Expert Reviews

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OBSTETRICS

Human amniotic fluid: a source of stem cells

for possible therapeutic use
Margaret Dziadosz, MD; Ross S. Basch, MD; Bruce K. Young, MD

tem cells have become the focus

of great interest because of their
potential for therapy in a wide variety of
conditions. Conditions possibly treatable by stem-cell transplants include
genetic defects, tissue and organ
replacement, autoimmune disease, and
malignancies. A stem cell is an undifferentiated cell that is capable of
prolonged self-replication without differentiation. There is a spectrum of stem
cells that ranges from multipotent cells
that have the potential to differentiate
into 1 or 2 lineages, pluripotent cells that
may differentiate into many lineages,
and totipotent cells that have unlimited
differentiation potential. A totipotent
stem cell should have the potential to
differentiate into lineages for mesodermal, ectodermal and endodermal
tissues (such as osteogenic, neurogenic,
and hepatic lineages).1
Stem cells are characterized by the
presence of surface markers and transcription factors that are associated with
self-renewal without differentiation as
seen in early embryonic states. Some
examples of these surface markers are
From the Division of Maternal Fetal Medicine,
Department of Obstetrics and Gynecology
(Drs Dziadosz and Young) and the Department
of Pathology (Dr Basch), New York University
Langone Medical Center, New York, NY.
Received Sept. 1, 2015; revised Oct. 22, 2015;
accepted Dec. 31, 2015.
There was nancial support from The William
and Linda Haugland Foundation for laboratory
research cited, but they had no involvement in
study design, data collection, data analysis,
interpretation, or writing of this report or in the
decision to submit this manuscript for
publication.
The authors report no conict of interest.
Corresponding author: Bruce K. Young, MD.
Bruce.young@nyumc.org
0002-9378/$36.00
2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajog.2015.12.061

Stem cells are undifferentiated cells with the capacity for differentiation. Amniotic fluid
cells have emerged only recently as a possible source of stem cells for clinical purposes.
There are no ethical or sampling constraints for the use of amniocentesis as a standard
clinical procedure for obtaining an abundant supply of amniotic fluid cells. Amniotic fluid
cells of human origin proliferate rapidly and are multipotent with the potential for
expansion in vitro to multiple cell lines. Tissue engineering technologies that use amniotic
fluid cells are being explored. Amniotic fluid cells may be of clinical benefit for fetal
therapies, degenerative disease, and regenerative medicine applications. We present a
comprehensive review of the evolution of human amniotic fluid cells as a possible
modality for therapeutic use.
Key words: amniotic fluid, stem cell, therapy, transplantation

SSEA3, SSEA4, Tra-1-60, Tra 1-81,

CD117, and CD90. Some important
transcription factors that are associated
with embryos are Oct3, Oct4, Sox2,
Nanog, and Rex1. Flow cytometry has
been the primary technique for the
identication of stem cells by detection
of these markers.
Stem-cell therapy introduces stem
cells into selected tissue environments to
prevent and treat injury or repair
abnormal tissue. Stem cells from various
sources have been considered as potential therapeutic alternatives to organ
replacement and other therapies that
require transplantation. They can be
studied in vitro by modeling pathophysiologic processes of inammation,
disease, and treatments that would occur
in vivo. They are introduced intravenously or directly into tissue to study
the prevention of injury or the repair
of a damaged system or congenital
defect. Stem cells are well-known to exist
in embryonic tissue and bone marrow.
Additional sources of stem cells in
humans have been identied: umbilical
cord blood, umbilical cord cells,
placenta, amniotic membranes, amniotic uid, peripheral blood, and somatic
cells induced to pluripotency. For
induced pluripotent cells that are derived
from human somatic cells, Oct4, SOX2,

KlF4, and c-myc also have been used to

transduce somatic cells to develop a
pluripotent cell population. There has
been signicant controversy regarding
the various sources about safety in
transplantation, accessibility, and ethical
issues when human embryos are the
source, tumorigenesis with embryonic
and transduced cells, and expansion and
reliability of all these sources for clinical
use.2 Therefore, amniotic uidederived
cells might be a practical alternative
source because they are readily available,
grow well in culture, and avoid these
issues.

