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Journal of the Chinese Medical Association 76 (2013) 606e610
www.jcma-online.com
Review Article
Amniotic fluid may act as a transporting pathway for signaling molecules
and stem cells during the embryonic development of amniotes
Xinglong Tong*
Hebei Xinglong Institute of Pharmacological and Medical Science, Shijiazhuang, China
Received August 16, 2012; accepted March 11, 2013
Abstract
Amniotic fluid (AF) is formed at the very early stages of pregnancy, and is present throughout embryonic development of amniotes. It is wellknown that AF provides a protective sac around the fetus that allows fetal movement and growth, and prevents mechanical and thermal shock.
However, a growing body of evidence has shown that AF contains a number of proteins and peptides, including growth factors and cytokines,
which potently affect cellular growth and proliferation. In addition, pluripotent stem cells have recently been identified in AF. Herein, this article
reviews the biological properties of AF during embryonic development and speculates that AF may act as a transporting pathway for signaling
molecules and stem cells during amniote embryonic development. Defining this novel function of AF is potentially significant for further
understanding embryonic development and regenerative medicine, preventing genetic diseases, and developing therapeutic options for human
malignancies.
Copyright Ó 2013 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved.
Keywords: amniotic fluid; embryonic development; embryonic induction; stem cells
1. Introduction
2. Signaling molecules in embryonic induction
Animals that possess an amnion are referred to as amniotes.
The cavity between the amnion and the fetus is called the
amniotic sac and contains amniotic fluid (AF). The amniotic
sac is formed at very early stages and is present throughout
embryonic development.1 In humans, the amniotic sac and its
aqueous fluid appear 7e8 days after fertilization. It is widely
accepted that AF provides mechanical protection and nutrients.2 Based on current literature and our own studies,3,4 this
article hypothesizes that a potential important physiological
role of AF may be to act as a transporting pathway for
extracellular signaling molecules and stem cells during embryonic development.
A fertilized oocyte divides into billions of cells that
differentiate into diverse tissues and organs, resulting in the
formation of a new organism. Embryonic induction is thought
to play an important role in the development of tissues and
organs in most animal embryos, from the lower chordates to
the higher vertebrates. In principle, induction means any
process in which the developmental pathway of one cell
population is controlled by signals emitted from another.5
Embryonic induction is further subcategorized into primary,
secondary, and tertiary induction.5 Primary embryonic induction includes neural and dorsal mesoderm inductions. The
tissue interactions governing cell differentiation and morphogenesis throughout embryonic development are secondary and
tertiary inductions; for example, the induction of the lens is
classified as secondary induction. Induction is carried out
through signaling molecules (regulatory messengers) or
inducing factors.6 These molecules are secreted into the
extracellular space and target responding cells by specifically
* Corresponding author. Dr. Xinglong Tong, Hebei Xinglong Institute of
Pharmacological and Medical Science, 232, South Zhonghua Street,
Shijiazhuang 050056, China.
E-mail address: [email protected].
1726-4901/$ - see front matter Copyright Ó 2013 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved.
http://dx.doi.org/10.1016/j.jcma.2013.07.006
X. Tong / Journal of the Chinese Medical Association 76 (2013) 606e610
activating intracellular pathways.7 To date, many extracellular
signaling molecules and their corresponding intracellular
pathways have been identified, including a variety of growth
factors (ligands for tyrosine kinase receptor), transforming
growth factor beta (TGF-b), hedgehog, notch, wingless, and
retinoic acid.6,8e10 They control cellular proliferation, differentiation, and other functions during development.
Most of the signaling molecules are proteins, and some are
lipids or organic acids. It is generally accepted that these
molecules are secreted by specialized groups of cells into the
extracellular space and diffuse to neighboring cells. However,
how these molecules reach target cells over long distances is
not yet well understood.
3. AF may act as a transporting pathway for signaling
molecules reaching target cells over long distances
3.1. Bi-directional substance exchange between AF and
fetal tissues
In humans, the amniotic sac and fluid are formed during the
blastocyst stages and are present throughout all embryonic
stages.1,2 AF is a dynamic milieu that undergoes multiple developmental changes over the duration of fetal growth. In early
embryogenesis, AF mostly derives from the fetal extracellular
matrix. Thereafter, production of AF is predominately accomplished by the excretions of fetal urine and secretion of skin, oral,
nasal, tracheal, and pulmonary fluids.1 There is rapid bidirectional exchange between the fetus and the amniotic cavity
for solutes in AF by swallow, absorption, excretion, and secretion.
