Human amniotic epithelial cells: isolation, identification and multi-directional differentiation

doi:10.3969/j.issn.2095-4344.2017.09.020

Abstract

Abstract:

BACKGROUND: Human placenta is a stable source for human amniotic epithelial cells, which is becoming a cell source in the regenerative medicine that attracts widespread attentions.
OBJECTIVE: To establish the method of isolation, culture, and adipogenic, chondric and osteogenic differentiation of human amniotic epithelial cells.
METHODS: Trypsin-EDTA digestion was used to isolate human amniotic epithelial cells from human amnion tissue, which were then cultured and identified in vitro. The growth curve of the cells was observed in 12 days. Passage 1 human amniotic epithelial cells were induced to differentiate into adipocytes, chondrocytes and osteoblasts, and conventional cultured cells were used as controls. After 16 days induction, oil red O, Masson and alkaline phosphates staining methods were carried out, and adipogenic transcription factor, type II collagen, osteopontin, alkaline phosphatase mRNA expressions were detected using real-time fluorescene quantitative PCR.
RESULTS AND CONCLUSION: Human amniotic epithelial cells were successfully obtained from human amnion tissue. Immunofluorescence data showed the expression of epithelial cell surface marker CK19. Passage 1 cells had a strong ability to divide and proliferate. Compared with passage 1 ones, passage 2 cells showed a slight decrease in proliferation ability, and the proliferation ability of passage 3 cells was the worst. Red lipid droplets, brilliant blue cartilage matrix and reddish brown calcium nodes were detected by oil red O, Masson and alkaline phosphates staining after adipogenic, chondrogenic and osteogenic differentiation, respectively. With the time prolonged, the expressions of adipogenic transcription factor, type II collagen, osteopontin and alkaline phosphatase mRNA were increased. These results demonstrated that human amniotic epithelial cells could be isolated from human amniotic membrane by enzyme digestion method, and these amniotic epithelial cells could be induced to differentiate into differentiate into adipocytes, chondrocytes and osteoblasts.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Amnion, Epithelial Cells, Adipogenesis, Tissue Engineering

CLC Number:

R394.2

Li Ping, Wang Jiu-cun, Lu Yao, Xu Hui-li . Human amniotic epithelial cells: isolation, identification and multi-directional differentiation[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1420-1425.

Figures/Tables 1

2.1 人羊膜上皮细胞免疫荧光鉴定在荧光显微镜 633 nm激发光条件下，人羊膜上皮细胞经CK19染色后呈阳性，结果显示细胞具有上皮细胞特性(图1)。 2.2 人羊膜上皮细胞的细胞周期及生长曲线流式细胞仪对细胞周期分析结果，见表1，使用R语言进行单因素方差统计，差异有显著性意义(Pr < 0.05)。从中可以看出，随着传代次数的增加，细胞处于静止期(G0/G1期)的比例有所下降，细胞处于增殖期(G2/M+S期)的比例有所上升，提示随着细胞传代次数的增加，羊膜上皮细胞的分化潜能将下降。生长曲线显示，P1代细胞具有较强的分裂增殖能力，约在第2天进入明显的对数生长期，在第6天进入平台期；P2代细胞与P1代相比增殖能力略有下降；P3代细胞增殖能力最差，且细胞无明显的对数生长期，见图2。 2.3 人羊膜上皮细胞向成脂、成骨、成软骨细胞诱导分化鉴定人羊膜上皮细胞能成功诱导成为成脂细胞、成骨细胞和成软骨细胞(图3)。成脂诱导油红O染色后，实验组胞浆中大量的圆形光亮红色脂滴被洗脱出，阴性对照组胞浆中有少量红色脂滴形成；成骨诱导碱性磷酸酶染色后，实验组胞浆中大量红棕色沉淀被洗脱出，阴性对照组胞浆中无红棕色颗沉淀物；成软骨诱导Masson染色后，实验组胞浆中有亮蓝色圆形物被洗脱出，阴性对照组胞浆中无亮蓝色圆形物质；以上结果证明人羊膜上皮细胞具有向成脂、成骨、成软骨细胞诱导分化潜能。 2.4 人羊膜上皮细胞向成脂、成骨、成软骨细胞诱导过程中目的基因的表达人羊膜上皮细胞成脂、成软骨、成骨诱导8，12 d后，荧光定量PCR检测发现成脂转录因子、Ⅱ型胶原蛋白、骨桥蛋白、碱性磷酸酶的mRNA表达均显著上升，见图4。"

References

[1] Sakuragawa N,Thangavel R,Mizuguchi M,et al.Expression of markers for both neuronal and glial cells in human amniotic epithelial cells.Neurosci Lett.1996;209(1):9-12.

[2] Sakuragawa N,Elwan M,Uchida S,et al.Non-neuronal neurotransmitters and neurotrophic factors in amniotic epithelial cells: expression and function in humans and monkey.JpnJ Pharmacol.2001;85(1):20-23.

[3] Uchida S,Inanaga Y,Kobayashi M,et al.Neurotrophic function of conditioned medium from human amniotic epithelial cells.J Neurosci Res.2000;62(4):585-590.

