Mammalian yolk sac-an alternative source of stem cells

Priscilla Avelino Ferreira Pinto1* , Vitória Mattos Pereira2 , Lina Castelo Branco Motta3 , Matheus Ferreira de Almeida2 , Tiago Gonçalves dos Santos2 , Vanessa Cristina Oliveira4 , Luciana Cristina Machado4  & Carlos Eduardo Ambrósio5  1Physiotherapist, MSc, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo (USP), São Paulo, SP, Brazil 2Undergaduate in Veterinary Medicine, Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, USP, Pirassununga, São Paulo, Brazil 3Biologist, MSc, Criocord Banco de Sangue de Cordão Umbilical e Placentário, Fortaleza, Ceará, Brazil 4Biologist, DSc, Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, USP, Pirassununga, São Paulo, Brazil 5Veterinarian, DSc, Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, USP, Pirassununga, São Paulo, Brazil


Introduction
The placenta, a highly vascularized organ formed by the fusion of fetal membranes with the endometrium, allows the exchange of oxygen, nutrients, and waste between the fetus and the mother, in addition to being related to immunoprotection and fetal growth (Dzierzak & Robin, 2010;Myren et al., 2007). Some research has been conducted to elucidate placental development in different species of mammals and identify cell lines for use in regenerative medicine (Carter & Enders, 2016;Umezawa et al., 2019;Vanover et al., 2017).
The yolk sac (YS) is one of the four placental membranes present in mammals. It is responsible for the nutrition of the embryo during the first trimester of mammalian pregnancy, i.e., the period when vascular communication has not been established yet (Burton et al., 2002). YS plays an important role in protein transport and synthesis and is involved in the absorption of amino acids (King & Enders, 1970) and vitamins, which are fundamental to the development of the embryo in the early stages of pregnancy (Brent & Fawcett, 1998;Zohn & Sarkar, 2010). It is also the first hematopoietic site of the embryo (Palis & Yoder, 2001) as demonstrated by the presence of hematopoietic and mesenchymal stem cells (Favaron et al., 2014;Mançanares et al., 2015;Oliveira et al., 2017). YS is also responsible for the production of macrophage progenitors in adult tissue (Stremmel et al., 2018).
Stem cells derived from fetal tissues have been the target of several studies, owing to higher capacity of differentiation and proliferation compared to that of stem cells derived from adult tissues (Bobis et al., 2006). Previous studies on the isolation, culture, and characterization of stem cells from the YS in different species have shown that YS-derived stem cells do not exhibit Major Histocompatibility Complex II (MHC II) expression, allowing greater capacity for histocompatibility. Furthermore, in some species, stem cells are positive for pluripotency markers (Borghesi et al., 2019;Cremonesi et al., 2011;Saulnier et al., 2016). However, stem cells derived from fetal tissues have an advantage over embryonic stem cells as they do not result in teratoma formation (Vanover et al., 2017). Therefore, investigations on fetal membrane-derived stem cells enable tissue regeneration and treatment of immune diseases in several animal species.
Thus, this review aims to describe the development, function, and possible dysfunctions of the YS in different species and consolidate findings regarding the isolation, in vitro culture, and application of YS-derived stem cells in regenerative medicine.

Development function and dysfunction of YS in different species
The YS is composed of two layers, i.e., the endoderm of the hypoblast and the extraembryonic mesoderm. It is responsible for the primary nutrition of the mammalian embryo (Hafez, 2017), embryonic hematopoiesis, and innate immunity, which includes the formation of "blood islands", the sites responsible for the first erythrocytes (Palis & Yoder, 2001), progenitors of macrophages (Stremmel et al., 2018), and some B lymphocyte strains (Yamane, 2018). The YS also plays a role in the synthesis, transport, and absorption of proteins and immunoglobulins from the mother King & Enders, 1970). The function of the YS depends on several factors, such as the type of placenta ( Figure 1).
