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Amniotic fluid characteristics and its application in stem cell therapy: A review

Abstract Amniotic fluid (AF) is a clear yellow fluid that surrounds the fetus during pregnancy. The amniotic sac consists of 2 layers: the amnion and the chorion. Osmotic and hydrostatic forces cause the maternal plasma to pass through the fetal skin and generate the AF. AF allows the fetus to grow inside the uterus, supports it from injuries, retains consistent pressure and temperature, and enables the exchange of body chemicals with the mother. At first, it consists of water and electrolytes but after the 12-14 th wk the liquid also contains carbohydrates, proteins, lipids, phospholipids, urea, hormones, and some biochemical products. AF appearance is characterized by the grade of cloudiness and the number of flakes of the vernix. The volume of AF increases with the fetus's growth. Its appearance depends on the gestational age. In addition to differentiated cells, stem cells are also found within the AF. These cells express embryonic-specific cell markers and bear high self-renewal capacity and telomerase activity. AF stem cells possess the potential to differentiate into osteogenic, cardiac, skeletal muscle, lung, neuronal, kidney, bone, cartilage, ovarian and hepatic cells in vitro. They represent a great promise in regenerative medicine for the reconstruction of bio-artificial tissues and organs in vivo. The purpose of this paper was to briefly review the development and function of AF and the application of its stem cells in cell therapy.


Introduction
Amniotic fluid (AF) is a complex physiological and dynamic biological fluid that surrounds the fetus during pregnancy. It provides mechanical protection and the nutrients required for fetal growth and wellbeing. After about 12 days of pregnancy, an amniotic membrane forms and lines the fetal cavity, while contemporary AF begins to fill the amniotic sac (1). In this review, we explain the features of AF stem cells (AFSCs). We focus on their ability to differentiate in vitro and their potential application for tissue regeneration in vivo.

AF sources
In the first trimester of pregnancy, the osmolality of the AF and maternal plasma are the same, which suggests that the maternal plasma generates the AF (2). Until the 22-25 th wk, the fetal skin is non-keratinized which allows AF to penetrate the skin and the surfaces of the placenta, amnion, and umbilical cord, which are freely penetrable to water and solutes (3). After the 24 th wk, the surfaces of the nose and mouth exchange fluid, but this is not the main origin of AF (4). In the latter half of pregnancy, the main origins of AF are from fetal urine production, pulmonary excretion, fetal swallowing, transmembranous movement among the amnion and chorion, and intramembranous movement between the fetal blood and the placenta (Table I) (5-9).

AF function
AF has vital functions such as nourishment, and antimicrobial and mechanical protection of the fetus during pregnancy. The most important roles of AF are as follows: 1. Shock absorber: AF protects the fetus from mechanical trauma (10).
2. Body parts development: AF helps the growth of the external body parts such as fingers and toes and the development of the lungs and the digestive systems by circulating freely within the womb and lubricating each part of the body. 3. Antibacterial activity: AF plays a significant role in the baby's natural immune system. It contains different antimicrobial substances including lactoferrin, lysozyme, bactericidal/permeability-increasing protein, a-defensins (HNP1-3), psoriasin (S100A7), calprotectin, cathelicidin (LL-37), and secretory leukocyte protease inhibitor, which contribute to the natural immune system in babies. These antimicrobials protect the baby from various bacteria, protozoa, fungi, and viruses (11).
Biochemical products are interchanged between the mother and fetus. The value of some biochemical markers such as ß2microglobulin, glucose, and uric acid present significant correlations with gestational age and creatinine, while other biochemical markers like urea, potassium, and phosphorus present mild correlations with age and creatinine. ß2-microglobulin, glucose, and uric acid are significant indicators of the function and maturation of fetal kidneys ( Figure 1) (14). From the 14 th -16 th wk of gestation, the AF is composed of some growth factors that promote the growth, attachment, polarity, and migration of cells in culture. We previously demonstrated that AF was able to advance the trans differentiation of retinal pigmented epithelium cells into rod photoreceptors, retinal progenitor, and retinal ganglion cells (16)(17)(18). At the end of the 32 nd wk of pregnancy, the mean macroscore is 0; the AF is clear and does not have any flakes. By the 36 th wk, the average macroscore is 1-2 as the AF has some flakes and carries a trace of cloudiness. After that the score increases until the 40 th wk of pregnancy; by this wk the mean score is 8 ± 2.6 as the AF contains a moderate amount of flakes and is cloudy. The mean macroscore is about 8 ± 3.2 in the 41 st wk, and thereafter the score increases until 9.3 ± 2.5 in the 42 nd wk. By this wk it has a large number of flakes and is very cloudy