[4] Parolini O,Alviano F,Bagnara GP,et al.Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells.Stem Cells.2008;26(2):300-311.

[5] Strom SC,Skvorak K,Gramignoli R,et al.Translation of amnion stem cells to the clinic. Stem Cells Dev.2013;22 suppl 1:96-102.

[6] Terada S,Matsuura K,Sakuragawa N,et al.Inducing proliferation of human amniotic epithelial (HAE) cells for cell therapy.Cell Transplant.2000;9(5):701-704.

[7] Rooney I,Morgan B.Characterization of the membrane attack complex inhibitory protein CD59 antigen on human amniotic cells and in amniotic fluid.Immunology.1992; 76(4):541-547.

[8] Rebmann V,Pfeiffer K,Grosse-Wilde H,et al.Detection of soluble HLA-G molecules in plasma and amniotic fluid.Tissue Antigens.1999;53(1):14-22.

[9] Miki T,Lehmann T,Cai H,et al.Stem cell characteristics of amniotic epithelial cells. Stem Cells.2005;23(10):1549-1559.

[10] Miki T,Lehmann T,Cai H,et al.Stem cell characteristics of amniotic epithelial cells.Stem Cells.2005;23(10):1549-1559.

[11] Yang S,Sun HM,Yan JH,et al.Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.J Neurosci Res.2013;91(7): 978-986.

[12] Okawa H,Okuda O,Arai H,et al.Amniotic epithelial cells transform into neuron-like cells in the ischemic brain.Neuroreport.2001;12(18):4003-4007.

[13] Takahashi N,Enosawa S,Mitani T,et al. Transplantation of amniotic epithelial cells into fetal rat liver by in utero manipulation.Cell Transplant.2002;11(5):443-449.

[14] Miki T,Grubbs B.Therapeutic potential of placenta-derived stem cells for liver diseases: Current status and perspectives.J Obstet Gynaecol Res.2014;40(2):360-368.

[15] Marcus A,Coyne T,Rauch J,et al.Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane.Differentiation.2008; 76(2):130-144.

[16] Fliniaux I,Viallet JP,Dhouailly D,et al.Transformation of amnion epithelium into skin and hair follicles. Differentiation. 2004;72(9-10):558-565.

[17] He Y,Alizadeh H,Kinoshita K,et al.Experimental transplantation of cultured human limbal and amniotic epithelial cells onto the corneal surface.Cornea.1999; 18(5): 570-579.

[18] Wei J,Zhang T,Nikaido T,et al.Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice.Cell Transplant.2003;12(5):545-552.

[19] Singh M,Ananthula S,Milhom DM,et al.Osteopontin: a novel inflammatory mediator of cardiovascular disease.Front Biosci. 2007;1(1):214-221.

[20] Diao H,Iwabuchi K,Li L,et al.Osteopontin regulates development and function of invariant natural killer T cells.Proc Natl Acad Sci USA.2008;105(41):15884-15889.

[21] Holm L,Bockermann R,Wellner E,et al.Side-chain and backbone amide bond requirements for glycopeptides stimulation of T cells obtained in a mouse model for rheumatoid arthritis.Bioorg and Med Chem.2006; 14(17): 5921-5932.

[22] 曹慧,张忠慧,许时婴.鸡胸软骨酶解产物的表征及对类风湿关节炎大鼠的免疫调节作用[J].食品科学,2012,33(3):243-247.

[23] Armani A,Mammi C,Marzolla V,et al.Cellular models for understanding adipogenesis, adipose dysfunction, and obesity. J Cell Biochem.2010;110(3):564-572.

[24] Lehrke M, Lazar MA. The many faces of PPARgamma. Cell. 2005;123(6):993-999.

[25] 蒋金航,马云,王新庄．PPARγ基因调控脂肪细胞分化的研究进展[J].中国畜牧杂志,2014,50(9):91-95.

[26] Jin H,Yang Q,Ji F,et al.Human amniotic epithelial cell transplantation for the repair of injured brachial plexus nerve: evaluation of nerve viscoelastic properties. Neural Regen Res. 2015;10(2): 260-265.

[27] Enosawa S,Sakuragawa N,Suzuki S.Possible use of amniotic cells for regenerative medicine.Nihon Rinsho.2003;61(3):码396-400.

[28] Kakishita K,Elwan M,Nakao N,et al.Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: a potential source of donor for transplantation therapy.Exp Neurol.2000; 165(1):27-34.

[29] Sankar V,Muthusamy R.Role of human amniotic epithelial cell transplantation in spinal cord injury repair research. Neuroscience. 2003;118(1):11-17.

[30] Kakishita K, Nakao N,Sakuragawa N,et al.Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions.Brain Res.2003;980(1):48-56.

[31] Heeger P.Amnion and chorion cells as therapeutic agents for transplantation and tissue regeneration: a field in its infancy. Transplantation.2004;78(10):1411-1412.

[32] Bilic G,Zeisberger S,Mallik A,et al.Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy.Cell Transplant.2008;17(8):955-968.

[33] Giachelli CM,Bae N,Almeida M,et al.Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92(4):1686-1696.