Considering the case of epitheliochorial placenta, such as in equine species, the YS plays a fundamental role in the survival of the embryo, as it is responsible for regulating the storage and production of progesterone by the endometrium at the beginning of the embryonic phase. At later stages of pregnancy, the YS fuses with the chorion, giving rise to the vitelline chorion placenta, which is responsible for fetal nutrition during the first three months of pregnancy (Hafez, 2017;Raeside et al., 2004). Similarly, in swine species, the nutrition function occurs until day 15 of embryo development, and important proteins, such as albumin and alpha-fetoprotein, are found in the YS tissue, indicating protein biosynthesis (Tiedemann & Minuth, 1980). In contrast, in the cotyledonary placenta, as occurring in ruminants, the YS is present in a transient and ephemeral manner, until day 50-70 of gestation. It is responsible for the secretion of proteins and metabolites that are vital for survival and embryonic development, such as myoinositol that acts as a growth factor . In contrast to that in humans and other domestic animals, the YS of dogs and cats does not regress during the course of embryonic development. In these species, the YS reaches a prominent size in relation to the embryo after 20 days of gestation, being completely vascularized by vessels derived from the umbilical cord (Miglino et al., 2006). In carnivores, after day 24 of embryonic development, the placental membranes become visibly distinct from each other, with the YS appearing as a red structure connected to the central region of the inverted T-shaped embryo with two long projections that persists until birth (Miglino et al., 2006;Wenceslau et al., 2011). Erythropoiesis-characterized by the production of megakaryocytes and primitive erythrocytes-has also been detected in carnivore YS (Fratini et al., 2016). The carnivore YS is responsible for serum protein biosynthesis before hepatic maturity, a phenomenon fundamental during embryo growth and development (Miglino et al., 2006).
In the hemochorial placenta-present in rodents and humans-the function of YS tissue is elucidated in greater detail owing to the importance of human reproduction and the common use of rodents as animal models for embryological studies. The rodent YS is responsible for the formation of new vessels that enables the transport of gases and nutrients, and production of cholesterol-essential for growth-and growth factors that regulate the growth of vessels and embryonic and placental development; YS is the main source of insulin, which regulates the transfer and synthesis of nutrients (Freyer & Renfree, 2009). Primordial cells originating from the YS have also been known to be present in the fetal liver (Yamane, 2018). Studies on the mRNA expression profiles have demonstrated that the rodent YS mimics the functions of synthesis and distribution of proteins in the fetal liver before its development. There is also a rhythmic secretion of binding proteins that transport molecules; this process is extremely important for the transport and distribution of tyrosine in the fetus (Thomas et al., 1990).
The role of human YS is similar to that of rodent YS. In addition to hematopoiesis and embryonic nutrition, the YS is also responsible for protein biosynthesis, producing proteins critical to embryonic development, such as albumin, alpha-fetoprotein, and transferrin, and forms a plexus of vessels that aid the distribution of nutrients (Dzierzak & Robin, 2010;Giacomini et al., 2017;Gulbis et al., 1998). In humans, the YS is related to protein transport and production associated with embryonic growth (Gulbis et al., 1998). In addition, in birds, as there is no maternal-fetal connection, the YS has plays a central role in embryonic nutrition and growth, and has also been associated with the regulation of fetal metabolism through the modulation of thyroid hormones (Seifert, 2014).
A recent study demonstrated the presence of the ATP binding cassette and single solute carrier proteins, which are involved in transporting cholesterol and metals. These transporters are conserved across species, and are present in human, mouse, and chick YS (Lin et al., 2015). Moreover, these transport proteins are associated with multidrug resistance and absorption (Lin et al., 2015).
Furthermore, a correlation has been identified between the health of the YS and the life prospects of the embryo. In cases where the mother has diabetes-characterized by an irregular glycemic curve-vasculopathy of the YS is a likely outcome, possibly causing changes in the embryo or even embryonic death (Dong et al., 2016). In addition, the size of YS correlates with the proper development of the embryo (Brent & Fawcett, 1998), but it is impossible to separate the cause from the effect in vivo. Furthermore, the loss of bovine clones during early embryonic development is associated with abnormal YS development (Mess et al., 2017).