A previous study demonstrated that intrafetally injected technetium Tc 99m rapidly enters and is concentrated in the amniotic
cavity in fetal sheep at 131 days’ gestation. Meanwhile, intraamniotically injected technetium Tc 99m rapidly enters into the
fetal circulation.11 To characterize the substance exchange between the AF and the fetal tissues, we injected phenol into the
amniotic sacs of chick embryos at 16 days, and then detected the
concentrations of phenol in the AF and fetal tissues. The results
showed that phenol started to occur at 2 minutes in fetal tissues
after injection of phenol into the AF, including the lung, liver,
kidney, leg muscle, wing, brain, and eye. The concentration of
phenol reached peak levels in these tissues at 5 minutes, began to
decline at 6 minutes, and remained at stable levels at 10 minutes.
The phenol concentrations from high to low order were revealed
in the liver, lungs, heart, kidneys, leg muscles, wings, brain, and
eyes. In contrast, the phenol level in the AF displayed an inverse
pattern of changes compared to that observed in fetal tissues. It
first rapidly decreased, then increased when levels declined in
fetal tissues, and then reached a stable level at around 10 minutes.
This result directly demonstrated that the solutes of AF rapidly bidirectionally exchange with all fetal tissues.
3.2. The composition of AF reflects the dynamic status of
the developing fetus
AF contains electrolytes, carbohydrates, lipids, proteins,
amino acids, lactate, pyruvate, enzymes, and hormones. We
607
simultaneously measured the levels of the individual components, including electrolytes and organic substances in human
AF, umbilical cord serum (UCS), and maternal serum (MS)
from the same pregnant woman at 15e42 weeks’ gestation.3
The results showed that the level of primary electrolytes,
such as sodium, chloride, and anion gap in AF were at almost
the same level as in UCS and MS. The levels of organic
substances in AF, including total protein, glucose, triglyceride,
cholesterol, and various enzymes, were markedly lower than
in UCS and MS. In addition, the osmotic pressure of AF was
slightly lower than blood osmotic pressure. The data suggest
that the composition of AF is not a simple filtrate from the
blood, and AF is an independent fluid pool.
Almost all of the metabolites produced by fetal tissues have
been found in AF. We have detected >100 metabolites,
including cortisol, acetone, cholesterol butyrate, margaric
acid, and H3-acetyl carnitine, etc., in AF using the gas
chromatography-mass spectrometry (GC-MS) technique (unpublished data). Many fetal tissue-specific products have also
been detected in AF and serve as biomarkers for fetal diseasespecific conditions.12,13 Simultaneous measurement of cytokines in AF and matching maternal sera has revealed that the
concentrations of most cytokines are quite different from the
maternal blood.14 Proteomic analysis of AF also shows a
significantly different composition from the maternal
plasma.15 These data indicate that the composition of AF is
primarily representative of the fetal profile and reflects the
physiological status of the developing fetus.
3.3. AF is indispensable for fetal development
Clinical data have demonstrated that abnormal AF volume
is always associated with an increased risk of abnormal
development or even death of the fetus.16 Our study showed
that replacement of 1.5 mL of AF with an equal volume of
37 C isosmotic saline in chick embryos at Day 14e15 definitely caused the death of all chick embryos. When the
replacement was performed at Day 16e18, the exact time of
chick leg and wing development, chicks were born alive, but
unable to walk or move their wings. These phenomena suggest
that AF plays an important role in embryonic development.
A large number of proteins and peptides in AF have been
identified, and evidence has shown that most of these proteins
or polypeptides have low molecular weights.17 Among these
proteins or peptides, an increasing number are identified as
growth factors, tumor markers, and cytokines, which possess
potent bioactivity.18e22 The concentrations of these molecules
in AF show a dynamic pattern during embryonic development.
We monitored the levels of biomarkers including carcinoembryonic antigen, ferritin, cancer antigens 125 and 199, and
alpha fetoprotein in human AF, and found that these proteins
displayed different dynamic changes from those in the umbilical cord and maternal serum as gestation advanced.3 Lei
and Du23 reported that the levels of epidermal growth factor in
the human AF began to increase in the middle of pregnancy,
ultimately reaching levels exceeding 4.5 mg/L before birth.