Alternative sources of stem cells

Embryonic stem (ES) cells are totipotent
but have been shown to form tumors in
immunodecient mice. ES cells grow as
teratocarcinomas in vivo and frequently
acquire chromosomal aberrations.3,4
They have restricted differentiation
capacity and have been shown to yield
both genetic and epigenetic abnormalities in culture. Signicant ethical issues
arise regarding the use of embryos as
well.
Cord blood cells are expanded
easily but are primarily hematopoietic
lineage cells with <1% multipotent cells.
There is substantial clinical experience
with cord blood transplantation for

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hematologic indications and growing

interest in other applications.5,6 Umbilical cord and placental stem cells exist in
the middle ground between ES cells and
adult stem cells and might yield desirable
expansion with lack of tumorigenesis.
However, expansion and culture studies
are limited at present.
Bone marrow is the most common
source of adult stem cells for clinical
transplantation. Restricted lineage, low
proliferation rates because of limited
telomerase expression, and restricted
differentiation potential limit their clinical application so far. They are, however,
less tumorigenic than ES cells and do
not have ethical concerns. There is
extensive clinical experience with their
use, establishing standard protocols for
transplantation.
Amniotic uid is readily available,
is noncontroversial, and is obtained
routinely by amniocentesis. It is analyzed
for prenatal genetic diagnosis in the second trimester, for evidence of fetal pulmonary maturity in the third trimester,
and for infection at any gestational age. It
is made up of a heterogeneous population
of cells that routinely is retrieved clinically
and cultured for genetic studies. It has
been shown that a signicant percentage
of cells that are obtained from amniocentesis exhibit stem-cell markers. These
cells are cultured easily, expanded, and
remain viable over many passages, tolerating cryopreservation very well.2,7-10
A great deal of research has been
performed on second-trimester amniotic uid cells (AFC), but there is little
known about whether third-trimester
AFC closely resemble those of the midtrimester.11 You et al11 retrieved amniotic uid at the time of elective cesarean
delivery at term. AFC were isolated in
culture and found to be positive for
surface markers CD29, CD73, CD90,
and CD105 as well as Oct-4. Proliferation potential was veried, and differentiation into osteogenic lineage was
reported as successful. A more recent
study aimed to characterize human AFC
from the early third trimester, where 3
samples were analyzed from gestations
from 28-34 weeks. Third-trimester AFC
expressed comparable levels of Oct-4
and Nanog, but lower levels of SOX2

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and Rex-1. Samples were also successfully differentiated to adipocytes,
osteoblasts, chondroblasts, myocytes,
and neural-like cells, although thirdtrimester samples showed poor differentiation potential to myocytes and
stronger potentiation to neural lineage.12
Further studies on not only thirdtrimester amniotic uid but also AFC
at term are warranted, because these
ndings are promising but inconclusive.

AFC
In early studies, amniotic uid from
pregnant ewes was isolated and expanded
to mesenchymal, broblast/myobroblast cell lineages. These cells were noted
to proliferate signicantly faster than
surrounding cells, showed little cell
death, and could be isolated consistently
from amniotic uid.13 AFC showed
stem-cell potential when they were found
to contain Oct-4, which is a known
marker specic for human embryonic
stem cells that are associated with
maintenance of the undifferentiated state
and pluripotency.14,15 Besides their rapid
proliferation, human AFC have been
differentiated successfully into all embryonic germ layers, thereby demonstrating pluripotency. After multiple
passages in culture, human AFC
remained chromosomally stable and did
not form teratomas or undergo malignant change.2,7 These qualities of human
AFC suggested signicant advantages as
a potential source of cells for clinical
transplantation.
ES cell molecular markers are molecules that specically are expressed by
stem cells and are critical to the characterization and identication of their
pluripotency. There are a wide range of
cell-surface proteins, transcription
factors, and molecular markers indicative of stemness (Table). These surface
markers are usually glycosphingolipids
or membrane proteins. Research on
stem cells has used the technique of ow
cytometry. Flow cytometry separates
cells using their cell surface markers,
thereby identifying viable cells. This
process then permits the formation of
clonal cultures from specically identied cells. Flow cytometry can also be
used to identify transcription factors in

