Mesenchymal Stem/Stromal Cells Derived from Human and Animal Perinatal Tissues—Origins, Characteristics, Signaling Pathways, and Clinical Trials

Abstract

Mesenchymal stem/stromal cells (MSCs) are currently one of the most extensively researched fields due to their promising opportunity for use in regenerative medicine. There are many sources of MSCs, of which cells of perinatal origin appear to be an invaluable pool. Compared to embryonic stem cells, they are devoid of ethical conflicts because they are derived from tissues surrounding the fetus and can be safely recovered from medical waste after delivery. Additionally, perinatal MSCs exhibit better self-renewal and differentiation properties than those derived from adult tissues. It is important to consider the anatomy of perinatal tissues and the general description of MSCs, including their isolation, differentiation, and characterization of different types of perinatal MSCs from both animals and humans (placenta, umbilical cord, amniotic fluid). Ultimately, signaling pathways are essential to consider regarding the clinical applications of MSCs. It is important to consider the origin of these cells, referring to the anatomical structure of the organs of origin, when describing the general and specific characteristics of the different types of MSCs as well as the pathways involved in differentiation.

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Review
Mesenchymal Stem/Stromal Cells Derived from Human and
Animal Perinatal Tissues—Origins, Characteristics, Signaling
Pathways, and Clinical Trials
Magdalena Kulus 1, Rafał Sibiak 2,3 , Katarzyna Stefa ´nska 2, Maciej Zdun 4, Maria Wieczorkiewicz 4,
Hanna Piotrowska-Kempisty 4,5, J˛edrzej M. Ja´skowski 6, Dorota Bukowska 6, Kornel Ratajczak 1, Maciej Zabel 7,
Paul Mozdziak 8and Bartosz Kempisty 1,2,8,9,*


Citation: Kulus, M.; Sibiak, R.;
Stefa´nska, K.; Zdun, M.;
Wieczorkiewicz, M.;
Piotrowska-Kempisty, H.; Ja´skowski,
J.M.; Bukowska, D.; Ratajczak, K.;
Zabel, M.; et al. Mesenchymal
Stem/Stromal Cells Derived from
Human and Animal Perinatal
Tissues—Origins, Characteristics,
Signaling Pathways, and Clinical
Trials. Cells 2021,10, 3278. https://
doi.org/10.3390/cells10123278
Academic Editor: Joni H. Ylostalo
Received: 5 October 2021
Accepted: 19 November 2021
Published: 23 November 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1
Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun,
87-100 Torun, Poland; magdalena.kulus@umk.pl (M.K.); kornel@umk.pl (K.R.)
2Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland;
75094@student.ump.edu.pl (R.S.); k.stefanska94@o2.pl (K.S.)
3Division of Reproduction, Department of Obstetrics, Gynecology, and Gynecologic Oncology,
Poznan University of Medical Sciences, 60-535 Poznan, Poland
4Department of Basic and Preclinical Sciences, Institute of Veterinary Medicine,
Nicolaus Copernicus University in Torun, 87-100 Torun, Poland; maciejzdun@umk.pl (M.Z.);
maria.wieczorkiewicz@umk.pl (M.W.); hpiotrow@ump.edu.pl (H.P.-K.)
5Department of Toxicology, Poznan University of Medical Sciences, 60-631 Poznan, Poland
6Department of Diagnostics and Clinical Sciences, Institute of Veterinary Medicine,
Nicolaus Copernicus University in Torun, 87-100 Torun, Poland; jmjaskowski@umk.pl (J.M.J.);
dbukowska@umk.pl (D.B.)
7Division of Anatomy and Histology, University of Zielona Gora, 65-046 Zielona Gora, Poland;
m.zabel@wlnz.uz.zgora.pl
8Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC 27695, USA;
pemozdzi@ncsu.edu
9Department of Anatomy, Poznan University of Medical Sciences, 60-781 Poznan, Poland
*Correspondence: bkempisty@ump.edu.pl
Abstract:
Mesenchymal stem/stromal cells (MSCs) are currently one of the most extensively re-
searched fields due to their promising opportunity for use in regenerative medicine. There are many
sources of MSCs, of which cells of perinatal origin appear to be an invaluable pool. Compared
to embryonic stem cells, they are devoid of ethical conflicts because they are derived from tissues
surrounding the fetus and can be safely recovered from medical waste after delivery. Additionally,
perinatal MSCs exhibit better self-renewal and differentiation properties than those derived from
adult tissues. It is important to consider the anatomy of perinatal tissues and the general description
of MSCs, including their isolation, differentiation, and characterization of different types of perinatal
MSCs from both animals and humans (placenta, umbilical cord, amniotic fluid). Ultimately, signaling
pathways are essential to consider regarding the clinical applications of MSCs. It is important to
consider the origin of these cells, referring to the anatomical structure of the organs of origin, when
describing the general and specific characteristics of the different types of MSCs as well as the
pathways involved in differentiation.
Keywords: perinatal mesenchymal stem/stromal cells; MSCs differentiation; signaling pathways
1. Human and Animal Perinatal Tissues—Anatomical, Histological, and
Cellular Characteristics
Perinatal tissues are an extremely rich source of various cell types. The placenta is an
organ that is irreplaceable for the development of the fetus, because it is a multicellular
barrier. The placenta is also unique in terms of the origin of the cells that form it, because it
is composed of cells of maternal and fetal origin, that are genetically distinct organisms [
1
].
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Cells 2021,10, 3278 2 of 39
The necessary exchange of nutrients, metabolites, and endocrine regulation must take
place simultaneously with the maintenance of immunological tolerance between the two
organisms [
2
]. It replaces some of the inactive organs in the fetus (lungs, liver, endocrine
glands). The placenta is formed after successful fertilization and implantation of the
embryo in the mammalian uterus. There is a distinction between the maternal-uterine part,
which is the endometrium, and the fetal part. The membrane of maternal origin is referred
to as decidua. Decidualization occurs at different rates depending on the type of placenta,
and is stimulated by factors secreted by the blastocyst (histamine, prostaglandins). The
decidua basalis is the part of the endometrium immediately adjacent to the fetal bladder
and is involved in the formation of the chorioallantoic placenta. The decidua capsularis
separates the fetal bladder from the uterine cavity, and the decidua parietalis connects the
decidua basalis and decidua capsularis.
Fetal membranes arise from the zygote, and function as accessory organs. The fetal
membranes consist of the yolk sac (saccus vitellinus), amnion, chorion, and allantois. As the
early stage embryo migrates through the fallopian tube where fertilization occurred, the
cells divide to form a morula (morus) and then a blastocyst. The embryonic node (massa em-
bryonica) is where the embryo proper forms, and the peripherally arranged flattened cells
make up the trophoblast [
3
]. The trophoblast cells have microvilli where the cells of the
tubular epithelium of the endometrium fuse. The trophoblast cells contain proteolytic
enzymes (zinc-containing metalloproteases) that degrade the endometrial epithelium, al-
lowing the blastocyst to penetrate deep into the endometrium [
4
]. Initially, the trophoblast
has two layers: an inner layer (cytotrophoblastus) and an outer layer (syncytiotrophoblastus).
The inner layer consists of highly proliferating mononuclear cells, and the outer layer
is formed by cell fusion and has invasive capacity and is responsible for anchoring the
blastocyst in the uterus [
5
]. It will then develop into the chorion and participate in the
construction of the placenta.
The yolk sac forms as the first fetal membrane. The wall of the yolk sac is trilaminar
(saccus vitellinus trilaminaris) due to the ingrowth of the extraembryonic mesoderm between
the trophoblast and the extraembryonic endoderm. The yolk sac has hematopoietic func-
tions because the first blood cells and blood vessels are formed in it [
6
]. Primary germ
cells (gonocytes) also appear in the wall of the yolk sac, which then migrate to the gonadal
primordia [
7
]. In the further development of the embryo, the yolk sac usually disappears
and the established pedicle, together with the yolk vessels and the surrounding mesoderm,
become part of the umbilical cord.
The membrane that directly covers the embryo is the amnion, which is formed around
day 7 of embryonic development. The space between the amnion and the embryo, the
amniotic cavity, is filled with amniotic fluid (liquor amnioticus) [
8
]. The amnion consists
of ectodermal epithelium and mesenchymatous tissue and is generally not vascularized.
The amniotic epithelium is composed of large, polygonal flat cells, the surfaces of which
are covered with microvilli [
9
]. These cells may exfoliate into the amniotic fluid. Prenatal
diagnosis utilizes exfoliated epithelium by collecting fluid through a puncture of the
amniotic cavity (amniocentesis) [
10
]. Between individual amniotic epithelial cells, on
their lateral surfaces, intercellular spaces are formed. They are filled with microvilli
and protuberances, named amniotic water vacuoles because of their appearance. These
cells contain numerous lipid droplets and glycogen grains. The basement membrane of
the amniotic epithelium contains numerous reticular fibers, passing into mesenchymatic
tissue, which is rich in fibroblastic cells and collagen fibers [
11
]. This tissue shows great
strength. Amniotic fluid is produced by epithelial cells, and in addition serous fluid is
infiltrated from the mesenchyme through the intercellular spaces. The amount of fluid
changes during pregnancy, and the main components are water (99%), saccharides, proteins,
urea, and also exfoliated cells or fetal downy hairs (lanugo). Amniotic fluid is constantly
and rapidly exchanged. Resorption occurs by the amniotic epithelial cells and by the
fetus [
12
]. Amniotic fluid has important functions in providing the embryo with a watery
environment, protection from injury, amortization, and metabolism.

Cells 2021,10, 3278 3 of 39
The chorion lies in direct contact between the amnion and the endometrium, forming
an integral part of the placenta. It arises from the trophoblast, which merges with the
extraembryonic mesoderm [
13
]. The surface of the chorion is covered by characteristic
villi, which come into close physical contact with the uterine endometrium [
14
]. The shape
of these villi varies depending on the type of implantation and placenta, and the animal
species. The chorion does not produce blood vessels and vascularization comes from
the allantois or yolk sac. The chorion over a large area fuses with the allantois to form
the chorioallantois [15].
The last fetal membrane to form is the allantois, which is of endo- and mesodermal
origin. It arises from the posterior part of the primary intestine. Already at an early stage,
hematopoietic islands and blood vessels form in the wall of the allantois, which make up
the initial formation of the umbilical artery and vein [
16
]. The intraembryonic part of the
allantois merges with the bladder valve and then disappears to form the urachus and then
the median umbilical ligament [
17
]. The main role of the allantois is to supply blood vessels
to the chorion, forming the placental circulation. In some animals, the allantois also has
a role related to excretion of metabolic products, in which case it is well developed and
large in size [
18
]. In humans and rodents, it has a residual form as the diverticulum and
caulis allantoicus.
The umbilical cord (funiculus umbilicalis) extends from the ventral wall of the embryo
and connects the embryo to the placenta. It includes the yolk and umbilical blood vessels,
the yolk duct, and the caulis allantoicus, surrounded by the dermal cord. The yolk duct
quickly overgrows and becomes a solid string of cells with yolk vessels. The allantois duct
is lined with a flat monolayer of epithelium [
19
]. Anastomoses may be formed between
the vessels and the course of the vessels forms a spiral, providing great flexibility [
20
]. The
mesoderm of all the ducts running in the umbilical cord fuses together and develops into a
mucous connective tissue (tela mucoidea connectens), otherwise known as Wharton’s jelly.
It contains abundant intercellular substance, rich in glycosaminoglycans, collagen, elastic
and reticular fibers, and fibroblasts. Externally, the umbilical cord is surrounded by a thin
monolayer of epithelium (periderm) of ectodermal origin [
21
]. The umbilical cord may
contain nerves that receive sensory stimuli related to tissue tension [
22
]. The cord varies
in strength depending on the species, but is easily broken during birth. Bleeding from
the cord vessels is not abundant due to contraction of the strong arterial muscle layer and
rupture of the venous connection to the placenta.
Among mammals, a wide range of placental strategies can be observed based upon
the different gestational and environmental needs of the fetus [
23
]. Mammalian placentas
are mostly classified into two types: yolk sac placenta and chorioallantoic placenta. The
yolk sac placenta is a trilaminar yolk sac attached to the uterine tissue, which usually plays
a role during the early post-implantation period. In most mammals, with the exception
of rodents and rabbits, the yolk sac placenta becomes reduced after the first trimester of
pregnancy [
24
]. Thus, impaired structural and functional development of the yolk sac
contributes to embryo/fetal toxicity and teratogenicity in rats [
25
]. The chorioallantoic
placenta is formed from the endometrium of the mother and the trophectoderm of the
embryo, and is the principal placenta in mammals during middle to late gestation [24].
There are two main classifications of chorioallantoic placentas. The first, based on the
distribution of villi over the surface of the chorion, divides placentas into diffuse, multi-
cotyledonary, zonary, and discoid. In a diffuse type, such as those of pigs [
26
], camel [
27
],
lemurs, and lorises [
28
], the surface is covered with villi that interdigitate with crypts in the
uterine epithelium. The villi may aggregate into bundles, forming microcotyledons, such
as those observed in horses. In turn, most ruminants produce cotyledonary placentas. Each
cotyledon is a small disk, with their numbers varying from a few in deer (oligocotyledonary)
to many in bovids (polycotyledonary) [
29
]. Furthermore, the zonary placenta is typical for
carnivores, forming a belt around the chorionic sac [
24
]. Finally, the discoid placenta is
characterized by a roughly circular area. This type of placenta is found in most primates,
including humans, as well as in rodents and rabbits [24].