AF volume (AFV)
AFV can be measured directly at the time of cesarean or by uterine hysterotomy, or indirectly by dye-dilution techniques or sonography.
Because direct and indirect techniques are time-consuming and invasive and may require laboratory support, AFV is commonly estimated by ultrasound (20). The advantages of sonography are that it is easy to carry out and teach, and it can be reproduced (21).
At the 12 th wk of gestation, the average AFV is about 60 ml and by the 16 th wk, it becomes 175 ml.
In the 20 th and 30 th wk, there is a volume of 300 and 600 ml, respectively. AFV increases regularly during pregnancy until 400-1200 ml at 34-38 wk.
After 38 wk, AFV decreases to 800 ml by the 40 th wk. After 43 wk, this volume diminishes to 250 ml (22).
Oligohydramnios occurs when there is an AFV < 200-500 ml, which is detected by ultrasound (23). This can be due to idiopathic underproduction or loss of fluid. Underproduction can be related to urinary tract obstruction, dysfunctional kidneys, maternal dehydration, or abnormal placental function (5). Loss of AF might happen due to rupture of surrounding membranes (24). Maternal hydration affects idiopathic oligohydramnios (25), so oral hydration is a noninvasive, easily accessible intervention to treat it (26). Oligohydramnios can cause fetal lung hypoplasia and contracture malformations (27). It can also increase the risk of non-reassuring fetal heart rate patterns, cesarean delivery, meconium aspiration, neonatal intensive care unit admission, and mortality (28). It has been estimated that 13.0% and 9.8% of pregnancies with oligohydramnios are associated with chromosomal anomalies and renal anomalies, respectively (29). The gestational age influences oligohydramnios management.
If discovered after 37 wk of gestational age, it cannot induce premature labor. But it causes a significant increase in the risk of the fetus needing an operative delivery. It is increased the induction rate (56% ) and cesarean rate (57%), as well as a higher risk of perinatal mortality (30

Amniotic fluid stem cells (AFSCs)
AF is bordered by the amniotic membrane and the embryo's skin on one side, and the embryonic urinary tract, digestive tract, and respiratory ducts on the other side. AF has different embryonic cells from the 3 embryonic germinal layers (37).
The fetal origin of these cells is represented by karyotype analysis (38). AF has fully differentiated cells, precursor and multipotent stem-like cells (39).
In 1993, hematopoietic progenitor cells were found in the AF before the 12 th wk of gestation.
In 1996, stem cells were found to be present in AF when they were cultivated in the supernatant of rhabdomyosarcoma cell lines and expressed dystrophin as a skeletal muscle protein (40).

Characteristics of AFSCs
AFSCs grow easily, have extensive selfrenewal capabilities and appear phenotypically and genetically stable in culture (46). They can present with various morphologies from a fibroblast-like shape to an oval-round shape.  (Table III) (58-62).  Engineered constructs using AFSCs promote the regeneration of damaged tissues (Table IV) (67-77).

Neural tissue regeneration
AFSCs have been reported to be able to Amniotic fluid and stem cell therapy mice brains. When this has been studied, they are indistinguishable from surrounding murine cells (65). AFSCs that have been transplanted into the striatum of normal and ischemic rats have differentiated into neurons as well as astrocytes (68). AFSCs in fibrin glue or matrigel have been delivered into the crushed sciatic nerve where they promoted peripheral nerve regeneration (81). AFSCs promisingly promote peripheral and central nerve regeneration.