Some studies have demonstrated that in early gestation,
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X. Tong / Journal of the Chinese Medical Association 76 (2013) 606e610
proteins could diffuse across unkeratinized fetal skin.24 In late
gestation, proteins within AF can be absorbed by the fetal
gastrointestinal tract and transported to the whole fetal
body.25e27 The dynamic pattern and evidence for their transporting to the fetus imply that proteins in AF are required for
fetal development.
The human AF proteome of a 16e18 week normal pregnancy was profiled by Cho et al15 and the function of the fluid
was analyzed by proteomic analysis. A total of 842 proteins
and peptides were identified, functions of which included
cellular movement, development of organs, and cellular
growth and proliferation. It is known that the morphology and
biological properties of embryonic cells are similar to those of
tumor cells. We found that AF or proteins extracted from
human AF at 20 weeks’ gestation inhibited H22hepatocarcinoma growth in a mouse model primarily
through inducing tumor cells to adopt highly differentiated
states.4 A similar result has been reported by others with
mouse AF.28 The studies indicated that AF or AF-derived
proteins or peptides were involved in the inducement of
cellular differentiation. Moreover, we found the levels of total
protein, glucose, and triglycerides in human AF were much
lower than those in fetal and maternal sera.3 For example, the
average concentration of total protein in AF was 4.87 g/L,
whereas it was 39.46 g/L and 61.55 g/L in fetal and maternal
sera, respectively. The glucose level in AF was 1.29 mM,
3.45 mM in fetal, and 3.58 mM in maternal serum. Triglyceride and cholesterol were almost undetectable. These data
suggest that AF may not act as a nutrient transporting pathway
like it does in the blood circulation.
3.4. Hypothesis on the novel function of AF
Based on the characteristics of AF mentioned above, this
article speculates that AF may act as a pathway for transporting signaling molecules to distant target cells. Embryonic
development is a rapid process involved in extensive molecular expression and signaling transduction. Therefore, an independent and intact signaling delivering pathway is essential
for embryogenesis. In amniotes, the allantois is part of the anal
membrane, and forms an axis for the development of the
umbilical cord. The stalk connecting the allantois to the fetal
bladder eventually forms the bladder ligament after birth.
Trophoblasts are cells that form the outer layer of the blastocyst and provide nutrients to the embryo. They develop into a
large part of the placenta.
The fetal blood circulation transporting nutrients to all of
the fetal tissues is unlikely to act as the pathway delivering
signaling molecules. All of the molecules involved in
embryogenesis are highly efficient, and they work at low
concentrations. If these signaling molecules enter into fetal
blood circulation, they would first go into the placenta, where
substance in fetal blood exchanges with maternal circulation.
This long distance circulation may delay these molecules from
timely performing their actions, and they may also be diluted.
In addition, they would be cleared out by the maternal
immunological system in the placenta. Moreover, embryos do
not form a blood circulation system at early stages. In contrast,
AF is formed at very early stages of embryonic development
and present through all embryonic stages. AF does not directly
connect to the blood vascular system. All of the dissolved
solutes in AF constantly directionally exchange with the fetus
tissues. The composition of AF is a dynamic indicator of the
embryonic metabolism and most of the identified proteins or
peptides in AF have activities associated with cellular growth
and proliferation. Those characteristics strongly support the
view that the AF may act as a transporting pathway for
signaling molecules in embryonic development.
4. AF may also act as a transporting pathway for stem
cells
4.1. Debate about the origin of stem cells
The origins of fetal and adult stem cells are not yet well
defined.7 Many researchers have reported that stem cells isolated from bone marrow can differentiate into specialized
cells, such as liver, muscle, or neural cells. Mesenchymal stem
cells have been isolated from adult bone marrow; therefore,
many scholars believe that both adult and fetal stem cells are
originally produced by the bone marrow and then transferred
to different tissues by the circulatory system.29e31 Others
believe that tissues retain a small proportion of undifferentiated or partially differentiated cells as progenitor cells, which
act as a repair system for the body by replenishing adult tissues.7 Although these two hypotheses are based on rational
reviews and research data, this article suggests that both fetal
and adult stem cells originate from a unique cell mass. This
cell mass specifically produces stem cells that differentiate
into diverse progenitor cells at different stages during embryonic development. Those diverse progenitor cell subtypes
enter the amniotic cavity, then migrate through the AF and
implant into specific tissues, where they will be maintained for
life. Here we refer to the hypothesis as “the migration and
implantation of stem cells by amniotic fluid”.
