.5 reduced after culture. This is evidence that the cultured Viable 0 HAECs and amniotic tissue are likely to contain neural 1 2 3 4 5 6 7 stem cells that can differentiate and the number of neural [7] Time of culture (day) stem cells decreases after culture . Figure 5 Growth curve of rat glial cells cultured in different culture conditions. Data are expressed as the mean values of three samples A collected at each time point in a group. In human amniotic epithelial cell conditioned medium, the growth of cultured cells increased exponentially at 2-3 days, while cells could not survive in serum-free RPMI1640 medium. 0.24 ab ab a 0.20 a B 0.16 Absorbance 0.12 I II III IV V VI Figure 6 Effect of conditioned medium on the proliferation of rat glial cells by 3-(4, 5-Dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide assay. C A total of 16 cell samples were cultured in each culture medium for 5 days. Data are expressed as mean ± SD. aP < 0.01, vs. serum-free culture medium; bP < 0.01, vs. fetal bovine serum culture group (one-way analysis of variance). I: Control group; II: serum-free culture group; III: fetal bovine serum culture group; IV: one-third HAEC conditioned medium group; V: two-thirds HAEC conditioned medium group; VI: complete HAEC conditioned medium group. HAEC: human amniotic epithelial cell. Figure 7 Survival and migration of human amniotic MTT assay also showed that the nutrition effects of the epithelial cells at 1 month after transplantation into the rats HAEC conditioned medium were concentration- (Hoechst33342 staining, fluorescence microscopy). dependent ( Figure 6). (A) Needle tract in corpus striatum (arrow; ×
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