Studies On Isolation, Culture, Characterization And Differentiation Potential Of Equine Amniotic Epithelial Cells

The amnion epithelial cells(AECs) is a type of cells possessed with stem cell characteristics and derive from superseded placenta amnion. AECs are not only have multi-directional differentiation potentiality, but possess immune exemption potency that immunological rejection and tumorigenesis are not generate post transplant because of terminal transferas telomere defected, and AECs are gained readily and not cause ethics dispute, which make it become one hot spot of cell researches. To apply AECs curing some serious disease is the final objective of clinical researchers. Furthermore, successful clinical application and treat are base on safe and reliable animal experiment. At present, human amnion epithelial cells are studied chiefly and adopted to treat liver disease, pulmonary tuberculosis, heart disease, diabetes, nervous system disease and so on. Therefore, isolating, culture and assessing stem cell characteristics, differentiation in vivo and direction location induction and differentiation in vitro of horse amnion epithelial cells are proceed system research in this study.Cells from amnion membrane including Human Amniotic Mesenchymal Cells(HAMCs) and Human Amniotic Epithelial Cells(HAECs). Recent reports indicate that HAECs and HAMCs express stem cell markers and have the ability to differentiate toward all three germ layers. cell surface antigens on HAECs include CD29, CD105, CD73, stage-specific embryonic antigens(SSEA) 3 and4, and tumor rejection antigens 1-60 and 1-81(TRA 1-60、TRA 1-81). Surface markers thought to be absent on hAECs include SSEA-1, CD34,and CD133. In addition to surface markers, HAECs express molecular markers of pluripotent stem cells, such as octamer-binding protein 4(OCT-4) and Nanog. Recent research have found that cells from amnion membrane display stem cells characteristical, In theory they can differentiate into various tissue cells. These characteristics of the amniotic cells have made them a replacement of other stem cells and received extensive attention.First of all, isolating and culture of equine amnion epithelial cells.There is no information on equine amniotic epithelial cells(eAECs) digestion experiments reported in isolated and cultured in vitro methods. In this experiment human amniotic epithelial cells isolated digestion method reference established equine amniotic epithelial cells(eAECs) digestion experiments separation and isolation and culture of Trypsin and TrypLE influence in the process of doing eAECs related research. Experimental results show Trypsin digestion and TrypLE are isolated from equine amniotic epithelial layer of primary cells, but the separation is more conducive to digestion TrypLE equine amniotic epithelial cells, high cell viability obtained for subsequent cell subculture less affected, more subsequent cell culture studies, identification and differentiation potential suitable. Experiments using 90% FBS +10% DMSO cryopreserved cells coolant system cells better, the cell suspension was mixed and frozen vials placed into the freezer compartment of isopropyl alcohol, under conditions of-80 ℃ overnight, the next day to prepare for long-term storage in liquid nitrogen into experimental use. Experiment found that amniotic epithelial cells appeared in the three cell types in vitro, epithelial cells(epithelioid cells), mesenchymal-like cells(mesenchymanl-like cells). Trypsin digestion and isolated by long equine amniotic epithelial cells in the early generations of the first generation and culture doubling time than cells isolated Tryp LE doubling time and doubling time between subsequent cells passaged no significant difference in the in vitro cultured MA amniotic epithelial cells average doubling time was 1.25 days.Secondly, assessment the stem cell characteristics of equine amnion epithelial cells.Cell immunofluorescence and RT-PCR were performed to detect respectively the expression of embryonic stem cell of horse AECs marker proteins SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and totipotency related genes OCT-4, SOX-2, Nanog. The observed SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 immunoreactivity of horse AECs were predominant. RT-PCR experiments confirmed that the genes of Oct-4, Sox-2, Nanog were expressed. By FASC analysis, horse AECs expressed the following cell surface antigens including CD44, CD90 and CD105, but negatived for CD45.These results indicated that the equine AECs have the multi-directional differentiation potentiality of stem cell properties.Finally, directional induction and differentiation of equine amnion epithelial cells in vitro. Experimental in vitro studies by the three germ layers spontaneous differentiation and to SCID mice were injected with equine amniotic epithelial cells(eAECs) experiments in vivo differentiation of amniotic epithelial cells of the equine(eAECs) differentiation potential. In vitro suspension culture equine amniotic epithelial cells, 20 days after the discovery formation of embryoid bodies by RT-PCR detected the equine amniotic epithelial cells in vitro spontaneous differentiation expressed ectoderm, iconic mesoderm and endoderm three germ layers genes β-Tubulin III, GATA-4 and α-Fetoprotein. SCID mice were injected subcutaneously at a equine is found between 30 to 90 days after the occurrence of amniotic epithelial cells teratoma; the SCID mice in the control group was given a subcutaneous injection of embryonic stem cell commercialization, the mice were sacrificed after 90 days examination revealed teratoma formation.The results show that the equine isolated amniotic epithelial cells(eAECs) below immunogenicity of mouse embryonic stem cells, more suitable for clinical treatmentIn this study equine amnion epithelial cells were successfully gain and possessed with stem cell characteristics which have the multi-directional differentiation potency in vivo and in vitro. And the results in this research supply with the theory knowledges of AECs in stem cell characteristics and differentiation in vivo and in vitro, and establish rationale and provide practice base for clinical exploratory development. The equine amnion epithelial cells have low immunogenicity and non-tumorgenicity in vitro. To sum up, the ability of equine amnion epithelial cells to successfully engraft and survive long-term in various organs and tissues,makes sure its significant impact in the fields of cell-replaced therapy and regenerative medicine.