Cells 2021,10, 3278 4 of 39
The second classification is based on the number of tissues separating maternal and
fetal blood. In the hemochorial type of placenta, the trophoblast invades the uterine
epithelium, stroma, and maternal arterial walls to come into direct contact with maternal
blood [
23
]. There are hemomonochorial (higher primates, e.g., human; hystricomorph
rodents, e.g., guinea pig), hemodichorial (rabbits), and hemotrichorial (most myomorph
rodents, such as rats and mice) placentas, with one, two, and three trophoblast layers
in the interhaemal barrier, respectively [
24
,
28
]. Furthermore, there is a clear difference
between higher primates and lemurs or lorises (lower primates), with the former (similarly
to pigs) producing the epitheliochorial type of placenta. This is the most superficial type of
placenta, lacking significant invasion of the uterine lining [
26
,
28
]. Apart from these two
types, i.e., hemochorial and epitheliochorial, there is also the endotheliochorial type. In this
case, the maternal uterine epithelium and connective tissue disappear after implantation,
and the trophoblasts come into direct contact with the maternal endometrium. This type
occurs in all four major clades of eutherian mammals (Euarchontoglires, Laurasiatheria,
Xenarthra, and Afrotheria), including carnivores [24].
2. Mesenchymal Stem/Stromal Cells—Origin, Cellular and Molecular Characteristics,
and Signaling Pathways Involved in Differentiation
Cells with stem-like potential have been of continued interest to researchers in various
fields for many years. Division and classification are still being attempted, and molecular
characterization appears to be the most appropriate. Enabling mesenchymal stem/stromal
cells (MSCs) cells to be used in regenerative medicine, especially for musculoskeletal
diseases, degenerative diseases, or incurable conditions, is very promising. MSCs also
promote immunomodulation as they can both inhibit and stimulate the immune system and
express many immunosuppressors [
30
,
31
] and influence autophagy processes [
32
]. They
also exhibit anti-apoptotic [
33
] and antioxidant [
34
] effects, which promote the treatment
of neuromuscular soreness [
35
]. In recent years, there has been an abundance of progress
in research related to the isolation and culture of multipotent stem cells derived from
various human and animal tissues. The most commonly used sources of MSCs are: bone
marrow, adipose tissue, cord blood, peripheral blood, muscle tissue, placenta, and amniotic
fluid. According to the most general definition of MSCs, they need to exhibit the ability to
adhere to plastic surfaces; express specific differentiation clusters, such as CD73, CD90, and
CD105; and have the ability to differentiate into osteogenic, chondrogenic, or adipogenic
lineage cells
in vitro
. Unlike cells of the hematopoietic lineage, they do not express CD14,
CD34, CD45, and HLA-DR [
36
]. Additionally, MSCs can express other markers such as
nestin (Tuj-1) for neural cells [
37
], smooth muscle
α
-actin, smooth muscle myosin heavy
chain for muscle cells [
38
], or transforming growth factor-
β
(TGF-beta) receptor [
39
] and
integrins [
40
]. It should be emphasized that cells expressing neural specific markers in
in vitro
cultures often present only the transient neuron-like morphology. Inducing full
neuronal functionality remains an elusive goal. Cultured cells fail to generate functional
polarity and form new signals passing neuronal synapses [
41
]. Undoubtedly, significant
work remains to understand the biology of MSCs.
Initially, the discovery and identification of pluripotent embryonic stem cells (ESCs) [
42
]
revealed the existence of cells that can self-renew indefinitely and differentiate into all three
embryonic germ layers [
43
,
44
], revealing a wide field of applications. Ethical issues have
limited the availability of new ES cells lines [
45
]. An alternative source of stem cells has
proven to be adult tissues, which contain a certain pool of multipotent cells. The use of
adult stem cells is devoid of ethical considerations, they are widely available, and there
is a lower risk of tumorigenesis [
46
,
47
]. Hematopoietic stem cells (HSCs), which have
the ability to differentiate into all lineages of the blood and immune system, have been
distinguished. HSCs have found applications in the treatment of blood disorders and
leukemia [48].
A promising source consists of MSCs isolated from adult tissues, which
in vitro
differentiated into adipogenic [
49
], chondrogenic [
50
], osteogenic [
51
], or even neurogenic
lineages [
52
]. The cells taken from the patient are multiplied, differentiated, and have

Cells 2021,10, 3278 5 of 39
therapeutic applications. This would involve no need for xenotransplantation, as the
cells would come from the same patient. However, tissue harvesting itself is associated
with rather painful and invasive procedures and the possibility of infection at the harvest
point [
53
]. In addition, the patients themselves who require cell therapy are not in well
enough health to perform the procedure. The clinical condition of donor patients is also
not without significance. It appears that the exhaustion of physiologically occurring MSCs
in patients with primary osteoarthritis occurs. In biomechanical joint damage, subchondral
bone populations of MSCs are not impaired [
54
–
56
]. It is also important to establish
the ability to proliferate and differentiate, which are key processes in the harvesting of
MSCs. It appears that cells obtained from adult tissues exhibit different potentials [
57
],
and the capacity for proliferation and differentiation decreases during
in vitro
culture
after successive passages [
58
]. Differentiation of cells toward the tissues from which
they originate is overall more successful, as evidenced by the superior mechanisms of
tissue-specific MSCs [
59
]. It was also found that the differentiation capacity, as well as the
self-renewal potential, is dependent on the physiological state of the donor (age, health
status), its genetics, and the influence of environmental conditions [
60
–
64
]. For example,
the MSCs obtained from young rats presented faster growth correlating with the levels of
proliferating cell nuclear antigen and higher glucose utilization compared to older ones [
65
].
The impact of telomere erosion in MSCs was not insignificant here either [
66
]. MSCs can
be derived from perinatal tissues, which include the placenta [
67
], umbilical cord [
68
–
70
],
or the cord blood itself [
71
]. In addition, fetal tissue [
72
] and the surrounding amniotic
fluid [
73
] are promising sources for stem cell derivation. In principle, stem cells derived
from perinatal and fetal tissues should have a greater potential for self-renewal and the
ability to proliferate and differentiate; however, many sources have reported that they
show considerable diversity [
53
,
74
], the details of which are given in the following sections
describing the characteristics of individual perinatal MSCs.
Many authors have suggested that an attempt should be made to rename the MSC
because the term “mesenchymal stem cells” has been overused by groups commercializing
administration of “MSCs” for therapeutic purposes. However, the commercial entities do
not perform differentiation of the “MSCs”, and the therapeutic effect of the administered
compound is based mainly on local action and secretion of active factors [
75
]. Indeed,
traumatically altered tissues call induce repair mechanisms. MSCs respond to these signals,
migrate [
76
], and act by secreting active factors (immunomodulatory, trophic, regener-
ative) [
77
,
78
]. This translates into a local therapeutic effect that is, in fact, based on the
specific stem cells of a given patient that have been activated by the exogenous MSCs
administered [
79
,
80
]. Therefore, the first proposal for a new name for these cells in general
is “medicinal signaling cells”, which reduces the misleading of patients that they are re-
ceiving typical stem cells that will produce new tissue [
81
]. There has also been a proposal
(from the International Society for Cellular Therapy (ISCT)) that fibroblast-like cells, which
are plastic-adherent, should be called multipotent mesenchymal stromal cells regardless
of origin [
82
]. Attempts to undertake a uniform classification and nomenclature change
related to MSCs have been ongoing for several years. The acronym “MSCs” is proposed to
remain in the nomenclature, but the authors of individual studies should carefully specify
the origin and demonstrate the functional properties of MSCs [
83
]. However, it is important
to consider that stem/stromal cells remain the most widely used terms to describe MSCs.
Nevertheless, the wide variation in differentiation potential, the lack of standardized acqui-
sition procedures, and the absence of a single universal marker significantly limit standard
clinical application. A major step is the collection itself, the isolation of MSCs from a donor,
which influences the final cell population [
84
]. Most often, these are simple procedures in-
volving mechanical fragmentation of the harvested tissue or enzymatic digestion, followed
by seeding and attachment of the cell suspension into culture dishes. The explant-derived
method involves breaking the tissue fragments into small pieces to facilitate diffusion of
nutrients and gases, and placing these fragments in the culture medium. MSCs proliferate
and colonize on the surface of the bottom of the dish. Enzyme-mediated isolation involves