322 American Journal of Obstetrics & Gynecology MARCH 2016

cell nuclei that are related to stem-cell

behavior. However, in contrast to the
process of identication of cell surface
markers, the cells are no longer viable
and cannot be cultured after transcription factor identication.
Human amniotic uidederived stem
cells were identied as expressing
Oct-4, SOX2, Nanog, Rex1, cyclin A,
and mesenchymal markers that include
CD90, CD105, CD73, CD166, CD133,
and CD44.15-19 There is a higher percentage of stem-cell transcription factors Oct-4, Nanog, and SOX2 in AFC
from uid that is obtained from 15-17
weeks gestation vs later gestational ages.
Cell surface markers do not appear to
vary with gestational age among the
second-trimester samples, but individual samples expression varies greatly
and may overshadow this effect.10 The
ability for adipocyte, osteocyte, and
neuronal cell generation were also
demonstrated.20,21 De Coppi et al21
postulated that CD117 was a marker
for selection of AFC from human
amniocentesis cultures. However, subsequently, it was shown that CD117 is
actually a clonal marker that was present in only approximately 0.5-2% of
AFC in cultures that used the same
media.10,22 Another variety of human
AFC, mesenchymal stem cells have
been identied with distinct populations of varying differentiation
potential.23
Our studies on human AFC have
shown the presence of CD117, 133, 90,
15, 44, 29, 9, 73, as well as SSEA1, SSEA3,
SSEA4, Tra-1-60, Tra-1-81, Oct4, Rex1,
Nanog, and SOX2.24-28 Our laboratory
selected SSEA4, Tra-1-60, and CD90 for
subsequent investigations because they
were the most highly expressed ES cell
markers in our patient samples. These
studies have also shown clonal populations bearing all 3 markers, combinations of 2 different stem cell markers
(eg, SSEA4/CD90, SSEA4/TRA-1-60,
and Tra-1-60/CD90), and populations
with just 1 marker.10 Clones with
different combinations of markers may
vary in their properties of stemness. This
is an area for further investigation that
has not yet been explored. Thus, there is
a mixture of cells with varying potential

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TABLE

Human embryonic stem-cell markers24-28

Marker

Characteristic

Classification

Known activity

SSEA-1 (CD15/Lewis x)

EC, ES

Carbohydrate-associated; lactoseries
oligosaccharide antigen

Increase with differentiation

SSEA-3 (glycolipid GB5)

EC, ES, EG

Carbohydrate-associated; globoseries
carbohydrate antigen

Decrease with differentiation

SSEA-4

EC, ES, EG

Carbohydrate-associated glycoprotein

Decrease with differentiation

Nanog

ES

Homeobox family, DNA-binding

transcription factor

Decrease with differentiation

Oct-4 (POU5F1)

EC, ES, EG

Octamer-binding transcription factor

Most recognized marker totipotent ES

Rex-1 (Zfp42)

EC, ES

Zinc finger family

Maker of undifferentiated cells

SOX2

EC, ES

SOX family, DNA-binding transcription

factor

Specifies germ lineage at implantation,

regulates proliferation, differentiation

TRA-1-60

EC, ES, EG

Surface antigen, epitope expressed

on high molecular weight proteoglycan

Tumor recombinant antigen

TRA-1-81

EC, ES, EG

Surface antigen, epitope expressed

on podocalyxin

Tumor recombinant antigen

CD9 (MRP1)

ES

Surface marker, tetraspanin family

Cell adhesion, migration, T-cell stimulation

CD29 (B1 integrin)

ES

Surface marker, intregrin family

Cell adhesion, recognition, embryogenesis

CD44

EC, ES

Surface marker, glycoprotein

Cell interactions, adhesion, migration

CD45 (PTPRC)

ES

Protein tyrosine phosphatase

receptor; leukocyte common antigen

Cell growth and differentiation,

mesenchymal, hematopoetic cells

CD73

ES

Surface marker, enzyme

Converts adenosine monophosphate

to adenosine

CD90 (Thy-1)

EC, ES

Surface marker, GPI-linked

glycoprotein

Mesenchymal stromal cells, hematopoietic

stem cell, neuron, T-cell activation

CD105

ES

Surface marker, endoglin,

transmembrane glycoprotein
of zona pellucida

Differentiation of smooth muscle,

angiogenesis, neovascularization

CD117 (c-kit)

