| Researchers try to find out new resource of stem cell because of the ethical argument of embryonic stem cells. In 2003, Oct-4 positive cells were isolated in amniotic fluid, which showed that there might be multipotent stem cell in amniotic fluid. In 2007, Paolo demonstrated that they found a few stem cells in amniotic fluid,which was named human amniotic fluid stem cell (hAFS cell). hAFS cells that express embryonic and adult stem cell markers can differentiate into cell types of three embryonic germ layer and display specialized functions. hAFS cells can be obtained easily and do not damage the mother body and the embryo, which may offer an alternative approach for the therapy of cell and tissue engineering. In this study, hAFS cells were isolated from human amniotic fluid at the 2nd or 3rd trimester of gestation. Effects of the characteristics of amniotic fluid on the culture of hAFS cells were evaluated and culture system were established. At the same time, the biological characteristics of hAFS cells were detected by immunocytochemistry, RT-PCR and flow cytometer. hAFS cells were induced to differentiate to cardiomyocytes and neural cells.(1) Effects of the characteristics of amniotic fluid on the culture of hAFS cells Human amniotic fluid samples were obtained at the 2nd or 3rd trimester of gestation. Cells were isolated after centrifugation and were cultured with amniotic fluid stem cell culture medium (ACM). The cell attachment time and numbers were recorded to investigate the factors that affect the growth of hAFS cell primary culture. The results showed that the cell attachment time in the 2nd trimester of gestation (4.7±0.6 d) was significantly different with that in the 3rd trimester of gestation (6±0.5 d) (P<0.05), suggesting that cells collected from the fluid of 3rd trimester of gestation need longer time to attachment. We also find that blood contamination can significantly affect the time of cell attachment. The attachment time of maroon group (10.8±0.3 d) was significantly different with the clear group (6±0.5 d) and yellow group (6.3±0.6 d) (P<0.05). The effects of volume of amniotic fluid were also investigated on numbers and time of cell attachment. The volume of amniotic fluid did not affect cell attachment time, but affected the cell attachment numbers to a certain extent (P<0.05). The present studies systematically examine factors affecting on the primary culture of human AFS cells and provide useful data for AFS cell research.(2) The isolation, culture and detection of hAFS cells Human amniotic fluid samples at the 2nd or 3rd trimester of gestation were cultured and hAFS cells were isolated by the manual dissociation. The biological characteristics and the differentiation potential of hAFS cells were studied by the methods of RT-PCR, immunocytochemistry and flow cytometer. hAFS cells showing fibroblastoid-type were obtained and were positively express specific makers of embryonic stem cells, such as Oct-4, hTERT, Nanog, SSEA-1, SSEA-4 and CD117, but not SSEA-3. hAFS cells were stained positively for mesenchymal stem cells markers, including CD29, CD44, CD73, CD90 and CD105, but negatively for hematopoietic stem cells and hematopoietic lineage markers, including CD34, CD133 and CD45. hAFS cells were also positive for HLA-ABC and weakly positive for HLA-DR. hAFS cells maintain the proliferative potential and normal karyotypes even after expansion to several populations. In the suspension culture, hAFS cells could form embryoid bodies which were alkaline phosphatase (AP) positive and expressed fgf5,ζ-globin andα-fetoprotein which were the specific makers of three germs. hAFS cells proliferation rates were compared under two different medium, containing either FBS or KSR. The results showed that hAFS cells can be expanded in the absence of animal serum.(3) Differentiation of hAFS cells into cardiomyocyteshAFS cells were isolated from human amniotic fluid samples at the 3rd trimester of gestation. The induce effect were compared with different induce conditions. The results showed that the differentiated cells were positive forα-actin, Tbx5, Nkx2.5, GATA4 andα-MHC. The average optical density ofα-actin positive cells in conditional medium was higher than RA and DMSO treatments (P<0.05). In the same medium, the induce effect in EB group was well (P<0.05). The conclusion showed that the induce effect of hAFS cells differentiation into cardiomyocytes through the formation of embryoid bodies under the conditional medium was advantageous over other induce conditions.(4) Differentiation of hAFS cells into neural cellshAFS cells were isolated from human amniotic fluid samples at the 3rd trimester of gestation. The induce effect were compared with different induction conditions. The results showed that the differentiated cells were smaller,cytoplasmic contraction,cone-shaped or triangular,which were positive for Nestin, NSE, fgf-5 and CD56. Under RA medium, the average optical density of Nestin and NSE positive cells in monolayer group was higher than in EB group (P<0.05). Underβ-Me medium, no NSE positive cells were observed and the induce time should be changed in 3~4 hours. The conclusion showed that the induce effect of hAFS cells differentiation into neural cells under RA medium in monolayer group was advantageous over other induce conditions.
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