Cardiac regeneration
AFSCs can be differentiated into smooth

Skeletal muscle regeneration
Differentiation of AFSCs into skeletal myogenic cells and skeletal muscle regeneration has been confirmed. These cells express desmin, troponin I, and α-actinin as skeletal cell-specific markers in vitro (84)(85)(86). Transplantation of the cells into cardiotoxin-injured mice has demonstrated that they differentiate into myogenic precursor cells and aggregate with host myofibrils (87).
Transplantation of the muscle progenitor cells that differentiated from AFSCs into the denervated external urethral sphincter enhances urodynamic action and expression of myogenic-related markers (71). These findings demonstrate that AFSCs are one of the most hopeful cells to treat skeletal muscle degenerative diseases such as Duchenne muscular dystrophy.

Lung epithelial regeneration
Transplantation of human AFSCs into an injured murine lung has been shown to cause differentiation into pulmonary lineages that express bronchiolar and alveolar markers and are associated with lung mucosal cells (72). It seems that AF is a valid resource for cell-based therapy for lung-related disorders.

Kidney regeneration
AFSCs can differentiate to renal fate in an ex vivo system. When they are transplanted into a mouse embryonic kidney they aggregate with the renal tissue, take part in nephrogenesis, and express molecular markers apecific for primary kidney differentiation like ZO-1, GDNF, and claudin (88). Injection of the cells into a damaged kidney has been shown to lead to ameliorates acute tubular necrosis in a mouse cognate model (73).
When nephrectomy model rats were treated with AFSCs, a remarkable decrease was seen in proteinuria, macrophages, blood pressure, and expression of α-smooth muscle actin (89).

Hepatic regeneration
AFSCs can be induced to differentiate into hepatocyte cells that express albumin, hepatocyte nuclear factor 4, alpha-fetoprotein, hepatocyte growth factor receptor, and the multidrug resistance membrane transporter MDR120, and secrete urea (90). Transplantation of AFSCs into liver fibrosis mouse models has revealed that the cells fuse with the recipient liver cells and improve hepatic function (74).
AFSC transplantation into fulminant hepatic failure mouse models can improve liver function and increase survival rates (91). AF is a promising origin of progenitor cells for liver cell transplantation and hepatocyte regeneration.

Bone and cartilage regeneration
AFSCs have been used to develop bone constructs using tissue engineering principles.
AF has been validated as an effective source for functional repair of extensive bone defects.

Discussion
After the 10 th -20 th gestation wk, free diffusion happens between the fetus and AF through the placenta, fetal skin, and umbilical cord (4). At first, AF is mainly composed of water from the mother's body (98%) and the remaining 2% is salt and cells from the baby. But after the 12 th -14 th wk, it is also composed of carbohydrates, proteins, lipids, phospholipids, growth factors, and urea, which after the 12 th -14 th wk that help the development of the fetus (99). During this period, AF composition is similar to that of fetal plasma, thus the analysis of AF composition can provide useful would represent worth information about of the pathological or physiological status of the fetus (1, 100).
AF also contains comprises a heterologous cell population from all 3 embryonic germ layers (101).
It has been proposed stem cells that the biological and therapeutic characteristics of AF stem cells were be studied (99). These cells can be easily obtained during amniocentesis with low risk for the mother and the fetus, and grow in vitro (89).

Conclusion
In this review, we described AF as a fluid that surrounds the baby in the uterus. We explained the sources of AF and its vital functions in fetal development. We illustrated that it is composed of nutrients, hormones, and biochemical products.
The appearance and volume of AF depend on gestational age. Finally, the survival of stem cells in the AF, their high proliferation rate, the substantial potential of differentiation, normal karyotype, and low immunogenicity were discussed. We elaborated on the potential of AFSCs to promote the regeneration of various tissue defects in vitro and in vivo.