Despite these recent discoveries, there is a lack of understanding regarding the consequences of YS dysfunction and the mechanism by which YS absorbs nutrients. It is also important to emphasize that most pregnancy losses occur in the first months of gestation. Hence, this underestimated structure could be involved in some of the causes.

YS-derived stem cells
Several studies presented in the literature describe the isolation of stem cells from the YS (Figure 2), to identify cells related to hematopoiesis or the development of the immune system (Bian et al., 2020;Cumano et al., 1993;Sousa et al., 2017;Weiskopf et al., 2016;Zambidis et al., 2005). However, few studies have been conducted to isolate and culture cells for cell therapy (Table 1). Thus, this review analyzes articles in which hematopoietic cells and MSC lines from the YS tissue of different species were cultured and characterized.

YS-derived hematopoietic stem cells (HSCs)
During the initial development of the embryo, the first blood cells appear in the mesoderm of the YS. In the mesoderm, vascular islands of stem cells are formed, and these primitive multipotent stem cells are later colonized by hemangioblasts. The hemangioblasts can differentiate into either endothelial or hematopoietic cells and renew themselves such that a pool of cells is maintained for the continuous production of all blood cells (Hyttel et al., 2009). This relationship between endothelial cells and hematopoietic cells indicates that they originate from a common precursor, i.e., the hemangioblast (Choi et al., 1998;Hyttel et al., 2009).
The term hemangioblast was most noted when cells from endothelial and hematopoietic lineages were found to share the expression of a number of different genes (Young et al., 1995).
The first blood cells to be formed in the YS are nucleated erythrocytes. These primitive erythrocytes evolve in a few days and give rise to enucleated erythrocytes that enter the circulation (Mikkola & Orkin, 2006). Subsequently, hematopoietic cells are found in the aorta gonad mesonephros region (AGM), which appears after the development of the YS. The hematopoietic cells present in the AGM begin to colonize the fetal liver and later become permanently lodged in the bone marrow. The transition from the YS to the AGM is defined as primitive hematopoiesis, and after the lodging of hematopoietic cells in the bone marrow, it is defined as definitive hematopoiesis (Hyttel et al., 2009;McGrath & Palis, 2005;Mikkola & Orkin, 2006). These cells constitute the hematopoietic microenvironment existing in the marrow cavities of long and flat bones as well as in the spleen, liver, lymph nodes, and thymus (Robb et al., 1995;Shivdasani et al., 1995).
The term hematopoiesis can be defined as the ability to generate definitive mature cells through primitive cells (Hyttel et al., 2009;McGrath & Palis, 2005;Mikkola & Orkin, 2006). The hematopoietic system is an integrated network of cells that initiates the continuous cycle of differentiation of a small population of HSCs, which are pluripotent and produce all the heterogeneous functional cells of the blood and the immune system, thereby supporting a monophyletic theory of blood origin (Gasper, 2000). The HSCs are the only cells in the hematopoietic system that exhibit extensive proliferative potential and the ability to continuously differentiate into all cells of the lymphohematopoietic system until death (Gasper, 2000;Herzog et al., 2003).
HSCs are defined as cells with a high capacity for self-renewal and proliferative potential, which allow for differentiation into progenitor cells of all blood cells and the reconstitution of the hematopoietic population from a single cell. They comprise 0.05% to 0.1% of the human bone marrow and circulating hematopoietic cells (Grotto & Noronha, 2003). HSCs can differentiate into all elements of the blood (red blood cells, leukocytes, and platelets) while maintaining the capacity for self-renewal that allows the maintenance of a sufficient number of stem cells for the production of these elements throughout an individual's life.
