Derivation, Identification Of Human Amniotic Fluid Stem Cells

ObjectiveTo study the source of stem cells in amniotic fluid (amniotic fluid-drived stem cells, AFS) the exact source. Currently, the identification of sources of stem cells in amniotic fluid is no good way, but to determine whether is the AFS. In this experiment, stem cells isolated on the basis of its biological properties through the cell surface expression of stem cell factor and biological characteristics; by cell morphology and in situ immunohistochemistry to Mingque amniotic fluid derived stem cells, the exact source of purification for the next step to improve the method provides experimental basis, ultimately have a large number of AFS. Intrauterine intervention for the next time after the birth of clinical treatment or experimental basis and theoretical basis.MethodsUnder informed consent, by B ultrasound-guided paracentesis were collected 16-24 weeks of pregnancy the amniotic fluid 18-20ml, in situ after 2 weeks of culture, until up to 50% confluence, will be part of the culture bottle P1 The cells were divided into two parts, in the inverted microscope, the flask into two, with imports scraper to scrape respectively, into the new culture flask for passage, denoted P21, P22. The blade scraping cells to cell damage large, so continue to pass alternative method of trypsin digestion, a bottle in the inverted microscope for a variety of cell morphology, growth curve, and analysis of building embryonic karyotype; other Immunohistochemical bottle to do research OCT-4, CD117 expression; another part of the P1 cells used for in situ immunohistochemistry to detect amniotic fluid stem cell surface markers such as vimentin vimen, broad-spectrum cytokeratin CK, CD44, CD29, CD34, CD45 expression. Results1,In situ by cell culture, cell morphology under inverted microscope according to the difference in the amniotic fluid cells were divided into four categories, namely, the island-like cells, E cells, amniotic fluid cells and F-specific cells.2,Several passages, the cells showed no growth of colonies, but showed overlap-like growth to the main spindle, arranged closely with each other ill-defined. More uniform than the primary form, vigorous growth. Can be continuously passed 10 generations and found no cell aging.3,The amniotic fluid cell growth curve was S-shaped, P10 cells than P3, P5 cell growth slow. Growth curve is characterized by: about 2 days after inoculation for the potential adaptation period of about 3 days starting from the logarithmic growth phase, reached its peak on day 8, after entering a plateau, indicating a strong in vitro proliferation.4,Inverted suspension P5 amniotic fluid cells cultured for 3 days, embryoid bodies can be seen under a microscope-like structure. Have to prove their differentiation potential of the three germ layers, pending further study.5,Taken on behalf of the amniotic fluid cells for each chromosome karyotype analysis, which are normal diploid karyotype, indicating its stable inheritance.6,Do take P10 Immunohistochemical results showed that the expression of OCT-4, CD117, suggest amniotic fluid contains stem cells.7,The island-like cells expressed CK, Vimen, CD44, Oct-4, weak expression of CD117, did not express CD34, CD45; E-like cells expressed CK, Vimen, CD44, Oct-4, did not express CD117, CD34, CD45; amniotic fluid specific cell surface CK, Vimen, CD44, if the expression of OCT-4, did not express CD117, CD34, CD45; fibroblast-like cells Vimen, CD44, Oct-4, CD117, did not express CD34, CD45.Conclusions and significance1,Through this experiment proved that the expression of amniotic fluid containing CD44, Vimen mesenchymal stem cells, stem cell factor expression of CD117 stem cells, the expression of OCT-4 pluripotent stem cells.2,By their biological characteristics, proven amniotic fluid-derived stem cells in vitro has a strong proliferative capacity, differentiation potential and stable genetic characteristics.3,Epithelial cells and fibroblast-like cells in amniotic fluid derived stem cells are the exact source.4,Clear amniotic fluid derived stem cells from amniotic fluid stem cells to provide experimental basis for clinical application.