The Isolation And Identification Of Human Amniotic Epithelial Cells And The Differentiation To Insulin-secreting Cells

Chapter 1 The Isolation and Identification of Human Amniotic Epithelial CellsObjectives:We aimed to purify the hAECs isolated from human amnion and to identify the stem cell related characteristics of them so that we could establish a cell bank for further studies.Methods:We defined human amnions as classⅠ,Ⅱ,Ⅲdepending on the transparence and cleanliness of the amnions.Trypan-blue staining was used for detecting the ratio of living cells.The growth curve was drawn for judging the characteristics of cells isolated from different placentas of different classes.RT-PCR,immunocytofluorescental analysis and AKP dyeing were used respectively for identifying the stem cell characteristics of hAECs.The cell samples of different passages were collected and then semi-quantity RT-PCR was performed to examine the pluripotency-related genes expression,such as Oct-4,nanog,TERF1, THY1,LEFTYA,and Rex-1.Immunocytofluorescental analysis was taken to test the expression of stem cell related markers,such as TRA-61~+, TRA-80~+,SSEA-1~+,SSEA-3~+ and SSEA-4~+.AKP dyeing was performed to detect the AKP positive cells.Chromosome Giemsa band analysis was undertaken to evaluate the genetic stability of hAECs cultured continuously in vitro for 10 passages. Results:1.We have collected 62 amnion samples,and established a hAE cell bank with frozen cell to the amount of 300 tubes.2.Anmion of classⅠis the most abundant with a hAECs yield of(11.253+2.3)×10~7 cells/dm~2.3.Trypan-blue staining showed that the ratio of living cells was more than 75%.4.Almost all of the totipotent genes expressed in each passage of hAECs.THY1 gene expression up-regulated in P0,P1, P2 gradually;LEFTYA,Sox2,Oct-4 and TERF1 gene showed the same levels among 3 passages.5.Immunocytofluorescent analysis showed that most of hAECs were SSEA-4~+,part of them were positive weakly to TRA-1-60,TRA-1-81.6.AKP dyeing suggested that AKP~+ cells existed in P0,P1,P2 hAECs.7.Chromosome Giemsa band analysis showed the chromosomes maintained normal,even cultured for 10 passages.Conclusions:1.We established a miniature hAE cell bank for further studies.2.There are no obvious biological characteristic differences among P0,P1,P2 hAECs. Chapter 2 The Differentiation of Human Amniotic Epithelial Cells to Insulin-secreting CellsObjectives:To investigate the induction differentiation from hAECs into insulin-secreting cells by using culture medium supplied with Nicotina-mide and cytokine N2.Methods:P1 hAECs was seeded in cell dish coated with agar powder.RT-PCR was performed to testing the expression of pancreatic development-related genes such as insulin,glucagon,pdx1,nkx6.1 and ngn3.Flow cytometry was used to detect the PDX1~+ cell. Immunofluorescence was performed to testβ-cell specific marker Insulin. Glucose-stimulated insulin release experiment was taken to test the reaction ability of induced hAECs.Semi-quantity RT-PCR was used to evaluate the chronological gene expression changes during a 14-day inductive course,such as pdx1,ngn3,pax6,nkx6.1,insulin and glucagon. RT-PCR was performed to detect the expression of adhesion factor-related gene E-cadherin.Results:1.RT-PCR results showed that pdx1,nkx6.1,insulin,pax6, Isl1,ngn3 were expressed in hAECs after induction.2.PDX1~+ cell number amounted to 21.1%±3.4%after 3 days induction.3.Insulin~+ cells number achieved 13.64%±1.6%after 2 weeks.4.The average insulin release of hAECs reached about 59.43±6.0 vU/ml.5.The gene chronological expression analysis showed that the expression of insulin gene was first up-regulated in the first week and subsequently down-regulated,while the glucagon gene expression showed down regulation after the crest-time at day 10.Expression tendency of pdx1, nkx6.1 gene was similar to that of insulin gene.ngn3 were steadyly expressed in all of the samples.6.E-cadherin gene expressed in hAECs induced for 8 days,but not expressed in hAECs induced for 14 days.Conclusions:hAECs could be differentiated into insulin-secreting cells in vitro by using the induction system.