Cells 2021,10, 3278 6 of 39
incubation and enzymatic digestion of the harvested tissue to release individual cells from
the extracellular matrix (ECM), followed by centrifugation and placement of the resulting
cell pellet in the culture medium. Enzymatic isolations are considered to be more efficient,
but the explant method provides higher homology with better proliferation and viability
rates [
85
,
86
]. It is suggested that is the superior properties of the explant-derived cells are
associated with less stress on the cells [87].
The therapeutic effects of MSCs are related to the secretion of paracrine factors, in-
cluding cytokines, growth factors, mRNAs, miRNAs, and signaling lipids. Much recent
research has focused on nanoparticles derived from the cell membrane of MSCs, called
exosomes [
88
,
89
]. These vesicles are secreted outside the cell, mediating cell-to-cell com-
munication. MSC-derived exosomes are a tantalizing possibility for innovative cell-free
therapies, which could have advantages over whole-cell therapies. Extracellular vesicles
(EVs) can occur as exosomes released extracellularly by fusing with the cell membrane. EVs
are also microvesicles that bud from the plasma membrane, and sometimes occur as apop-
totic bodies of varying sizes [
90
]. In relation to secretory capacity, therapeutic properties are
associated with immunomodulatory and trophic effects at the site of injury/disease [
91
],
and EVs are able to penetrate the blood–brain barrier [92].
With recent advances in the study of exosome-based therapies with MSCs, it is impor-
tant to assess their localization, tracking, and monitoring after transplantation
in vivo
[
93
].
One method is magnetic resonance imaging (MRI), which allows the localization of ex-
osomes labeled with contrast agents (such as ultra-small superparamagnetic iron oxide
nanoparticles) [94].
2.1. Different Origins of MSCs—Sources, Cell Characteristics, Possible Applications
MSCs can be isolated from any vascularized tissue, and studies have indicated that
pericytes (perivascular cells) may be the source, as the gene expression profile of adipose
tissue pericytes is remarkably similar to that of adipose tissue stem cells [
95
,
96
]. Bone
marrow is rich in stem cells that show potential to differentiate into osteoblasts. Initiating
the differentiation process through the release of transforming growth factor β1 (TGF-β1)
leads to the development of osteocytes [
97
]. Research on the clinical application of various
procedures using MSCs in bone regeneration has been ongoing and advanced for several
years [
98
–
100
]. Supplementation of growth factors to promote bone repair by MSCs is also
currently being investigated, particularly in relation to tissue-engineering scaffolds [
101
].
MSCs also provide opportunities for the treatment of intervertebral disc degeneration.
Although they exhibit high regenerative potential, chondrogenic differentiation, and anti-
inflammatory effects, the specific nature of the intervertebral disc structure (including lack
of blood vessels, low pH and glucose levels, and hypoxia) largely limits the application of
this therapy [102].
Stem cells derived from adipose tissue have great potential for treating orthopedic
conditions [
103
,
104
]. Adipose-derived stem cells (ASCs) have been shown to have multidi-
rectional differentiation properties, including into adipocytes, osteocytes, and chondrocytes,
and they have been shown to express MSCs markers (CD 29, CD44, CD90) with a neg-
ative expression of hematopoietic markers (CD31, CD34, CD45). The demonstration of
cartilage repair in an animal model was also significant [
105
]. MSCs can also be isolated
from oral tissues such as dental pulp, gingiva, dental follicles, alveolar ligaments, and
others. These cells are used in tissue engineering as they show potential for multidirec-
tional differentiation and are easy to obtain and show regenerative potential [
106
]. MSCs
derived from dental tissues, in addition to their ability to differentiate into the mesodermal
lineage, have been shown to undergo ectodermal-neurocyte and endodermal-hepatocyte
differentiation [
107
]. MSCs also show remarkable potential in regenerative processes and
wound healing. MSCs extracted from the basal layer of the epidermis and hair follicles
have been shown to promote skin healing, new blood vessel formation, and endothelial
transformation. The mechanisms of these processes are not entirely clear [
108
]. MSCs
in the treatment of cutaneous wounds inhibit inflammation, promote angiogenesis, and

Cells 2021,10, 3278 7 of 39
accelerate wound closure, and their action is based on paracrine mechanisms [
109
]. MSCs
have also been shown to affect extracellular matrix remodeling [
110
,
111
]. Standard sources
of MSCs (adipose tissue or bone marrow), as well as perinatal tissues, are effective sources
to treat wounds [112,113].
Recently, there has also been research into the possibility of using MSCs from different
sources to treat female infertility of various backgrounds [
114
]. Additionally, numerous
literature data have indicated a high stemness potential of human [
115
], as well as ani-
mal [
116
], ovarian granulosa cells (GCs). GCs, co-forming the ovarian follicle, are classified
as mulipotent cells [
117
,
118
] and play a key role in oocyte maturation through their regular
contact. Their ability to differentiate into osteoblasts, chondroblasts [
119
], mioblasts [
120
],
or even cells from the neurogenic lineage [
121
] has been experimentally demonstrated.
However, a recent study showed that although there was a comparatively high expression
of stemness markers of cells isolated from ovarian follicles, the osteogenic and adipogenic
differentiation capacity in elderly patients was inferior to young ones [122].
Due to the many limitations in the use of adult MSCs, perinatal MSCs could be appli-
cable. They show higher plasticity and proliferation capacity than adult MSCs [
123
]. They
also differ from embryonic stem cells (ESCs) in that they express pluripotent markers and
have active telomerase, but at a much lower level than ESCs [
123
]. As a result, their use is
not associated with the risk of tumorigenesis, as in the case of ESCs [
124
,
125
]. Immunolog-
ical aspects of the transplanted cells are also important [
126
]. Perinatal MSCs should be
immunologically neutral due to the absence of intracellular HLA class II and poor expres-
sion of HLA class I compared to adult MSCs [
127
]. Perinatal cells are also distinguished
from adult MSCs by their greater capacity for osteogenesis
in vitro
, as well as
in vivo
[
128
],
exhibiting greater osteogenic potency [
129
]. Once perinatal MSCs are obtained, they are
centrifuged and cultured in a medium containing serum. Upon adhesion to the plastic
substrate, they form colonies and the cells assume a spindle shape, resembling fibroblasts.
Perinatal MSCs should not express hematopoietic and endothelial markers [
130
]. How-
ever, these are only general definitions of MSCs, which have been verified by subsequent
findings in the stemness field. Simply showing expression of molecular markers should
not determine that the cells in concern are pluripotent. Proteins, meaning products rather
than the genes themselves, are actually responsible for the mechanisms and abilities of
pluripotency [
131
]. For example, it is the amount of Oct-4 protein that determines the main-
tenance of pluripotency and also the direction of cell differentiation [
132
,
133
]. Therefore,
phenotypic characteristics, as well as the degree and efficiency of differentiation of the cells
studied, should be used to demonstrate their pluripotent abilities.
2.2. Differentiation of MSCs—Regulatory Factors and Signaling Pathways
Genetic regulation and transcription factors are involved in the differentiation of MSCs.
In addition, the microenvironment itself can promote proliferation and differentiation, in
addition to providing conditions for growth [
134
–
136
]. Differentiation towards osteogen-
esis, adipogenesis, or chondrogenesis is, in theory, straightforward for MSCs as they are
derived from the same embryonic lineage. However, theory alone does not correlate to the
actual laboratory results. The differentiation abilities are highly dependent on the source of
the starting cells, the primary cell population, and also the direction of differentiation [
137
].
A mixture of dexa-methasone (Dex) isobutyl-methylxanthine (IBMX) and indomethacin
(IM) is used to induce adipogenesis (Figure 1). Confirmation of a properly occurring differ-
entiation process has been provided by Oil Red O staining of neutral triglycerides and lipids
in cells and visualization of lipid droplets [
138
]. Osteogenic differentiation is performed
by stimulation with dexa-methasone (Dex),
β
-glycerophosphate (
β
-GP), and ascorbic acid
phosphate (aP) [
139
,
140
]. Alkaline activity and calcium accumulation were analyzed to
confirm the process. TGF-
β
2 and TGF-
β
1 are used for chondrogenesis [
141
]. Pathways
involving TGF-
β
, PPAR-gamma, Smad3, and SOX9 are involved in differentiation into
the mesodermal lineage [
142
–
144
], whereas ectodermal differentiation into the neurogenic
lineage involves the Notch-1 pathway and the protein kinase A (PKA) pathway [
145
–
147
].

Cells 2021,10, 3278 8 of 39
Hydrocortisone, DMSO, BHA, and KCL, as well as bFGF, NT-3,
β
-mercaptoethanol (
β
-ME),
BDNF, and NGF are used to induce differentiation [
148
,
149
]. Evaluation of differentiation
was based on Tuj-1,
γ
-aminobutyric acid (GABA), MAP-2, neurofilament 200, among oth-
ers [
150
]. Signaling pathways involving TGF-
β
, fibroblast growth factor (FGF), and bone
morphogenetic protein (BMP) are involved in endodermal differentiation of MSCs [151].
Figure 1.
Human mesenchymal stem/stromal cells (MSCs) differentiation and signaling pathways. Abbreviations:
aP—ascorbic
acid phosphate;
β
-GP—
β
-glycerophosphate;
β
-ME—
β
-mercaptoethanol; BDNF—brain-derived neurotrophic
factor; bFGF—basic fibroblast growth factor; BHA—butylated hydroxyanisole; BMP—bone morphogenetic protein;
Dex—dexa
-methasone; DMSO—dimethyl sulfoxide; FGF—fibroblast growth factor; IBMX—isobutyl-methylxanthine; IM—
indomethacin; KCL—potassium chloride; NGF—nerve growth factor; Notch1—notch homolog 1;
NT-3—neurotrophin-3
;
PPAR—gamma-peroxisome proliferator-activated receptor gamma; Smad3—mothers against decapentaplegic homolog 3;
SOX9—transcription factor SOX9; TGF-
β
—transforming growth factor-
β
. Created with BioRender.com (accessed on
5 October 2021).
Research continues to refine the procedures involved in the processes of stem cell
differentiation. Transcription factors for differentiation, such as osterix/Sp7 (Osx), runt-
related transcription factor 2 (RUNX2), and Dlx5 are known to play an important role
in osteogenic differentiation [
152
,
153
]. They influence the course of various signaling
pathways that play a role in osteogenic differentiation processes, including WNT [
154
],

Cells 2021,10, 3278 9 of 39
BMP [
155
], and Akt [
156
]. They may act as regulators as they both activate and inhibit
the differentiation of MSCs. Additionally, a recently published study demonstrated the
accelerating effect of using nanocomposites that activate the WNT/
β
-catenin pathway in
osteogenic differentiation of MSCs [157].
It is noteworthy that the signaling pathways and factors involved in cell differentiation
in a specific cell line described above are not obligatory and may proceed in different ways.
For example, the study by Brady et al. [
158
] compared factors that promote chondrogenesis
using human MSCs derived from perinatal and adult bone marrow. TGF
β
3 was shown
to induce SMAD3 phosphorylation in adult BM-MSCs but not in fetal BM-MSCs. It was
also observed that the induction of chondrogenesis in adult BM-MSCs occurred under
the influence of TGF
β
3 but not by BMP2. Conversely, differentiation of fetal BM-MSCs
was induced by BMP2 but not TGF
β
3. Further, fetal BM-MSCs stimulated chondrogenesis
simultaneously when TGF
β
3 and BMP2 were used. It was shown that they produced
tissue with proteoglycan and type II collagen content similar to that produced by adult
BM-MSCs treated with TGFβ3 alone [158].
3. Mesenchymal Stem/Stromal Cells Derived from Human and Animal Perinatal Tissues
MSCs are present in tissues of fetal origin in both animals and humans. Over time, their
isolation from tissues of fetal origin has become feasible using well-described laboratory
protocols. Isolation of stem cells from fetal material does not raise ethical dilemmas due
to the fact that these tissues are treated as medical waste immediately after delivery. As
a result, the material for laboratory analyses is readily available [
159
]. Research material
can be taken from tissues obtained from invasive diagnostic and treatment procedures
throughout the pregnancy, planned terminations, and after the full-term vaginal delivery
or cesarean section. The studies focused on perinatal stem cells are conducted worldwide.
Several countries, such as the U.S. or China, released their specific cellular and gene
therapy guidance and established regenerative medicine products’ regulations [
160
,
161
].
Each European Union member state currently has its own specific regulations targeted
on embryonic stem cells and perinatal tissues research. Perinatal stem cell clinical trials
must be approved by the local bioethical commissions and be conducted in line with the
EU clinical trials registration law [
162
,
163
]. Cells of fetal origin, also known as perinatal
stem cells, are derived from extraembryonic structures, such as the placenta, umbilical
cord, and amniotic fluid [
164
,
165
]. Perinatal tissues are also widely used as a reservoir of
hematopoietic progenitor cells obtained from the umbilical cord blood [
166
]. Furthermore,
it is believed that the perinatal stem cells obtained at the very early stages of pregnancy
(first trimester) possess a higher regenerative potency compared to cells isolated from
full-term or near-term feto-maternal tissues. Several authors reported that they detected
the higher expression of pluripotency genes in the samples collected at the earlier stages
of pregnancy [
167
–
169
]. However, the others found no differences in their expression
throughout the gestation [170].
Over the recent years, numerous studies have focused on perinatal stem cells appli-
cations in clinical practice and analyzed their characteristics. In most cases, to reach the
appropriate number of cells before the MSCs transplantation, they should be cultured
in vitro
for several passages. It raises the concern about the occurrence of replicative senes-
cence, which could affect the clinical effectiveness of cellular therapies. Importantly, it
has been noted that the perinatal MSCs seem to be less susceptible to senescence, along
with the passaging, in comparison to MSCs derived from other adult tissues—i.e., bone
marrow-derived MSCs. That feature is in favor of their application in more extensive
clinical trials [171–173].
The terminology used to describe particular cellular populations is unclear and needs
to be standardized. Most authors have proposed their own nomenclature based on the
origin of the cells. Silini et al. introduced a novel broad definition of perinatal derivatives
(PnD), which includes all birth-associated tissues, the cells they are composed of, and all
the factors secreted by the mentioned cells along with the conditioned media [174].