Owing to the plasticity of the hematopoietic system, many studies have been conducted on HSCs. Hematopoietic cells in the YS have previously been described in species, such as mice, goats, and bovines. Yamasaki et al. (2011) characterized the population of HSCs in the YS of rat embryos through the expression of CD45 and c-kit (CD117) markers. Gestational ages were measured based on the crown-rump length. High expression of CD117 and low expression of CD45 were noted at E9.5 to E14.5, and these cells could form colonies. The cell culture protocol used has been described briefly. The YS of embryos between E9.5 and E14.5 was excised from ICR mice and digested with 1 mg/mL dispase II for 20 min at 37 °C. The isolated cells were added to Hank's balanced salt solution containing 10% (v/v) fetal calf serum and 250 μg/mL DNase I. The expression levels of the CD45 and c-kit markers were determined. Pessolato et al. (2012) microscopically analyzed the YS of sheep and noticed that it comprised three layers, i.e., endoderm, mesoderm, and the mesothelium which included mesenchymal cells and various blood islands that are surrounded by endothelial cells. They identified and characterized hematopoietic cells from the sheep YS (gestational age 14 to 25 days). In culture, the HSCs exhibited non-adherent morphology and formed colonies in the methylcellulose matrix the day after they were plated. The expression of LMO2 at gestational day 25 was observed in the vitelline membrane. Oliveira et al. (2017) characterized the hematopoietic cells of the YS. The bovine embryos were divided into different groups based on the gestational stages (25 to 50 days of gestational age). In culture, hematopoietic cells showed supernatants, exhibited the formation of cell clusters, and the more advanced gestational age groups could not be maintained in culture for an extended period. After cryopreservation, these cells exhibited the same morphology as that before freezing and could form colonies after 14 days when plated in a methylcellulose matrix. Flow cytometry revealed the expression of the hematopoietic markers CD34, CD90, and CD117, and decreased expression of CD117 and CD34 in the more advanced gestational age groups. qPCR revealed the presence of hematopoietic progenitor genes, GATA3 and LMO2 .
Cell culture of bovine and sheep YS follows the same protocol. The YS is washed with phosphatebuffered saline-L-without calcium or magnesium-supplemented with antibiotics (5% penicillinstreptomycin) and macerated enzymatically with 0.5% collagenase IV for 1 h. Subsequently, the enzymes are inactivated by the addition of the culture medium, Stem Pro®-34 SFM (serum-free medium). The cells are plated in 24-well plates and incubated at 37 °C with 5% CO 2 and relative humidity at approximately 80%.

YS-derived mesenchymal stem cells
Mesenchymal stem cells (MSCs) were initially described by Miao et al. (2006) as cells adhering to plastic in vitro that had the ability to differentiate into different cell lines of the mesodermal line upon stimulation (Dominici et al., 2006). In addition to the ability to differentiate, these cells are of great importance in regenerative medicine owing to their ability to mediate the immune response in injured tissues, e.g., immunomodulation for tissue regeneration (Miao et al., 2006). However, their characterization and clinical use remains challenging (Rossi & Bonfim, 2020).
Initially, MSCs were derived from bone marrow; however, it is known that this cell population is small and decreases with age. Placental attachments, such as the chorion and the amniotic membrane serve as well-characterized alternative sources of MSCs (Ambrósio et al., 2020;Jones & Jauniaux, 1995); however, few studies have investigated the culture of MSCs derived from the YS (Jang et al., 2013).