EC, ES

Surface marker, stem-cell

factor receptor, tyrosine kinase
receptor

Present on hematopoietic progenitor

cells, role in gametogenesis

CD133

EC, ES

Surface marker, Prominin-1,

glycoprotein

Organizer of cell membrane topology

CD166

EC, ES

Surface marker, immunoglobulin

Cell interaction and adhesion

Stage-specific embryonic
antigens (cell surface associated)

Transcription factors

Cluster of differentiation markers

EC, embryonic carcinoma cells; ES, embryonic stem cells; EG, embryonic germ cells; GPI, glycosylphosphatidylinositol.
Dziadosz. Amniotic uid for possible therapy. Am J Obstet Gynecol 2016.

for differentiation into lineages from all

3 germ layers, likely in addition to cells
that are pluripotent.
As yet, it is not clear which clones will
have different potentials for differentiation, but clonal isolation of a single

pluripotent clonal cell line is not essential for AFC differentiation. Because
of the heterogeneous nature of AFC,
it may not be important to isolate a
pluripotent stem-cell clone to build a
framework with which to move towards

translational use in therapy.10 Our laboratory studied 37 different samples of

human amniotic uid. A total of 81
cultures were obtained for study from
doubling each culture sample from 2-8
times. With every culture, there was

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consistent inducibility to osteogenic,

chondrogenic, and neurogenic lineage
without the need for isolation of a specic clone with either a single marker or
combination of markers.10 Individual
variation in the percentage distribution
of cells that express different markers
does not appear to be a limitation
because every sample has a wide variety
of lineage precursors. Amniotic uide
derived cells have been reprogrammed
easily into induced pluripotent stem
cells, which supports this concept.21
This is in contrast to the concerns
raised by Ekblad et al.29 They focused on
selecting a single CD117-positive pluripotent cell as proposed by Zia et al22 and
were unsuccessful in enriching the
CD117 population. Thus, it is likely that
clonal selection is unnecessary for clinical use.

Translational properties
Amniotic uid is retrieved from the
amniotic sac at amniocentesis. It is
common to use 3-5ecc aliquots of amniotic uid. In our laboratory, this small
amount yields an average of 100,000 cells
per cubic centimeter with 60-90%
viability. Once AFC are isolated from the
amniotic uid, they are passaged and
doubled in an average of 36 hours.
Therefore, in 72 hours or 2 passages,
approximately 1.6 million cells become
available. Of this total number, approximately 75% are viable or 1.2 million and
up to 99% exhibit stem-cell markers.10
This is more than sufcient for laboratory studies. For clinical purposes, at
least 5 cc of amniotic uid may be
necessary, depending on the number of
cells that are required for transplantation
and the time window for a given treatment. This will have to be determined in
future clinical trials.
Culture characteristics of AFC include
longevity. Our laboratory has shown
proliferation capacity of up to 20 passages, which can expand a cell population from a single sample aliquot to up
to >10 billion cells.10 Cell lines have
been propagated more than 250 times
with retention of telomere length and a
normal karyotype.21 Their cryopreservability and inducibility to multiple lineages have been reported by different

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groups.7,10 This differentiation into
multiple fates has been documented via
messenger RNA lineage specic genes
and by histochemistry and histology.19
AFC have been reported to have a low
immunogenic prole with minimal
expression of HLA-ABC and no HLADR antigens.10,23 This could reduce the
risk of rejection and of graft-vs-host
disease. However, our recent work in
collaboration with Carriere et al (unpublished data) has demonstrated the
presence of HLA-DR antigens with the
use of DNA technology in contrast to
previous reports. This raises the question
of the need for HLA matching with other
than autologous transplantation. Thus,
it is possible that some immunologic
matching would be recommended for
clinical transplantation.