Cells 2021,10, 3278 10 of 39
3.1. Animal Perinatal Mesenchymal Stem/Stromal Cells
The studies conducted on animals paved the way for a better understanding of
perinatal MSCs, enabling the first clinical trials in humans. Bailo et al. began the era of
perinatal MSCs in modern medicine by isolating and transplanting the human placenta-
derived MSCs to swine and rats [175].
Subsequently, many further studies were undertaken to determine the conditions for
isolation and to characterize the morphology, immunophenotypes, and other properties of
animal perinatal MSCs. Bartholomew et al. confirmed that the collection of umbilical cord
tissue and umbilical cord blood for stem cell isolation is a safe procedure for mare and foal
pairs in the equine model. They did not observe any changes in time to stand and nurse,
nor in hematological parameters in foals or time to pass the placenta for mares [
176
]. It
was found that the equine umbilical cord-derived MSCs are highly proliferative, spindle-
shaped cells with the potential to differentiate to osteogenic, chondrogenic, and adipogenic
derivatives [
177
–
179
]. Furthermore, their immunophenotype analyses revealed that they
are positive for vimentin, osteonectin, smooth muscle actin, and MHC I and do not express
CD31, CD18, MHC II, and the T-cell co-stimulatory molecule CD86 [177].
Shaw et al. examined the amniotic fluid obtained from pregnant ewes in early ges-
tation. They detected the presence of AFMSCs in all of the nine collected specimens (less
than 20,000 cells in 10 mL of amniotic fluid). Collected cells were cultured and harvested
for up to 20 passages. Cells doubled their count every 36 to 48 h. Analyzed ovine AFMSCs
presented the expression of CD44, CD58, and CD166 and were negative for the hematopoi-
etic markers CD14, CD31, and CD45 [
180
]. Colosimo et al. examined the properties of
in vitro
cultured ovine AFMSCs. The cells were cultured for 12 passages. They reported
that the ovine AFMSCs retained a high proliferation rate up to six passages. However, cells
maintained the prolonged expression of CD29, CD58, and CD166 surface molecules, as
well as Oct-4, TERT, NANOG, and Sox-2 pluripotency markers (up to 12 passages) [
181
]. It
was discovered that the ovine placental cotyledons are the next readily available source of
spindle-shaped, colony-forming, and plastic-adherent ovine perinatal MSCs. Cultured cells
showed the features of chondrogenic and osteogenic differentiation. They expressed the
CD29, CD44, and CD166 surface markers and were negative for hematopoietic progenitor
cell markers [182].
The studies conducted on dogs confirmed that the plastic-adherent perinatal MSCs
could be successfully isolated from the canine placental tissue, umbilical cord, and amniotic
membrane [
183
–
186
]. All isolated MSCs displayed a fibroblast-like shape in
in vitro
culture
conditions. Saulnier et al. reported that the placenta-derived MSCs exhibited the highest
proliferation rate in comparison to other types of perinatal MSCs. They discovered that all
cellular populations presented the expression of CD29, CD44, CD73, CD90, CD105, and
Sox-2, but did not show the expression of CD34, CD45, MHC II, NANOG, and Oct-4 [
185
].
The average
in vitro
canine perinatal MSC population doubling time ranged from 21–42 h
and depended on the tissue of their origin [
183
]. It is postulated that canine MSCs are
non-tumorigenic, as Borghesi et al. found no tumor formation features in nude mice after
the transplantation of MSCs derived from canine amniotic membranes [
184
]. Finally, in
strictly controlled conditions, all cell populations have the potential to differentiate into
adipocytes, osteo-, and chondroblasts [183–185].
3.2. Human Perinatal Mesenchymal Stem/Stromal Cells
Silini et al. have also proposed the systematized nomenclature and classification of
human perinatal tissues and cells. According to their definition, based on their location,
the following human perinatal MSCs could be distinguished: human amniotic membrane
mesenchymal stromal cells (hAMSC), human placental amniotic membrane mesenchymal
stromal cells (hPAMSC), human reflected amniotic membrane mesenchymal stromal cells
(hRAMSC), human chorionic mesenchymal stromal cells (hCMSC), human chorionic plate
mesenchymal stromal cells (hCP-MSC), human chorionic plate mesenchymal stromal
cells derived from blood vessels (hCP-MSC-bv), human chorionic villi mesenchymal

Cells 2021,10, 3278 11 of 39
stromal cells (hCV-MSC), human chorion leave mesenchymal stromal cells (hCL-MSC). The
umbilical cord-derived MSCs: human umbilical cord amniotic mesenchymal stromal cells
(hUC-AMSC), human umbilical cord Wharton’s jelly mesenchymal stromal cells (hUC-
WJ-MSC), human umbilical cord sub-amnion Wharton’s jelly mesenchymal stromal cells
(hUC-saWJ-MSC), and a lineage of human umbilical cord intermediate Wharton’s jelly
mesenchymal stromal cells (hUC-iWJ-MSC). Finally, the population of human amniotic
fluid mesenchymal stromal cells (hAF-MSC), human basal decidua mesenchymal stromal
cells (hBD-MSC), and human parietal decidua mesenchymal stromal cells (hPD-MSC) [
174
]
were also distinguished.
3.2.1. Placenta-Derived Mesenchymal Stem/Stromal Cells
Historically, participants of the First International Workshop on Stem Cells from
Placenta (2007) proposed the first systematic classification of human placental stem cells
based on their origin and characteristics. They distinguished the following placenta-
derived stem cell subpopulations: (1) human amniotic epithelial cells (hAEC), (2) human
amniotic mesenchymal stromal cells (hAMSC), (3) human chorionic mesenchymal stromal
cells (hCMSC), and (4) human chorionic trophoblastic cells (hCTC) [
187
]. Those cellular
subpopulations emerged from the amniotic and chorionic membrane tissue. The same
experts established the minimal criteria for the definitions of hAMSC and hCMCS. Both cell
lineages: (1) have a capacity to adhere to plastic in
in vitro
conditions; (2) form fibroblast
colony-forming units; (3) from
in vitro
passages 2 to 4; are positive for CD90, CD73, and
CD105 antigens and do not express the CD45, CD34, CD14, and HLA-DR surface antigens;
(4) have the potential to differentiate into one or more lineages, including osteogenic,
adipogenic, chondrogenic, and endothelial; and (5) have a fetal origin [
187
]. Subsequent
studies revealed that placenta-derived MSCs could also be isolated from chorionic villi
samples [188–191] and maternal decidua basalis [192,193].
In standard collection protocols, the samples of placental tissue are surgically dis-
sected. The chorionic and amniotic membranes are manually separated and minced into
small pieces. Subsequently, cells are isolated from the tissue by the enzymatic digestion
(dispase II and collagenase 2). In the next step, the solution is filtered and transferred
into culture dishes. Finally, the authors treat isolated cells with various basal culture
media supplemented with different fetal bovine serum concentrations (10–20%) and other
supplements, such as epidermal growth factor and antibiotics (Figure 2) [194–198].
Figure 2.
Perinatal mesenchymal stem/stromal cells: isolation and preparation techniques. Created
with BioRender.com (accessed on 5 October 2021).

Cells 2021,10, 3278 12 of 39
It has been reported that the hAMSCs have a fibroblast-like cell shape and do not
change their morphology in
in vitro
culture conditions up to five passages. Moreover,
they can be easily distinguished from the hAEC because they are three times larger than
hAEC [
199
]. Tested hAMSC cultures reached the senescence after 5–15 passages
[199–201]
.
Their immunophenotype analyses revealed the presence of the following antigens: i.a.
CD29, CD44, CD73, CD90, CD105, CD166, SSEA-3/4, CK18, HCAM-1, and HLA ABC,
and were negative for the CD14, CD34, CD45, TRA-1-60, VCAM-1, PECAM-1, and HLA-
DR—shown in Table 1[
199
–
201
]. Moreover, they exhibited the expression of the Oct-3/4,
GATA-4
, Rex-1, BMP-4, SCF, NCAM, nestin, HFN-4alpha, CK18, and vimentin genes,
but did not show the expression of BMP2, FGF-5, Pax-6, and telomerase reverse tran-
scriptase [
199
,
200
]. To date, no significant differences between the hAMSC obtained from
different regions of amniotic membranes have been found [174].
Table 1. Expression of cellular markers in various populations of perinatal mesenchymal stem/stromal cells (MSCs).
Cells Positive Expression (+) Negative Expression (−) References
Amniotic MSCs
CD29, CD44, CD73, CD90, CD105, CD166,
SSEA-3/4, CK18, HCAM-1, HLA ABC,
Oct-3/4, GATA-4, Rex-1, BMP-4, SCF,
NCAM, nestin, HFN-4alpha,
CK18, vimentin
CD14, CD34, CD45, TRA-1-60,
VCAM-1, PECAM-1,
HLA-DR, BMP2, FGF-5, Pax-6,
TERT
[199–201]
Chorionic membrane MSCs CD13, CD29, CD44, CD54, CD73,
CD105, CD166
CD3, CD14, CD34,
CD45, CD31 [202]
Chorionic villi MSCs CD44, CD73, CD90, CD105,
HLA-ABC, Sox-2
CD45, CD34, CD19, HLA-DR,
CD14, CD40, CD56, CD80,
CD83, CD86, CD275
[36,202]
Chorionic plate MSCs CD44, CD73, CD90, CD105, CD166, Oct-4,
NANOG, Sox-2
CD14, CD19, CD34, CD45,
HLA-DR [203–205]
Decidua MSCs CD44, CD90, CD105, CD146, CD166,
HLA-ABC
CD40, CD80, CD83, CD86,
HLA-DR [193]
Umbilical cord MSCs
CD13, CD29, CD44, CD73, CD90, CD105,
C10, CD49b-e, CD146, CD166, HLA-ABC,
NANOG, Rex-1, Sox-2
Uncertain expression:
Oct-3/4, SSEA-3, SSEA-4,
STRO-1, TRA-1-60, TRA-1-81
CD14, CD31, CD34, CD45,
CD51/61, CD64, CD106,
HLA-DR
[206–211]
Amniotic fluid MSCs CD73, CD90, CD105, Oct-4 CD45, CD34, CD31 [212]
Amniotic fluid stromal cells
CD29, CD44, CD73, CD90, CD105, SSEA-4,
Oct-4, MHC-I, NANOG, SSEA-3,
TRA-1-60, TRA-1-81
MHC-II, CD80, CD86 [167,213]
Human chorionic membrane, chorionic plate, chorionic villi, and chorionic leave
MSCs have been distinguished due to the place of their origin. However, these cellular
populations have similar cellular characteristics and immunophenotypes consistent with
classification criteria established by Parolini et al. [
187
]. Chorionic membrane MSCs present
a fibroblast-like morphology and plastic adherence capacity. The comparative analysis
revealed some morphological differences between the various types of perinatal MSC.
Araujo et al. reported that the chorionic MSCs are smaller than other perinatal MSCs
isolated from amniotic membranes, umbilical cord, or the decidua; however, they exhibited
similar proliferation capacity until passage 8 [
214
]. Chorionic membrane MSCs show the
expression of CD13, CD29, CD44, CD54, CD73, CD105, CD166 surface markers, and the
absence of CD3, CD14, CD34, CD45, and CD31, and are characterized by high cellular
plasticity [
202
]. Chorionic villi MSCs meet the minimal MSC criteria proposed by the
International Society for Cellular Therapy—specifically, they show an expression of CD44,
CD73, CD90, CD105, and HLA-ABC and lack expression of CD45, CD34, CD19, and HLA-