Some studies in mammals have shown that amniotic
epithelial cells generate stem cells.32,33 Other reports suggest
that stem cells originate from the yolk sac.34 Some researchers
believe that fetal stem cells are derived from the inner cell
mass,35 a view that is identical to our hypothesis. This article
proposes that, whatever the derivative source of stem cells may
be, it is unlikely that some stem cells would be maintained in
an undifferentiated state, while other cells differentiate into
specialized cell types during embryonic development. We
cultured stem cells in vitro and found that different stem cells,
including lipid, neural, and mesenchymal stem cells, had
uniform phenotype after treatment with inducers. In other
words, we never determined that some of the cells were
differentiated while others were maintained in an undifferentiated state after induction. Obviously, this “all or none”
phenomenon does not support the hypothesis that tissues retain
stem cells by themselves. In addition, it is known that bone
marrow is formed at the middle or late stages of embryonic
development, while fetal stem cells appear at early stages of
X. Tong / Journal of the Chinese Medical Association 76 (2013) 606e610
embryogenesis and are continuously produced until the end of
embryonic development. Therefore, it is unlikely that bone
marrow is an original source of stem cells.
4.2. A novel hypothesis on the origin of the stem cell and
its transporting pathway
This article speculates that both fetal and adult stem cells
may originate from a unique embryonic cell mass. This cell
mass produces and releases a variety of subtypes of stem cells
into the amniotic cavity at different stage of embryogenesis,
which travel through AF, implanting into and residing in all
tissues for a lifetime. The following evidence supports this
hypothesis.
Cells from three embryonic layers (mesoderm, endoderm,
and ectoderm) have been found in AF, including epithelial-like
cells, amniotic and trophoblast cells that produce estrogen and
chorionic gonadotropin, and mesenchymal fibroblast cells.36,37
Many scholars have isolated diverse progenitor cells from AF at
early to late embryonic stages.38 AF-derived stem cells (AFS)
express embryonic and adult stem cell markers, including
CD29, CD44, CD90, CD105, and CD73.39e41 AFS rapidly
proliferate and retain long telomeres and a normal karyotype
after over 250 population doublings in culture in vitro. They can
differentiate into cell types representing the three embryonic
germ layers, including cells of adiopogenic, osteogenic,
myogenic, endothelial, neuronal, and hepatic lineages after induction. Those characteristics fit the commonly accepted
criteria for defining pluripotent stem cells.42,43 AFS have similar
characteristics to mesenchymal stem cells derived from bone
marrow and lipid tissue, including a pyramidal morphology
typical of fibroblast-like cells.40 We injected AFS into the amniotic cavity of chick embryos at Days 15e18 and detected AFS
in the liver, lungs, heart, kidneys, leg muscles, and wings within
10 minutes of injection. Fewer stem cells were detected in the
brain, probably due to the bloodebrain barrier. The result implies a low capacity for self-repair when the brain is damaged
after birth. In addition, a study demonstrated that AFS could be
found in intestine, heart, liver, lung, spleen, and bone tissue after
intraperitoneal injection.44 These experiments indicate that
AFS are able to move freely into fetal tissues. Why do diverse
progenitor cells present in AF at different stages of embryonic
development? This article suggests that the reasonable explanation is that AF may play a role in transporting stem cells to
fetal tissues during embryonic development.
In conclusion, the evidence mentioned above has shown
that AF contains a number of proteins or peptides, including
growth factors or cytokines that possess potent bioactivity on
cellular growth and proliferation. Recently, a variety of
pluripotent stem cells have been identified in AF. For the first
time, this article speculates that AF may act as a transporting
pathway for signaling molecules and stem cells during the
embryonic development of amniotes. Defining this novel
function of AF is potentially significant for further understanding embryonic development and regenerative medicine,
preventing genetic diseases, and developing therapeutics for
human malignancies.
609
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