Isolation of MSCs from placental attachments is simpler because of the ease of obtaining these cells from extraembryonic structures, as this material is discarded during delivery (Filioli Uranio et al., 2011). In addition, the MSCs from placental attachments have an immunomodulatory capacity and can be transplanted (Weiss et al., 2008). MSCs derived from carnivore YS are highly plastic and are able to propagate in culture without any alterations in morphological and phenotypic characteristics or the loss of the ability to differentiate into multiple strains under specific conditions, thereby demonstrating a high potential for cell therapy (Motta, 2019;Wenceslau et al., 2011) Two protocols have been developed to isolate canine YS-derived MSCs. Wenceslau et al. (2011) cultured fragments of YS in three different media in a controlled environment (37 °C and 5% CO 2 ) to observe which would be more appropriate for cell proliferation. The solution containing α-minimum essential medium (α-MEM) supplemented with 15% fetal bovine serum (FBS), 1% glutamine, antibiotic (ATB) solution, and non-essential amino acids (NEAA) was demonstrated to be more efficient than the other media. After characterization, these stem cells were found to have MSC-like morphology, with many organelles and irregular membranes with expansive growth; they were also able to differentiate into chondrocytes and osteocytes and form colonies. Giacomini et al. (2017) and Motta (2019) used collagenase IV to isolate the MSCs from the tissue by cultivating the cells for 1 h in α-MEM supplemented with 15% FBS, 1% glutamine, ATB solution, and NEAA. These cells displayed an expansion in growth after the fourth day of culture, showing peak growth at the fifth passage, and were positive for the membrane marker, CD105, and negative for CD34 and CD45, which are characteristic markers of MSCs. In addition, these cells could develop colonies and differentiate into three cell lines, besides being 70% viable after freezing (Motta, 2019). To date, no study has been conducted on the isolation of feline YS-derived MSCs. Franciolli et al. (2020) demonstrated that is possible to isolate cells from the YS tissues of equine animals for up to day 40 of gestation. The process of isolation consists of mechanically macerating the YS tissue and culturing in presence of 1 mL FBS for 24 h at 37 °C in an atmosphere of 5% CO 2 . Thereafter, 5 mL of α-MEM supplemented with 20% FBS, 1% NEAA, 1% L-glutamine, and 1% ATB was added. The cells were cultured until passage 10 and had MSC-like morphology, could form colony-forming units, adhered to plastic, and could be differentiated into adipocytes, osteocytes, and chondrocytes. In addition, the cells expressed CD45, CD34, CD105, OCT3/4, Nanog, and Stro-1. The immunophenotypic diversity is explained by the isolation method selected by the authors, where selection between hematopoietic and mesenchymal cells was not performed. Bertassoli et al. (2015) isolated and characterized MSCs derived from the swine YS. The cells were collected from YS tissue at day 30 of gestation. The tissue was macerated and enzymatically processed with trypsin 2.5% for 5 min. After isolation, the cells were cultured in Dulbecco's modified Eagle medium, supplemented with 15% FBS, 2% NEAA, 3% L-glutamine, 280 mM HEPES, 0.5% sodium bicarbonate (NaHCO3), and 1% ATB. The cells were incubated at 37 °C in an atmosphere containing 5% CO 2 and cultured until passage 6. The cells exhibited MSC-like morphology and were positive for the CD90, CD105, CD117, vimentin, Stro-1, Oct-4, vascular endothelial growth factor, beta-tubulin, cytokeratin, Nanog, and PCNA and were negative for CD45 (Bertassoli et al., 2015;Kolf et al., 2007). In addition, the potential for chondrogenic, adipogenic, and osteogenic differentiation has been successfully demonstrated in stem cells obtained from swine YS (Seifert, 2014). Mançanares et al. (2015) developed the protocol to isolate, culture, and characterize MSCs from bovine YS. The YS was collected between days 20 and 50 of gestation. The tissue was isolated using a mechanical and enzymatic process (collagenase type IV for 5 min). After isolation, the cells were cultured in α-MEM, supplemented with 10% FBS, 1% NEAA, 1% BME amino acid solution, 1% β-mercaptoethanol, and 1% ATB and incubated at 37°C in an atmosphere containing 5% CO 2 . The cells were cultured until passage 11. From the sixth day of culture, the cells could form colony-forming units and presented MSC-like morphology.