Cells 2021,10, 3278 13 of 39
DR surface molecules [
36
]. Moreover, they are non-immunogenic, meaning they do not
express CD14, CD40, CD56, CD80, CD83, CD86, CD275 immune markers. The expression of
Sox-2 is the only detected pluripotency feature of CV-MSCs [
215
]. Perinatal MSCs isolated
from the chorionic plate are positive for CD44, CD73, CD90, CD105, and CD166; do not
exhibit the expression of CD14, CD19, CD34, CD45, and HLA-DR surface antigens [
203
,
204
];
and express the Oct-4, NANOG, and Sox-2 pluripotent stem cell markers [
205
]. It has been
reported that chorionic plate-derived stem cells possess significantly higher migration and
proliferation properties compared with other perinatal stem cells [203,205].
It is also postulated that tissue samples obtained from different individuals have
specific cellular characteristics. Tai et al. analyzed the differences in properties of placenta-
derived MSCs collected from various areas of the placenta—chorionic plate, amniotic
membrane, and decidual plate—in five patients. They discovered only a moderate hetero-
geneity in osteogenic and adipogenic differentiation potentials in samples obtained from
different placental regions in enrolled individuals. Similar heterogeneity was observed in
the tubulin acetylation measured in different samples [
216
]. Moreover, they discovered that
only the chorionic plate MSCs could decrease the proliferation of peripheral blood mononu-
clear cells (PBMCs) triggered by the phytohemagglutinin. All cell lineages decreased the
proportions of CD3+/CD8-/IFN-
γ
+ Th1 and CD3+/CD8-/IL17+ Th17 cells and elevated
the proportion of Treg in PBMCs [
216
]. Placenta-derived MSCs regulate trophoblast func-
tioning by promoting increased cell survival and protecting mitochondria from the effects
of oxidative stress and, as a result, facilitate trophoblast invasion in humans [217,218].
Decidua MSCs originate from the maternal part of the placenta. They could be
isolated from both decidua basalis and decidua parietalis tissue. It was found that MSCs
isolated from term decidua basalis are capable of differentiating into three mesenchymal cell
lineages [
193
]. Furthermore, Macias et al. reported that decidua-derived MSCs differentiate
into derivatives of all germ layers [
192
]. Decidua MSCs form a monolayer of plastic
adherent, fibroblast-like cells positive for MSC surface markers (CD44, CD90, CD105,
CD146, CD166) and HLA-ABC, and negative for hematopoietic and endothelial markers,
as well as the following molecules—CD40, CD80, CD83, CD86, and HLA-DR [193].
3.2.2. Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells
MSCs could be isolated from both the umbilical cord blood and umbilical cord tis-
sue [
174
]. Yang et al. identified the population of plastic-adherent, fibroblast-like, umbilical
cord blood-derived MSCs in ~25% of primary cultures. The detected cells were positive
for CD13, CD29, CD44, CD73 (SH3, SH4), CD90, and CD105 (SH2) and negative for CD14,
CD31, CD34, CD45, CD51/61, CD64, CD106, and HLA-DR. The isolated cells presented
features of adipogenic, osteogenic, and chondrogenic differentiation [
219
]. Nonetheless,
the low presence of MSCs in the umbilical cord blood makes their isolation and cultures
non-effective for clinical use [219–222].
Isolation protocols for MSCs derived from umbilical cord tissue are similar to those
used to isolate chorionic and amniotic membrane MSCs. At first, the umbilical cord tissue—
Wharton’s Jelly—is manually separated from the cord blood vessels and cut into smaller
pieces. Then, the collected samples are directly placed in culture flasks (explant cultures)
or enzymatically digested (collagenase I/II, hyaluronidase, trypsin). In the next step,
the processed solution is filtered or centrifuged and transferred into culture dishes and
cultured in various media at 37
◦
C in a 5% CO
2
atmosphere [
197
,
223
–
225
]. Fortunately,
the isolation of MSCs from the umbilical cord tissue is much more efficient. Some authors
postulated that the two embryologically different MSCs populations (hUC-AMSC and
hUC-WJ-MSC) are present in the umbilical cord tissue. However, the direct connection
between the amniotic membrane and the inner connective tissue makes it difficult to isolate
the individual cellular subpopulations [
174
,
226
]. Cells isolated from Wharton’s Jelly share
the features of other perinatal MSCs. Interestingly, two morphologically distinct types of
hUC-WJ-MSC (small-sized subpopulation with a flat cell body, large-sized subpopulation)
have been visualized in primary cultures. It was found that the subpopulation of small-

Cells 2021,10, 3278 14 of 39
sized cells displayed higher expression of several surface antigens (CD44, CD73, CD90,
and CD105) [
206
]. Furthermore, those two cellular subpopulations exhibited different
cytoplasmic filament profiles (vimentin and cytokeratin filaments) [
207
]. Other reports
indicate that the umbilical cord-derived MSCs show a mostly fibroblastic morphology
and are positive for other cellular markers, i.e., C10, CD13, CD29, CD49b-e, CD51, CD146,
CD166, and HLA-ABC. In addition, umbilical cord MSCs express the NANOG, Rex-1, and
Sox-2 pluripotency gens. However, the expression of Oct-3/4, SSEA-3, SSEA-4, STRO-1,
Tra-1-60, and Tra-1-81 is uncertain due to the contrary results of previous studies [
208
–
211
].
Similar to other subpopulations of perinatal MSCs, umbilical cord-derived MSCs possess
high plasticity, and, under specific environmental conditions, could differentiate into all
germ layer derivatives [227–231].
3.2.3. Amniotic Fluid-Derived Mesenchymal Stem/Stromal Cells
The amniotic fluid is the next rich source of fetal-origin stem cells collected during
diagnostic amniocentesis, therapeutic amnioreduction, or cesarean section [
232
–
235
]. Stem
cells suspended in the amniotic fluid form a heterogeneous group of cells with distinct
properties and cellular characteristics. Similar to placental tissues, amniotic fluid is abun-
dant in multipotent amniotic fluid mesenchymal stem cells that shed from the placenta
and umbilical cord. It was shown that these cells are able to differentiate into osteoblas-
tic, bone-forming cells, demonstrated through alkaline phosphatase (ALP) activity and
calcium deposition in the extracellular matrix. The MSCs possess the ability to adhere to
plastic flasks, which makes them possible to isolate from the second and third trimester
amniotic fluid samples in standard culture conditions [
236
]. In addition, differentiation
toward osteoblasts was more efficient on a gelatin scaffold compared to monolayer cul-
ture [
237
]. The experimental observations confirmed the presence of highly proliferative,
colony-forming, spindle-shaped cells in amniotic fluid cultures obtained from full-term
cesarean sections. Immunophenotype analysis revealed that the hAF-MSC did not express
the hematopoietic and endothelial markers (CD45, CD34, CD31), and expressed the MSC
markers (CD73, CD90). The expression of CD105 was detectable, but, significantly lower
than observed in other MSC lineages. The expression of Oct-4 was significantly increased in
the freshly obtained samples in comparison to
in vitro
cultured cells [
212
]. Moraghebi et al.
have also reprogrammed the term hAF-MSC into the pluripotent stem cell with a similar
expression of Oct-4 and NANOG to human embryonic stem cell lines. The reprogrammed
cells had the potential to form teratomas and differentiate into hematopoietic and neural
cell lineages [212].
Amniotic fluid can be used as a source of cells with higher potency, known as the
amniotic fluid stromal cells (AFSCs). AFSCs have the capacity to differentiate into cells of all
three embryonic germ layers without forming tumors. That ability places them somewhere
between ESCs and MSCs. The plastic adherent AFSCs are isolated by the positive selection
for CD117 surface antigen [
232
,
238
]. Cloned, CD117-positive cells express CD29, CD44,
CD73, CD90, CD105, SSEA-4, Oct-4, and MHC-I molecules, and are negative for MHC-II,
CD80, and CD86 antigens. Whereas the first trimester AFSCs could express NANOG,
SSEA-3, TRA-1-60, and TRA-1-81, their expression was not detected in the second trimester
cells [
167
,
213
]. In addition, AFSCs have the potential to be reprogrammed into the induced
pluripotent stem cells [
232
,
239
]. The regenerative properties of amniotic fluid-derived
stem cells mainly depend on the significant paracrine activity of numerous peptides and
cytokines released into the surrounding of damaged tissues [240].
4. Signaling Pathways Involved in Fetal-Derived MSC Development and Differentiation
All trophoblast lineages are derived from the trophoectoderm cells of the blasto-
cyst [
241
]. After the implantation in the uterus, trophoectodermal cells become cytotro-
phoblasts. Human trophoblastic cells can be distinguished into three subpopulations. Both
extravillus cytotrophoblasts and syncytiotrophoblasts are derived from the undifferentiated
cytotrophoblast cells [242]. Trophoblastic cells were demonstrated to differentiate in vitro

Cells 2021,10, 3278 15 of 39
from human embryonic stem cells forming embryoid bodies [
243
]. Bone morphogenetic
protein 4 (BMP4), a member of the transforming growth factor
β
(TGF-
β
) superfamily, in-
duced such differentiation, as indicated by Xu et al. [
244
]. Transcriptomic studies revealed
that BMP4-treated embryonic stem cells exhibited increased expression of trophoblastic
markers, such as CG-
α
, CG-
β
(subunits of human chorionic gonadotropin), placental
growth factor, glial cells missing 1 (GCM1), the non-classical HLA class I molecule HLA-G1,
and CD9. On the contrary, the expression of genes associated with pluripotency, such as
POU domain class 5 transcription factor (POU5F1) or telomerase reverse transcriptase
(TERT), was decreased [244].
Cytotrophoblast proliferation is associated with hypoxic conditions (2% O
2
), as indi-
cated by
in vitro
studies, however, low levels of oxygen do not induce syncytialization [
245
].
Effects exerted by hypoxia are mediated by the hypoxia inducible factor 1 (HIF-1), which
is regulated by the tumor suppressor protein von Hippel-Lindau (VHL) via complex for-
mation. Under normoxic conditions, HIF-1/VHL complex is degraded [
242
]. Apart from
that, hypoxia induces expression of several genes, such as cyclin B1, focal adhesion kinase
(FAK), α5β1 integrin, p53, BAX, TGF-β, or MMP-2 [246].
Syncytialization is a process occurring at implantation when cytotrophoblast cells fuse,
which may be influenced by various factors, as indicated in
in vitro
studies, such as epider-
mal growth factor (EGF), granulocyte-macrophage stimulating factor (GM-CSF), human
chorionic gonadotropin (hCG), glucocorticoids, or estradiol. Syncytin, encoded by an enve-
lope gene of a defective endogenous human retrovirus, HERV-W, induces cell fusion and
syncytiotrophoblast formation, as indicated by
in vitro
studies by Frendo et al. [
247
]. Con-
nexin 43 was demonstrated to participate in syncytiotrophoblast formation as well [248].
Extravillus cytotrophoblasts comprise several subtypes based on their location, such
as cytotrophoblasts of cell columns, interstitial cytotrophoblasts, or endovascular cytotro-
phoblasts. Differentiation towards extravillus cytotrophoblasts occurs along the invasive
pathway, where the cells invade the endometrium [242].
Transcriptomic studies performed by Okae et al. [
249
] revealed that the genes related
to the wingless/integrated (Wnt) and epidermal growth factor (EGF) signaling pathways
were overexpressed in cytotrophoblast cells isolated from first-trimester placentas. This
led to the establishment of proliferative human cytotrophoblast cells in
in vitro
culture
via activation of Wnt and EGF and inhibition of TGF-
β
, histone deacetylase (HDAC),
and Rho-associated protein kinase (ROCK). Such cultured cells were able to give rise to
the three major trophoblastic lineages and, therefore, were designated ‘trophoblast stem
cells’. Their differentiation towards extravillus cytotrophoblast cells was dependent on
the addition of neuregulin 1 (NRG1), A83-01 (an TGF-
β
inhibitor), and Matrigel
®
to the
culture, which resulted in epithelial-mesenchymal transition and expression of HLA-G.
Trophoblast stem cells were also successfully differentiated towards syncytiotrophoblast
and the addition of forskolin (cyclic AMP agonist), EGF, and 3D culture conditions were
vital for this purpose [
249
]. In contrast, the derivation of mouse trophoblast stem cells is
dependent on activation of FGF and TGF-βand inhibition of Wnt and ROCK [250].
The development of the human umbilical cord starts after the implantation of the
blastocyst. Initially, the embryo is connected to endometrium through the trophoblast,
which develops into the connecting stalk, constituting the earliest sign of the umbilical
cord [
22
,
251
]. The umbilical cord’s connective tissue originates from the extraembryonic
mesoblast. Between 28 and 40 days post coitum, the expanding amniotic cavity compresses
the connecting stalk, the allantois, and the yolk sac and covers them with the amniotic
epithelium, forming the cord. Fetal blood vessels originate from the allantois around the
third week post coitum and subsequently develop into umbilical vessels [251].
Although stem cells are located in various compartments of the umbilical cord, the
stromal tissue, called Wharton’s jelly, provides cells that are the richest in stemness proper-
ties [
252
]. Moreover, these cells are mostly located in the proximity of umbilical vessels
in Wharton’s jelly, therefore, it seems that the perivascular region is a source of precursor
cells, Figure 3[253,254].

