Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

Since the first establishment of hESC in 1998 (Thomson et al., 1998) , hESCs have been cultured in conditions using mouse derived fibroblasts, such as mouse embryonic fibroblasts (MEFs) and STO as feeder cells. However, concerns remain regarding the potential risks of cell line contamination by unknown components secreted from these animal derived materials, therefore, studies have been performed to overcome these challenges (Soong et al., 2013) . For example, studies have examined the replacement of mouse derived fibroblasts with human tissue-derived feeder cells, such as human foreskin fibroblasts, fetal muscle fibroblasts (Richards et al., 2002) , umbilical cord derived mesenchymal stem cells (Ding et al., 2012) , marrow stromal cells (Havasi et al., 2013) , and placental cells (Park et al., 2011) . Additionally, researchers have also attempted the development of extracellular matrix and xeno-free medium, which can be used in feeder-free conditions for cultures without feeder cells . There have also been continuous efforts to maintain hESCs efficiently and stably and establish new cell lines in the developed culture conditions. Human amniotic fluid cells (hAFCs) have been used for prenatal genetic diagnosis to examine the possibility of fetal abnormalities, these cells can be easily obtained from pregnant women through second midtrimester amniocentesis (Prusa & Hengstschlager, 2002) . Although few studies have characterized the various types of cells found in amniotic fluids, cells in amniotic fluids have been shown to be capable of differentiating into three germ layer cells. Thus, these cells have the potential for applications as materials for studies of neuronal differentiation or as stem cells (Prusa & Hengstschlager, 2002; Kook et al., 2006) . Amniotic fluid derived stem cells have intermediate characteristics between hESCs and adult stem cells and express markers such as CD29, CD90, and CD105, which are expressed in bone marrow derived mesenchymal stem cells. These cells also express markers of the undifferentiated state of hES cells, including Oct-4, Nanog, SSEA-4, and TRA1-81 (Bajek et al., 2014) . In particular, Oct-4, a representative marker for undifferentiated hESCs, has been shown to be expressed in 90% of amniotic fluidderived stem cells (Prusa et al., 2003) . Moreover, hAFCs have been shown to have higher reprogramming efficiency for establishment of induced pluripotent stem cells (iPSCs) than somatic cells (Galende et al., 2010) . In addition, in studies using hAFCs as feeder cells, Kim et al. (2004) successfully cultured hESCs using hAFCs as feeder cells instead of mouse derived fibroblasts, which are more commonly used. Other reports have also described the maintenance of the undifferentiated state of hESCs using human amniotic fluid cell-derived stem cells as feeder cells without the addition of basic fibroblast growth factor (bFGF) (Ma et al., 2014) , and new hESC lines have been established using amniotic fluid-derived mesenchymal stem cells as feeder cells (Soong et al., 2013 ).