Preclinical models
The use of preclinical models has
been promising for future therapeutic
possibilities. Following reconstructive
engineering concepts that were pioneered by Fauza et al30,31 in 1998, when
gravid ewes fetal tissue was harvested
and manipulated for autologous transplantation for injured fetal bladder, skin,
and diaphragm, Kaviani et al13 engineered models with amniotic uid cells.
Amniotic uid was obtained from
pregnant ewes and expanded into
mesenchymal, broblast, and myobroblast cell lineages. Amniotic uid
mesenchymal stem cells transplanted to
fetal lambs with intentional skin wounds
yielded improved and more rapid healing time.33 Using a mouse model, Sun
et al34 differentiated human AFC into
keratinocytes that improved epidermal
regeneration at intentional excisional
wounds. Mesenchymal amniocytes that
were grown on seeded scaffolds inserted
to sites of diaphragmatic defects in fetal
lambs showed improved structural
repair over myocyte or acellular grafts.32
Recently, intraamniotic delivery of
neurally induced amniotic uid cells
for fetal lambs with spina bida was
performed. Clustering of the induced
cells in the neural placodes was seen,
supporting regenerative therapeutic
potential for transplanted AFC for the
treatment of neural tube defects.36

324 American Journal of Obstetrics & Gynecology MARCH 2016

AFC were used to engineer autologous

fetal tissue for body-wall defects,
myelomeningocele, bladder extrophy
defects, and congenital diaphragmatic
hernia, also shown in a mouse
model.35,37 Intratracheal injection of
human AFC into a rabbit model of
congenital diaphragmatic hernia also
showed promising effects on improved
lung density and function.38 Amniotic
uid cells have been shown to repair
injured urinary bladder detrusor muscle
and increase muscle strength in mice
that had been modeled for spinal
muscular atrophy.7,39 In mice with acute
tubular necrosis caused by either rhabdomyolysis or cisplatin toxicity, infusion
of AFC improved renal function as AFC
migrate to areas of ischemia.40,41 AFC
with renal progenitor phenotype have
demonstrated a nephroprotective effect
in an acute ischemia reperfusion model
using rats that resulted in reduced
creatinine level, less tubular necrosis,
and less long-term brosis.42
Scaffolds provide an improved environment for cell growth and organogenesis. They are also a fundamental
component for development of multidimensional structures. AFC are able
to form tissue-engineered bone from
printed scaffold constructs of osteogenically induced lineage cells in mice.13,21
The ability to create 3-dimensional
bone constructs in rabbits shows promise for craniofacial reconstruction.43,44
There has been evidence of inducibility
to cardiogenic lineage forming heart
syncytia that pulsate, and vascular
grafts have been produced by seeding
3-dimensional scaffolds.45,46 AFC systemic transplantation has also been
shown to be cardioprotective and
proangiogenic in a rat model of induced
cardiac ischemia.47
AFC induced to neural lineage and
exposed to nerve growth factor have
been shown to facilitate peripheral nerve
regeneration after sciatic nerve crush
injury in rats.48 Ischemic stroke mouse
and rat models have also been used
to study the benet of AFC therapies.49
No deformation of natural anatomy or
malignant process developed in treated
rat brains, and there was improvement
in not only cognitive ability but also in

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rat behavior after treatment.50 In mice,
intracerebroventricular administration
of AFC also signicantly reduced
neurologic sequelae and behavior decits after cerebral ischemia was induced
via middle cerebral artery occlusion.51
Animal models with human AFC have
been used to study neurodegenerative
diseases of demyelination and neuronal
loss mimicking Parkinsons disease. The
cells wholly integrate into mice brains,
becoming indiscernible from surrounding tissue. The transplanted stem cells
migrated towards areas of damaged
neural tissue and resulted in symptomatic improvement.21
Endodermic lineages have also had
promising results through AFC therapy.
Injured liver in mice that were treated
with AFC have shown the production of
mature hepatocytes and progenitors that
appear therapeutic.52 AFC have been
transplanted to animal models with
benecial effects on necrotizing enterocolitis, which is a severe gastrointestinal
disease that is common in premature
neonates.53 Although AFC have not been
differentiated successfully into lung tissue to date, the cells engraft and express
alveolar and bronchial markers in the
background of lung injury.54
Human AFC have been induced
easily to pluripotency for possible
autologous or homologous matched
transplantation.55,56 Our laboratory has
cultured human AFC in a serum-free
medium, which is a prerequisite for
clinical use and transplantation. Comparison of serum-free cultured cells with
the same patients cells grown in standard cultures showed retention of
stem-cell markers, similar proliferation
potential, and no difference in the ability
to be differentiated after cryopreservation into neural, osteoid, and cartilage
cell lineages.9 It seems likely that, even
without a cell line induced to pluripotency, AFC could be used for
transplantation.
Although in just the beginning phases,
human transplantation with fetal stem
cells has been promising. Fetal neural
tissue from human fetal ventral mesencephalic tissue has been reported as a
restorative treatment for patients with
Parkinsons disease with evidence of