Cells 2021,10, 3278 16 of 39
Figure 3.
Umbilical cord-derived MSCs—location and characteristics. Created with BioRender.com
(accessed on 5 October 2021).
This hypothesis, related to Wharton’s jelly MSCs, is consistent with the results ob-
tained by Crisan et al. [
255
]. The authors aimed to investigate the presence of multilineage
progenitors among perivascular cells, mostly pericytes, and isolated them from skeletal
muscle, pancreas, adipose tissue, placenta, umbilical cord, and other tissues. These cells
expressed NG2 (neural/glial antigen 2), CD146, and PDGFR
β
(platelet-derived growth
factor receptor
β
), while not expressing endothelial cell markers. When cultured over
a prolonged time period, perivascular cells exhibited expression of markers typical for
MSCs, such as CD44, CD73, CD90, and CD105, and were able to differentiate towards
chondrocytes, adipocytes, and osteocytes, suggesting that MSCs are derived from perivas-
cular cells [
255
]. Sarugaser et al. [
256
] isolated a nonhematopoietic human umbilical cord
perivascular cell population capable of bone nodule formation and expressing markers
typical of MSCs. Interestingly, umbilical cord perivascular cells are also PDGFR
β
+ and may
be recruited through the PDGFB signaling pathway, especially since amniotic fluid has been
demonstrated to contain both PDGFA and PDGFB, which could influence their migration
from the vasculature [
257
,
258
]. Takashima et al. [
259
] demonstrated that the earliest wave
of PDGFR
α
+ MSCs in the embryonic trunk was generated from Sox1+ neuroepithelium
partially through a neural crest pathway. Nevertheless, MSCs may also be isolated from
non-perivascular regions of the umbilical cord, such as the umbilical cord lining, however,
they could have simply migrated away from the vasculature [260].
Moreover, Wharton’s jelly-derived MSCs exhibit properties of both fibroblasts (the expres-
sion of vimentin) and smooth muscle cells (the expression of desmin, actin, and myosin) [
253
]
and, therefore, are regarded as myofibroblasts. As indicated by Nanaev et al. [
21
], the
level of differentiation of stromal cells towards myofibroblasts is dependent on the stage
of gestation, and the most differentiated cells are located in the perivascular zone of
Wharton’s jelly.
The amnion adheres to the umbilical cord and fetal skin and is extended from the edge
of the placenta. This fetal membrane is composed of an epithelial monolayer contacting the
amniotic fluid and four layers of connective tissue of mesodermal origin. The connective
tissue layer is composed of fibroblast-like mesenchymal cells and a collagenous extracellular
matrix. Importantly, the amniotic membrane regulates the volume and composition of
amniotic fluid, which consists of amniotic fluid-derived MSCs [
261
]. However, the cellular
component of the amniotic fluid changes during gestation, receiving cells shed from the
fetus or possibly containing cells derived from the placenta or the inner cell mass of the

Cells 2021,10, 3278 17 of 39
morula [
262
]. It has also been hypothesized that embryonic cell mass releases a variety of
stem cell types into the amniotic cavity, which are transported by the amniotic fluid and
implant various tissues [263].
Torricelli et al. [
264
] obtained small nucleated round cells from the amniotic fluid
before the 12th week of gestation, which were identified as hematopoietic progenitor cells
originating from the yolk sac. This is consistent with the results of Pieternella et al. [
265
],
who demonstrated that amniotic fluid-derived cells are of fetal origin, as indicated by
molecular HLA typing. Moreover, these cells exhibited properties of MSCs, such as
multilineage differentiation potential towards fibroblasts, adipocytes, osteocytes [
265
], and
chondrogenic lineage [
266
]. Ovine mesenchymal amniocytes were shown to give rise to
smooth and skeletal muscle cells after the treatment with promyogenic medium, which
resulted in expression of transgelin, calponin, and α-actin [267].
However, recently, third-trimester amniotic fluid was demonstrated to contain MSCs
of renal origin [
268
]. These cells were not only positive for pluripotency markers, such
as SSEA-4 (stage-specific embryonic antigen 4), c-kit, or TRA-1-60, but also expressed
the master renal progenitor markers: SIX2 (SIX homeobox 2) and CITED1 (CBP/p300-
interacting transactivator 1), as well as renal proteins, including PODXL (podocalyxin
like), LHX1 (LIM homeobox 1), BRN1 (POU class 3 homeobox 3), and PAX8. Moreover,
these cells exhibited renal functions, as demonstrated by albumin endocytosis assays,
and gene ontology terms revealed their involvement in pathways associated with kidney
morphogenesis [268].
Both renal and osteoblastic differentiation was reported to be dependent on mTOR
(mechanistic target of rapamycin) signaling cascade. Importantly, this pathway was demon-
strated to be fully active in amniotic fluid stem cells by Siegel et al. [
269
]. Blocking intercellu-
lar activity of mTOR via the inhibitor rapamycin or through siRNA resulted in diminished
embryonic body formation by amniotic fluid stem cells, which constitutes the principal
step in differentiation of pluripotent embryonic stem cells. Specifically, embryonic body
formation was reported to be dependent on two complexes, namely mTORC1, which regu-
lates mRNA translation via kinase phosphorylation, and mTORC2, which phosphorylates
and subsequently activates AKT [270].
5. Animal Models and Clinical Applications
Human and animal stem cells of fetal origin have been the subjects of numerous
studies that aimed to find their possible applications in daily clinical practice. The first
reports about the unique properties of cells derived from human amniotic membranes were
released almost two decades ago. Balio et al. described a breakthrough in the field of peri-
natal MSC transplantation. They successfully transplanted the human amnion and chorion
cells, obtained from term placentas, to neonatal swine and rats. They discovered that
the obtained cells did not induce allogeneic or xenogeneic lymphocyte proliferation [
175
],
making them viable therapeutic candidates.
5.1. Animal Models
Laboratory animals have been used in medical research for many years. Veterinary
medicine provides a tool to study transplantation mechanisms between basic science and
clinical human medicine. Most human diseases also affect animals, hence the etiopatho-
genesis and treatment are similar. Veterinary medicine, thus, represents a valuable field,
especially in regenerative medicine, where the development of animal-based protocols
could be transferred to human medicine. A key issue is the collaboration of researchers
from different fields, including physicians, veterinarians, biologists, geneticists, and others,
and working together in accordance with the “One Health” mindset [271].
5.1.1. Bone and Cartilage Diseases
With regard to bone repair processes, SCID mice were used that were subcuta-
neously implanted with MSCs from human placental chorion and MSCs from the decidua

Cells 2021,10, 3278 18 of 39
basalis [
272
]. In both cases, ectopic bone formation was observed at 8 weeks. The expression
of the markers osteopontin (OPN), osteocalcin (OCN), biglikan (BGN), and bone sialopro-
tein (BSP), which are characteristic of bone tissue, was also demonstrated. Placenta-derived
MSCs are, therefore, cells with bone-forming potential [
272
]. Similar findings were pre-
sented in a Wistar rat model, where, among MSCs from different sources (bone marrow,
Wharton’s jelly, umbilical cord, placenta, adipose tissue), human placental MSCs showed
the highest osteogenic potential and complete bone regeneration [273].
Human amniotic MSCs were differentiated into chondrocytes, as confirmed by the
expression of SOXs and BMPs, and then transplanted into non-cartilage tissues in mice
and on a collagen scaffold into defects in rat bone. Morphological changes to the trans-
planted MSCs, along with deposition of type II collagen, were observed, suggesting their
potential use in the treatment of osteoarthritis [
274
]. A rabbit model was also used to study
the treatment of cartilage damage, where placental MSCs were applied to a silk fibroin
biomaterial scaffold [
275
]. Damaged femoral condyles lacking articular cartilage were
analyzed after implantation of MSCs. Defect repair occurred within 4–12 weeks and no
more degeneration or inflammatory cell infiltration was observed thereafter. Another study
using a rabbit model for cartilage damage in the knee joint showed similar results [
276
].
MSCs from amniotic fluid were differentiated on chondrogenic medium with TGF
β
3 and
BMP2 and xenotransplantation allowed in vivo survival for 8 weeks [276].
5.1.2. Cardiac Diseases
Due to the fact that adult cardiomyocytes do not regenerate after injury, treatment of
heart failure continues to pose problems. Zhao et al. [
277
] investigated the feasibility of
using human amniotic MSCs to treat heart injury in a rat model. MSCs were cultured with
neonatal rat heart explants and then transplanted into infarcted rat hearts. During culture,
MSCs were stimulated with bFGF and activin A and shown to express Nkx2.5 and atrial
natriuretic peptide (specific for cardiomyocytes). The cardiac-specific myosin alpha heavy
chain gene was also detected. When cultured together with explants, MSCs integrated
and differentiated into the host tissue. After transplantation, MSCs were maintained for
2 months as similar to cardiomyocytes [
277
]. Animal models have also been used to study
the effects of MSCs on myocardial infarction healing [
278
,
279
]. MSCs from Wharton’s
jelly were transplanted into mouse [
279
] and mini-swine [
278
] models. Both studies
demonstrated decreased apoptosis in injured myocardium, cardioprotective effects, and
increased capillary density. Nevertheless, studies in the mini-swine model demonstrated
differentiation of MSCs into cardiomyocytes and endothelial cells. Human placenta-derived
MSCs were also used to study myocardial infarction in a porcine model. MSCs were
preconditioned with hyaluronan mixed with butyric and retinoic acid ester. Implantation
of these MSCs reduced scar size and increased capillary density and myocardial perfusion,
along with a reduction in fibrous tissue [280].
5.1.3. Neurological Disorders
MSCs isolated from Wharton’s jelly have been used to study Parkinson’s disease
using a rat model. The animals were induced to have forebrain lesions, causing movement
disorders. A reduction in motor deficits was observed in rats after MSC administration,
explained by the protection of dopaminergic neurons by growth and neurotrophic factors
secreted by MSCs [
281
,
282
]. Human placenta-derived MSCs were also used in studies
of Parkinson’s disease treatment in a rat model [
283
]. Almost normal motor function
was observed 24 weeks after MSC transplantation. Through immunohistochemical and
positron emission tomography (PET) analyses, dopaminergic differentiation of progenitors
was demonstrated, indicating that neuronal progenitors can differentiate eventually
in vivo
and alleviate motor defects [
283
]. A mouse model of Alzheimer’s disease (AD) was used
to study the effects of MSCs from human placenta [
284
]. These cells were given to mice in-
travenously and the first effects observed were improved spatial learning ability correlated
with fewer A
β
plaques in the brain. There was also a decrease in pro-inflammatory and

