Isolation and Characterization of Amniotic Fluid Stem Cells

The study was conducted in accordance with the guidelines on good clinical practice and with the ethical standards for human experimentation established by the 1964 Declaration of Helsinki. All subjects gave informed consent before being included in the study. The study protocol was reviewed and approved by Institutional Review Board of Siriraj Hospital, Mahidol University, Bangkok, Thailand [Protocol No. 103/2553(EC3)]. Amniotic fluid was collected from the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital. Briefly, amniotic fluid samples were obtained by amniocentesis performed for routine fetal genetic diagnosis at 16–20 weeks of gestation, centrifuged at 2100RPM for 5 min, and the pellet retrieved for cell line establishment. AFSC were cultured in MEMα medium (Gibco, Invitrogen, Carlsbad, MA, USA) supplemented with 15% embryonic stem cell qualified fetal bovine serum (ES-FBS), 1% L-glutamine, 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 20% Chang medium (Irvine Scientific, Santa Ana, CA, USA) at 37 °C with stable 5% CO₂ in normoxic conditions^{28 (link)}. Cell quantity, morphology and expression of Oct-4, CD29, CD34, CD44, CD45, CD73, CD90 and CD105 were evaluated. Chromosomal stability was assessed by karyotype (Supplementary data – Methods).