Obstetrics
prolonged symptomatic relief.57 Umbilical cord blood nucleated cells can
differentiate into cells of neural lineage.58,59 In another study, fetal autologous umbilical cord blood has been
transplanted into infants with neonatal
hypoxic ischemic encephalopathy via the
intravenous route. The study showed
that autologous transplantation of fetal
cells is feasible and that further clinical
trials should be pursued.5 Fetal allogeneic HLA-mismatched mesenchymal
stem cells have been transplanted to a
fetus with in utero diagnosis of severe
osteogenesis imperfecta. No immune
reaction was detected against transplanted cells at 9 months of life, and
the child has exhibited signicantly
decreased numbers of fractures from
the expected dismal prognosis of severe
osteogenesis imperfecta.60 A recent
study in mice has demonstrated immunoregulatory properties of human AFC
transplants that resulted in immunosuppression of T cells in regional lymph
nodes.61
There are limited clinical trials
currently ongoing that involve human
transplantation of human AFC for
therapeutic
interventions.
NuCell
(Nutech Medical Inc., Birmingham, AL)
is experimenting with an amniotic allograft, ReNu, that is composed of particularized amniotic membrane and cells
from amniotic uid and currently
is recruiting patients in a multicenter
study. The primary outcome involves
improvement of pain scores after direct
site injection for people with
osteoarthritis-causing knee pain (Identier NCT02318511).

Future developments
AFC collected at routine amniocentesis
could be banked and proliferated in
culture, as needed.62 AFC banking would
be a convenient source for autologous
therapies. Even if HLA matching should
be required, AFC could be used for
therapy in histocompatible HLA typematched recipients and possibly used
for fetal therapy. We are continuing to
investigate the HLA immunogenicity of
AFC to address this potentiality. In the
evolving eld of regenerative medicine
that aims to replace or regenerate cells,

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tissue, or organs to restore normal

function, AFC may be used to create
grafts for reconstructive therapy. There
are ongoing studies that are using
biocompatible lattices and other stemcell sources that would be readily
adaptable for AFC.63 They could be
transplanted in the prenatal or postnatal
period for diseases such as periventricular leukomalacia and neonatal
encephalopathy and traumatic brain
injury. Studies that are using cord blood
are already underway, and similar studies
with the use of AFC are feasible today.5,6
The establishment of a bank would be
ideal for possible therapy in such a wide
range of indications. The challenges will
be to obtain Food and Drug Administration approval to initiate clinical trials,
although basic preclinical work in
humans will continue. Among these, the
identication of a single pluripotent cell
and cloning of that population remains
to be accomplished.

Comment
There is enormous potential in the
growing eld of regenerative medicine,
clinical transplantation, and therapies
that target morbidities that are associated with dysgenetics and birth injury.56
AFC can be retrieved easily during
routine amniocentesis with minimal risk
to the mother and fetus. Advantages
include a high yield of cells per sample
retrieved, a high proliferation rate, a
differentiation potential into all embryonic germ cell layers, and the stability of
both genetics and phenotype. In fact,
this has been shown after 250 populations without change in karyotype.
AFC also have slow onset of senescence
and maintain normal telomere length.10
AFC show no evidence of tumor formation, including late passage cells.21,23
Low tumorigenic risk is essential for
therapeutic use. Disadvantages include
the small risk of the amniocentesis procedure including an estimated 0.1% risk
of pregnancy loss in mid trimester. In the
future, it may be ideal to collect amniotic
uid for AFC at term. Human AFC are
heterogeneous with diversity among
donors,29 but all amniotic uids have
large numbers of cells with multipotency. Further clinical trials must be

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explored, but clinical use remains a

promising possibility.
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