Cells 2021,10, 3278 19 of 39
an increase in anti-inflammatory cytokines in mice after MSC administration compared to
animals receiving saline. Improvement of AD pathology through paracrine processes and
immune modulation was indicated [284].
A rat model was also recently used to study the anti-inflammatory effects of Wharton’s
jelly MSCs in spinal cord injury [
285
]. The study used real-time polymerase chain reaction,
Western blotting, and ELISA and determined the expression levels of NLRP1, ASC, active
caspase-1, interleukin-1beta (IL-1
β
) and IL-18, and TNF-
α
. These factors are responsible
for the local inflammatory response. The results indicated decreased expression in rats
with injured spinal cords that received MSC transplantation and, in addition, the motor
functions of the animals were improved [
285
]. Interesting studies have also been conducted
in rodent models for the treatment of spinal cord injury using umbilical cord MSCs. The
spleen was shown to be an important organ mediating the effects of MSCs, through their
immunomodulatory action, stimulating, for example, specific inflammatory cytokines
that recruit immune cells. Splenectomized animals lost the ability to reduce spinal cord
hemorrhage and did not increase systemic IL-10 levels after MSC administration [
286
]. In
contrast, another study, also in a rat model with a damaged cord, compared the effects of
MSCs alone with Wharton’s jelly and conditioned medium [
287
]. Cells in culture exhibit
paracrine activity, hence the potential action of factors contained in conditioned medium
(CM). Rats with compressive lesions were administered MSCs and CM intrathecally. In
both cases, improvement and increased expression of genes related to axonal growth were
observed. However, when MSCs were used, expression of inflammatory markers was
demonstrated, which is a result of the inflammatory response to the transplant. In the
case of CM, no inflammatory response was shown, and, in addition, axonal sprouting was
improved and the number of reactive astrocytes decreased [
287
]. Similarly, treatment of
spinal cord injury in a rat model was studied using Wharton’s jelly MSCs. In the study
described here, MSCs were administered intrathecally at different concentrations and
repetitions. The results showed a positive but dose-dependent effect of MSCs on spinal
cord regeneration [288].
The experimental animals modeling multiple sclerosis were administered human
placenta-derived MSCs (hPMSCs) and low and high doses of extracellular vesicles from
hPMSCs (hPMSCs-EVs), as well as saline. High doses of hPMSCs-EVs resulted in improved
motor function. Both hPMSCs-EVs and hPMSCs reduced DNA damage in oligodendroglia
and also increased myelination in the spinal cord [
289
]. Research on the use of MSCs to
treat multiple sclerosis has also been conducted previously in a mouse model [
290
]. Human
placental MSCs (hPMSCs) were administered intracerebrally to mice with experimental
autoimmune encephalomyelitis in an MS model. Both survival and reduced disease severity
were observed, and the effects were attributed to a reduction in the anti-inflammatory
protein TSG-6 [290].
The rat model has also been used to study MSC therapy in neural tissue ischemia.
MSCs from Wharton’s jelly were transplanted into rats intracerebrally and were shown
to differentiate into glial cells and neuronal cells and also showed increased angiogenesis.
The regulation of beta1-integrin was shown to include an important role in the processes
that promote plasticity of MSCs when transplanted intracerebrally [
150
]. The effect of
human umbilical cord-derived stem cells on the treatment of neonatal hypoxic–ischemic
encephalopathy was also studied in a rat model [
33
]. Motor and cognitive functions were
shown to be improved and caspase-3 and Beclin-2 expression was decreased, suggesting
the potential for MSCs in the treatment of hypoxic–ischemic encephalopathy.
5.1.4. Organ Disorders
The rat and mouse model was also used to study the effect of MSCs from Wharton’s
jelly on liver [
291
] and lung [
292
] fibrosis. In both cases, the amount of collagen was reduced,
which improved organ function, and inflammation was also shown to be reduced. Similar
studies were also conducted later using intravenously administered human placental
MSCs with green fluorescent protein (GFP) expression in the treatment of liver fibrosis

Cells 2021,10, 3278 20 of 39
in a rat model [
293
]. Alleviation of liver fibrosis, reduction of collagen area, reduction
of TGF-
β
1 and
α
-SMA (markers of fibrosis) expression and improvement of rat organ
function were obtained. Liver regeneration by MSCs was examined using a rat model with
carbon-tetrachloride-damaged liver tissue [
294
]. Human MSCs derived from chorionic
platelets were used in this study. The expression of markers related to autophagy, apoptosis,
cell survival, and liver regeneration were analyzed. The results indicated that MSCs
induced tissue repair through HIF-1
α
-mediated mechanisms and autophagy [
294
]. The
effect of human MSCs from amniotic membrane on CCl
4
-induced cirrhosis in mice was
studied [
295
]. The isolated MSCs were injected into the spleens of mice, which were then
sacrificed after 4 weeks. Alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) levels in the blood of mice were evaluated and histological analysis of the liver was
performed. Reduced areas of liver fibrosis and improved blood parameters (ALT, AST)
were observed in mice after MSC injection compared to the control group. Activation
of hepatic stellate cells and apoptosis of hepatocytes were decreased, while regenerative
processes were promoted. The injected MSCs showed expression of hepatocyte-specific
markers (
α
-fetoproteinran and human albumin) [
295
]. Due to the high genetic similarity
of pigs to humans, as well as similar organ size, clinical studies on a porcine model are
particularly valuable. The study by Cao et al. [
296
] used Chinese miniature pigs with
acute liver failure that were implanted with human placental MSCs. Histological analysis
showed a reduction in liver inflammation, liver denaturation, and necrosis and also showed
promotion of liver regeneration; however, these observations applied only to the group of
pigs injected with MSCs via the portal vein. Those that received administration of MSCs
through the jugular vein or treatment of MSCs with X-rays prior to injection and the control
group did not show these changes. The authors suggested that the portal vein infusion
route with the help of B-ultrasound is more favorable compared to the jugular vein [296].
The isolated MSCs from cord blood were used to study colitis in a mouse model
[297,298]
.
The first study used NOD2-activated MSCs from cord blood that were injected intraperi-
toneally. It was shown that MSCs inhibited the inflammatory response and activated the
anti-inflammatory response in the colon [
297
]. In turn, the second study analyzed extracts
from MSCs injected intraperitoneally into mice. It was shown that the extracts strongly
inhibited the inflammatory process and increased the body weight of the animals. A shift
in the functional phenotype of macrophages from M1 to M2 was observed [298].
The mouse model was also used to study the effect of MSCs from human amniotic
fluid on the treatment of stress urinary incontinence [
299
]. MSCs were first differentiated
in vitro
for the myogenic direction, as confirmed by the expression of PAX7, MYOD, and
dystrophin. MSCs were labeled with silica-coated magnetic nanoparticles containing
rhodamine B isothiocyanate and then injected transurethrally into mice with gluteal nerve
injury. Nerve regeneration and neuromuscular junction formation were demonstrated by
expression of neuronal markers and acetylcholine receptor. Transurethral injection of MSCs
resulted in a return to normal histological structure of the urethral sphincter and also a
definite improvement in function, while lacking tumorigenicity and immunogenicity [
299
].
5.1.5. Ischemia and Wound Healing
MSCs isolated from the umbilical cord were tested in a mouse model of hind limb
ischemia. MSCs were isolated and cultured in vitro and then differentiated in endothelial
differentiation medium with VEGF and bFGF. Transplanted cells into the mouse hind limb
differentiated into endothelial cells, indicating the potential use of these cells in promoting
angiogenesis and reendothelialization [230].
Animal models involving mice were used to study wound healing using Wharton’s
jelly MSCs that were placed on a scaffold decellularized from amniotic membrane. Re-
sults showed accelerated wound healing, reduced scarring, and also hair growth on the
treated skin [300].

Cells 2021,10, 3278 21 of 39
5.2. The Application of Perinatal MSC in Human Clinical Trials
The PubMed database was searched for the relevant references from the last five
years until April 2021 to summarize the latest reports on the perinatal MSC application
in the completed clinical trials. We searched the PubMed database using the following
terms: “placenta mesenchymal stem cells”, “umbilical cord mesenchymal stem cells”, and
“amniotic fluid stem cells”. We set the article type to “Clinical Trial” and the species to
“Humans” for additional searching filters.
5.2.1. Placenta-Derived MSCs
Regenerative medicine opens novel perspectives for the clinical application of placenta-
derived MSCs. Soltani et al. analyzed the safety and efficacy of intra-articular injection of
placenta-derived MSCs in knee osteoarthritis treatment. Patients did not present any acute
and long-term severe adverse effects of MSC therapy. The first results were promising—
eight weeks after the procedure, a significant increase in knee flexion range of motion
and pain reduction was noted. Patients also reported a significant improvement in their
daily activity and quality of life. However, they did not observe any significant long-term
clinical improvements and chondral regeneration (24 weeks after the procedure) [
301
]. The
list of the most recent human perinatal MSC clinical trials is shown in Table 2. Winkler
et al. investigated the profile of safety and the potential dose-dependent benefits from the
intramuscular injection of placenta-derived MSCs in the patients who underwent the hip
arthroplasty that was used as a standardized injury model. The MSC administration
was well tolerated and did not cause any adverse effects. Interestingly, the patients
who received the lower dose of placenta-derived MSCs had better post-operative results.
They had significantly improved muscle strength and volume compared with placebo.
Furthermore, the administration of placenta-derived MSCs reduced the levels of surgery-
related inflammatory biomarkers [
302
]. Norgren et al. published the protocol of their
planned clinical trial that aims to assess the safety and the efficiency of placenta-derived
MSC intramuscular injections performed in patients with atherosclerotic critical limb
ischemia who are unsuitable for the standard revascularization [
303
]. Levy et al. reported
that the intrapenile placenta-derived MSC injection increased the penile peak systolic
velocity at 3 and 6 months after the procedure. At 3 months after the procedure, 3 out of
8 patients were able to achieve erection [
304
]. Finally, Zeng et al. described a successful
single-case report of placenta-derived MSC hydrogel application to treat the diabetic
foot ulcer [305].
Immunomodulatory properties of placenta-derived MSCs could be applied to the
treatment of chronic inflammatory diseases. Haller et al. performed the new drug’s
clinical trial for patients with chronic obstructive pulmonary disease. The drug contains
a mixture of anti-inflammatory molecules obtained from the exosomes isolated from
the placenta-derived MSCs. After three (one per week) inhalations with the new drug,
treated patients were found to have significantly increased spirometry parameters (FEV1,
PEF) and improvements in CT pulmonary images [
306
]. Hashemian et al. focused on
the possible implementation of the perinatal MSC transplantations in the treatment of
patients with COVID-19-induced ARDS. They discovered that the cell therapy was safe
and could be associated with reduced dyspnea and increased SpO
2
within 48–96 h after
the intervention [
307
]. Ringden et al., in their non-randomized trial, reported that the
decidua-derived MSCs could be used as a promising new tool for the treatment of severe
acute graft-versus-host disease [308].
5.2.2. Umbilical Cord and Amniotic Fluid-Derived MSCs
Similar to placenta-derived MSCs, umbilical MSCs have found numerous applications
in regenerative medicine. Several clinical trials focused on their utility in the treatment
of osteoarthritis. Patients treated with MSCs via intra-articular injections experienced a
significant decrease in knee pain and improvements in knee joint function and clinical
parameters, such as Western Ontario and Mc Master Arthritis Indexes. The MSC therapy

