The aim of regenerative medicine is currently underway to use the body’s own cells to repair diseased or damaged tissue. The use of stem cells from different tissues for regenerative medicine applications has increased over the past few years. Amniotic fluid (AF) represents rich sources of stem cells population deriving from either the fetus or the surrounding amniotic membrane that can be used for clinical therapeutic applications in the patients who develop organ failure that have resistance to current therapies. The AF was first studied at the beginning of the 20th century (1). Initial studies have been performed in order to detect fetal abnormalities during development the fetus (2). Isolation and identification of amniotic fluid- stem cells (AFSCs) dating back to the early 1990’s (3). The study of AFSCs has received significant attention of late for several reasons. First, AF is easily collected during the first trimester of pregnancy by scheduled amniocenteses for fetal karyotyping, prenatal diagnosis and detection a variety of genetic diseases (4). Second, expansion and storage of AFSCs is easy and achieved at minimal costs. Finally, these cells could be stored in cell banks and used in disease research, drug screening and genetic disorders.

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Human AF is a protective and nourishing watery liquid that providing mechanical support during embryogenesis and is constituted of about 98% water (4). Other ingredients include electrolytes, pigments, sugars, fats, amino acids, proteins, carbohydrates, enzymes, growth factors and cells (5). This volume and composition varies throughout pregnancy (5, 6). After formation of the amniotic cavity, 7~8 days after fertilization, this fluid starts to gather immediately. AF volume increases progressively and is then completely surrounding the embryo after 4 weeks of pregnancy. The average volume is 270 ml at week 16 which increases to 400 ml at week 20 of pregnancy and 800 ml at birth (5). At the beginning of pregnancy, the amniotic osmolarity is similar to the fetal plasma. After keratinization of the fetal skin, which usually occurs at week 24 of pregnancy, amniotic osmolarity decreases relatively to maternal or fetal plasma, mainly due to the inflow of fetal urine (7). Additional investigations have been recently focused on the cellular and molecular properties of amniotic derived cells and their potential use in pre-clinical models and in cell therapies (4).

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There are three major protocols for isolation of AFSCs from human amniotic fluid. The first one is based on single-stage method (8). In this method AF collected from second-trimester amniocentesis is centrifuged. The number of cells is counted by hemocytometer and mixed with an equal volume of culture medium, usually Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) and the cells allowed to adhere to a plastic culture plate at 10⁴ cells/cm² and incubated overnight at 37°C under 5% CO₂ (9). Culture medium is changed after 3~5 days to remove non adherent cells and twice weekly thereafter. The primary cells are cultured for 4~5 days until they reached confluence and are defined as passage “0”. In this step the heterogeneous morphological cell population appears and after several sub-culturing the fibroblastic like cells was dominated. The cells typically reach confluence in 4 to 6 days and the remaining cells are cryopreserved in cryopreservation media (10% dimethylsulfoxide, 90% FBS), frozen at −80°C for 24 h, and stored in liquid nitrogen the next day. The second one is immunoselection based on surface antigens. Ditadi was the first researcher to show that the c-Kit population cells extracted from the AF do have hematopoietic potential (10). At this time, Atala et al. (2) and Schmidt et al. (11) isolated CD117 positive cells by c-kit (a rabbit polyclonal antibody to CD117) and CD133 positive cells by CD133 magnetic beads, respectively. Their study showed these cells can be easily expanded in cultures and sub-population of CD133 positive exhibited similar characteristics of mesenchymal progenitors cells (11). Following these studies, Arnhold et al. sorted CD117 positive cells by magnetic associated cell sorting. They indicated that the percentage of CD117 positive cells was 3.2±1.03% of the whole cell population and demonstrated that these cells could differentiate to adipogenic, osteogenic, myogenic and neurogenic lineage (12). The third one is the two-stage culture protocol established by Tsai et al., using nonadhering AF cells of the primary amniocytes culture to isolate AFSCs. In this protocol, nonadhering AF cells are collected from supernatant of AFCs that cultured in serum-free changes medium (first stage). Then collected cells are plated for AFSCs culturing after the completion of fetal chromosome analysis (second stage) (13). This method has some advantages over the others. Major advantage of this culture protocol comparing to the other two is that instead of the adhering cells derived in AF it isolates from the nonadhering cells, which is being left in the incubator without any added nutrition for 7~10 days (13). As mentioned two-stage method is more superior compared to other methods, the use of this method have been proposed.

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The AFSCs are mainly composed of three heterogeneous groups of adherent cells, calcified based on their growth, morphological and biochemical characteristics that derived from the three germ layers (14). Epithelioid (E-type) cells that are cuboidal to columnar, derived from the fetal skin and urine, AF (AF-type) cells are originating from fetal membranes, and fibroblastic (F-type) cells are generated mainly from fibrous connective tissue. The percentage of these cells is 33.7%, 60.8% and 5.5%. Some studies have reported that AFSCs can be easily obtained from a small amount of AF (4, 15). Like other mesenchymal stem cells (MSCs), the AFSCs expressed CD73, CD90, CD105, CD29, CD166, CD49e, CD58, CD44 and HLA-ABC antigens and are negative for the hematopoietic markers such as CD14, CD34, CD45 and CD133, the endothelial marker such as CD31, and the HLA-DR antigen (Fig. 1) (4). These cells are able to differentiate along adipogenic, osteogenic, myogenic, endothelial, neurogenic and hepatic pathways (Table 1) (16–19). Additionally, the majority of these cells expressed the pluripotency markers such as the octamer binding protein 3/4 (Oct-3/4), the homebox transcription factor Nanog, and the stage-specific embryonic antigen 4 (SSEA-4) (20, 21). Similar to MSCs, AFSCs express MHC II at a very low level. Unlike human MSCs that are telomerase negative, low to moderate levels of the enzyme have been described in AFSCs (5, 22). Telomerase is an enzyme that maintains telomere sequences at chromosomal ends (23). Telomeres consist of TTAGGG repeats protect the ends of chromosomes from end-to-end fusion, recombination and deterioration (5). The presence of telomerase activity in both cultured and uncultured cells was found in 1999 (24). Another interesting finding have shown the presence of a population of Oct-4-positive cells in AF (25). AFSCs also express vimentin and alkaline phosphatase, which are markers of pluripotent stem cells (5, 26). Like other MSCs, AFSCs are attractive candidates for clinical applications, which were reviewed in Table 2. For instance some reports indicated that the AF can be a reliable and practical source of cells for the engineering of select fetal tissue constructs (27, 28). Another clinical application of AFSCs is the use as produce mineralized bioengineered constructs in vivo, functional repair of bone defects and bone engineering (2, 29–31), Neural tissue regeneration and nerve myelination (32–35), lung epithelial regeneration (36, 37), cardiac regeneration (38, 39) and kidney regeneration (21).

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Like other MSCs, AFSCs have advantages such as rapid cell proliferation, low or negligible immunogenicity. Many of these cells seem to express some of the same pluripotency markers. All of these features make them valuable for potential therapy applications. Thus far they have been used in pre-clinical settings to treat a variety of diseases such as osteogenesis imperfecta, congenital diaphragmatic hernia, Parkinson’s disease and cancer with encouraging results. Finally, their usefulness for AFSCs is very likely to expand their future clinical use even further. Human AFSCs could be isolated by several methods including immunoselection method based on surface antigens, single-stage and two-stage methods. Two-stage method has some advantages over the others. For example this method doesn’t interfere with the normal culture process for fetal karyotyping and also illustrated their ability to successfully differentiate into osteocyte, adipocyte and etc in vitro. As two-stage method is more superior compared to other methods, the use of this method have been proposed.

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