Cells 2021,10, 3278 22 of 39
was more efficient than the hyaluronic acid injections—its effect was stable over seven
years of follow-up [
309
–
312
]. He et al. analyzed the efficacy of intramyocardial grafting
of collagen scaffolds covered with umbilical cord-derived MSCs in patients with chronic
ischemic heart disease. They did not observe any significant benefits (reduction of the in-
farct size) from the cellular therapy compared with the control group [
313
]. Interestingly, it
was reported the umbilical cord MSC infusion was connected with the significant improve-
ment in the left ventricular ejection fraction in patients with heart failure [
314
]. Hashemi
et al. noted that the human umbilical cord-derived MSCs seeded on the acellular amniotic
membrane scaffolds improved the process of healing of chronic skin ulcers in patients with
diabetes [
315
]. In addition, the umbilical cord-derived MSC conditioned media could be
used in the novel protocols of regenerative medicine. Kim et al. reported that the topical
drugs with umbilical cord-derived MSC conditioned media improved the skin stratum
corneum and strengthened the skin barrier in patients with atopic dermatitis [
316
]. Finally,
it was found that the application of umbilical cord blood-derived MSC conditioned media
containing cream or serum reduced the total area of microcrusts and erythema in patients
treated with ablative CO2fractional laser [317].
The immunomodulatory properties of the umbilical cord-derived MSCs may success-
fully modify the course of chronic inflammatory and degenerative diseases. Therefore,
several studies have aimed to investigate their feasibility and efficacy in the treatment
of rheumatoid arthritis. MSC therapy was found to be safe and associated with reduced
levels of inflammatory parameters and disease activity [
318
–
321
]. Other factors, like cervus
and cucumis peptides and interferon-
γ
combined with MSCs, could improve the therapy
effects [
318
,
319
]. Large analysis revealed that the umbilical cord-derived MSC transplanta-
tions are safe and do not cause serious adverse effects in patients with chronic autoimmune
rheumatoid diseases, such as systemic lupus erythematosus, Sjögren’s syndrome, and
systemic sclerosis. However, Deng et al. declared that the MSC therapy has no signif-
icant positive effects in patients with lupus nephritis [
322
]. Riordan et al. reported no
serious adverse effects of MSCs therapy in patients with multiple sclerosis. Assessment
performed one month after the treatment revealed that the treated patients experienced
positive changes in the bladder, bowel, non-dominant hand, and sexual functions, as
well as in the results of walk tests and general quality of life [
323
]. Allogenic umbilical
cord-derived MSCs were also used to treat individuals with cerebral palsy and autism
spectrum disorders. The therapy combined with rehabilitation improved the gross motor
and comprehensive function in children with cerebral palsy, and was associated with
lower levels of inflammatory markers and better clinical outcomes in patients with autism
disorders [324,325].
Moreover, allogeneic umbilical cord-derived MSC infusions were found to be safe
in patients with Crohn’s disease. The randomized controlled trial outcomes revealed
that patients treated with MSCs had significantly reduced disease activity index and
corticosteroid dosage, compared with the controls, at 12 months after the cell therapy [
326
].
Some authors speculated that the unique immunomodulatory properties of umbilical
MSCs could modulate the immune response in allogeneic graft recipients. MSCs were
administered as supplementary induction therapy in patients who underwent kidney
transplantation. The applied procedure was found to be safe for enrolled patients. However,
there was no evidence of its efficacy in comparison with the control group treated with
standard immunosuppressive drugs [
327
]. However Shi et al. discovered that umbilical
cord-derived MSC modifies the course of acute liver allograft rejection. Their analyses
revealed that the MSCs transplantation increased the T-regulatory/T-helper 17 cells ratio,
TGF-β1, and prostaglandin E2 levels [328].
It was discovered that the umbilical cord-derived MSC infusions are safe and well-
tolerated in patients with severe COVID-19 infection. The results of the first clinical trials
indicate that the MSCs therapy could be potentially efficient in the management of COVID-
19 cases. The authors observed several improvements in the treated patients compared
with the placebo—in the CT imaging outcomes and clinical parameters, such as the results

Cells 2021,10, 3278 23 of 39
of 6 min walk tests. However, further large phase III clinical trials are needed to confirm
those mostly positive tendencies [
307
,
329
–
331
]. He et al. administered a single infusion
of umbilical cord-derived MSCs in patients with severe sepsis. They reported that the
experimental procedure was safe and well-tolerated. Nonetheless, to investigate its efficacy
in that indication, further studies are still needed [332].
Even though the animal models brought us the first promising outcomes, there have
been no studies that assessed the safety and efficacy of amniotic fluid-derived stem cells
in experimental treatment protocols in humans [
232
]. It was demonstrated that after the
appropriate preparation, the population of amniotic fluid-derived MSCs could be used as a
model in the studies focused on human genetic diseases. Squillaro et al. reported that they
silenced glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) genes in the amniotic
fluid-derived MSCs, creating the models of Gaucher and Fabry diseases. They found that
GBA and GLA silencing provoked impaired autophagy and DNA repair mechanisms and,
as a consequence, promoted apoptosis and increased senescence in the investigated cellular
populations. Moreover, the authors concluded that the perinatal cells collected from fetuses
affected by genetic diseases could be used as a readily available source of experimental
materials alternative to animal models [333].
Multiple phase I/II clinical trials confirmed the safety of perinatal MSC administration
in the regenerative medicine protocols and the treatment of various chronic diseases.
However, their efficacy should be investigated in further large randomized controlled trials.
Table 2. Perinatal MSCs in human clinical trials.
Condition/
Procedures Type of Study Number of
Participants Material First Author; Year;
Reference
Knee osteoarthritis
Randomized, double-blind,
placebo-controlled
clinical trial
20 Allogenic
placenta-derived MSCs Soltani; 2019; [301]
Hip arthroplasty
Randomized, double blind,
placebo-controlled,
phase I/IIa
clinical trial
20 Allogenic
placenta-derived MSCs Winkler; 2018; [302]
Erectile dysfunction
Prospective,
non-randomized,
single-arm
clinical trial
8Allogenic
placenta-derived MSCs Levy; 2016; [304]
Chronic obstructive
pulmonary disease
Prospective,
non-randomized,
single-arm
clinical trial
30
Allogenic placenta-derived
MSCs-derived product:
Exo-d-MAPPS drug
Harrell; 2020; [306]
COVID-19-induced
ARDS
Prospective,
non-randomized,
single-arm
clinical trial
11
Allogenic
placenta-derived MSCs
(5 cases)
Umbilical
cord-derived MSCs
(6 cases)
Hashemian; 2021; [307]
Acute graft-versus-host
disease
Prospective,
non-randomized,
single-arm
clinical trial
38 Allogenic decidua-derived
MSCs Ringden; 2018; [308]
Knee osteoarthritis
Single-arm,
open-label
clinical trial
29 Allogenic umbilical
cord-derived MSCs Dilogo; 2020; [309]

Cells 2021,10, 3278 24 of 39
Table 2. Cont.
Condition/
Procedures Type of Study Number of
Participants Material First Author; Year;
Reference
Knee osteoarthritis
Randomized,
placebo-controlled,
phase I/II
clinical trial
26 Allogenic umbilical
cord-derived MSCs Matas; 2019; [310]
Knee osteoarthritis
Open-label,
single-arm,
single-center,
phase I/II clinical trial with
7-year extended follow-up
7
Allogeneic human
umbilical cord
blood-derived MSCs
Park; 2017; [311]
Knee osteoarthritis
Randomized,
placebo-controlled
clinical trial
36 Allogenic umbilical
cord-derived MSCs Wang; 2016; [312]
Chronic ischemic
heart disease
Randomized, double-blind
clinical trial 115
Collagen scaffolds covered
with umbilical
cord-derived MSCs
He; 2020; [313]
Heart failure
Randomized, controlled,
phase I/II
clinical trial
30 Allogenic umbilical
cord-derived MSCs Bartolucci; 2017; [314]
Chronic diabetic
skin ulcers Randomized, clinical trial 5
Allogenic umbilical
cord-derived MSCs seeded
on biological scaffold
Hashemi; 2019; [315]
Atopic dermatitis
Prospective,
non-randomized,
single-arm
clinical trial
28
Topical drugs with
allogenic umbilical
cord-derived MSCs
conditioned media
Kim; 2020; [316]
Ablative CO
2
fractional
laser treatment
Randomized,
double-blinded, controlled
clinical trial
23
Umbilical cord
blood-derived MSCs
conditioned media
containing serum
Kim; 2020; [317]
Rheumatoid arthritis Randomized, controlled
clinical trial 119 Allogenic umbilical
cord-derived MSCs Qi; 2020; [319]
Rheumatoid arthritis
Randomized, controlled,
phase I/II
clinical trial
63 Allogenic umbilical
cord-derived MSCs He; 2020; [318]
Rheumatoid arthritis
Prospective,
phase I/II
clinical trial
64 Allogenic umbilical
cord-derived MSCs Wang; 2019; [320]
Rheumatoid arthritis
Open-label,
single-arm,
single-center,
phase Ia clinical trial
9Allogenic umbilical cord
blood-derived MSCs Park; 2018; [321]
Lupus nephritis
Randomized,
double-blind,
placebo-controlled
clinical trial
18 Allogenic umbilical cord
blood-derived MSCs Deng; 2017; [322]

Cells 2021,10, 3278 25 of 39
Table 2. Cont.
Condition/
Procedures Type of Study Number of
Participants Material First Author; Year;
Reference
Multiple sclerosis
Prospective,
non-randomized,
single-arm
clinical trial
20 Allogenic umbilical
cord-derived MSCs Riordan; 2018; [323]
Cerebral palsy Randomized, controlled
clinical trial 39 Allogenic umbilical
cord-derived MSCs Gu; 2020; [324]
Autism spectrum
disorder
Single-arm,
phase I/II
clinical trial
20 Allogenic umbilical
cord-derived MSCs Riordan; 2019; [325]
Crohn’s disease Randomized, controlled
clinical trial 82 Allogenic umbilical
cord-derived MSCs Zhang; 2018; [326]
Kidney transplantation
Multicenter, randomized,
controlled
clinical trial
42 Allogenic umbilical
cord-derived MSCs Sun; 2018; [327]
Acute liver
allograft rejection
Randomized, controlled,
clinical trial 27 Allogenic umbilical
cord-derived MSCs Shi; 2017; [328]
COVID-19
Randomized, double-blind,
placebo-controlled,
phase II
clinical trial
101 Allogenic umbilical
cord-derived MSCs Shi; 2021; [329]
COVID-19
Parallel assigned
controlled,
non-randomized, phase I
clinical trial
18 Allogenic umbilical
cord-derived MSCs Meng; 2020; [330]
COVID-19
Single-center
open-label, individually
randomized, standard
treatment-controlled
clinical trial
41 Allogenic umbilical
cord-derived MSCs Shu; 2020; [331]
Sepsis
Single-center,
open-label,
dose-escalation phase 1
clinical trial
15 Allogenic umbilical
cord-derived MSCs He; 2018; [332]
6. Conclusions
Due to the indicated properties of differentiation into other cell types, MSCs show great
potential for application in regenerative medicine. Additionally, the simplicity of obtaining
cells from perinatal tissues during routine procedures and minimal ethical concerns make
perinatal tissues one of the most valuable sources for MSCs. Promising results from clinical
trials indicate possible therapeutic use. It seems, therefore, necessary to describe a universal
and simple isolation protocol for the standard use of MSCs in medicine.
Funding: This research received no external funding.
Acknowledgments:
Support in part by the National Institute of Food and Agriculture, United States
Department of Agriculture Animal Health Project NC7082.
Conflicts of Interest: The authors declare no conflict of interest.

Cells 2021,10, 3278 26 of 39
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