Stem cells derived from bovine YS were positive for CD90 and CD105 and negative for CD79, CD44, and CD45, characteristic of MSCs (Kolf et al., 2007;Mançanares et al., 2015) and could be differentiated into osteocytes, chondrocytes, and adipocytes. In addition, the isolated stem cells could not form teratomas when transplanted into nude immunodeficient mice (BALB/c-Nu), making these cells suitable for use in regenerative therapies without the risk of teratoma formation. Furthermore, the YS MSCs of bovine embryos exhibited spontaneous formation of tubular structures in vitro similar to blood vessel-like structures without growth factors; expression of vascular endothelial growth factor revealed that the YS may play an important role in vascular development (Mançanares et al., 2019).
In humans, because the YS regresses during pregnancy, studies commonly evaluate the presence of MSCs in fetal attachments collected from other membranes, such as the amnion and chorion (Miao et al., 2006;Soncini et al., 2007). It is well established that cells cultured from the human placenta show differentiation capacity in osteogenic, chondrogenic, adipogenic, myogenic (Soncini et al., 2007), and neurogenic (Pop et al., 2015) strains. Miao et al. (2006) performed immunophenotypic characterization using flow cytometry, and found that cells derived from the amnion and chorion were CD44+, CD105+, CD29+, CD19-, CD106-, CD45-, CD34-, and HLA-DR-. Kadam et al. (2010) characterized the cells derived from the human chorion and found that they were CD44+, CD105+, CD117+, CD10-, CD34-, CD45, and CD166-, and revealed the ability of these cells to differentiate into cells similar to pancreatic islets. Wang et al. (2008) collected YS from human fetuses (between 25 and 40 days from volunteers who had opted to terminate the pregnancy). Each sample was dissected in a culture plate containing cold phosphate-buffered saline (PBS) under a microscope. For enzymatic dissociation of the tissue, collagenase type I (0.1%) was used in PSB with 20% FBS for 1 h at 37 °C. The dissociated cells were plated at a concentration of 5 × 10 3 cells/cm 2 in α-MEM supplemented with 10% FBS, 1% ATB, and 5 ng/mL bFGF. The cells were incubated at 37 °C in an atmosphere containing 5% CO 2 , and after 48 h, the non-adherent cells were removed. When the cells reached a confluency of 70-80%, they were detached from culture plates using trypsin/0.25% EDTA. The cells presented MSC-like morphology, as well as the ability to adhere to plastic and expand in the culture plate. With respect to differentiation into mesoderm strains, differentiation was observed only for the adipogenic and osteogenic lineages, and not for in vitro chondrogenic strains.
In addition to research on domestic animals and humans, research on MSCs in new animal models, including unconventional species, such as wild animals, can generate promising results with respect to cell dynamics in vitro and in vivo, which represents an important advancement in cell culture and use. Favaron et al. (2014) studied the culturing of stem cells derived from the YS of the New World mouse (Necromys lasiurus), a rodent from Latin America. The YS was collected from 15-16-day old fetuses and placed in culture plates after cell isolation, at 37 °C in an atmosphere of 5% CO 2 . The cells were cultured in Dulbecco's modified Eagle medium-high glucose supplemented with 10% FBS, 1% antibiotic, 1% L-glutamine and 1% NEAA. The culture medium was changed every 3 days and the cells were maintained until 70% confluence. The cultured cells remained in fibroblastic morphology and showed growth until P4, entering senescence and reaching P6. In addition, flow cytometry revealed that cells derived from the YS of the New World mouse were positive for CD90, CD105, CD73, and CD117 and did not express CD34 and CD45. Moreover, cultured cells could differentiate into adipogenic, chondrogenic, and osteogenic strains.

YS in regenerative medicine and cellular therapy
The ability of MSCs to differentiate into different cell lineages and to secrete diverse bioactive molecules responsible for mediating the immune response, i.e., immunomodulation has fueled interest in MSCs (Abumaree et al., 2017).
MSCs can stimulate cells of the immune system, both by direct cell-cell contact and through modulation of inflammation. These cells induce the release of extracellular vesicles with paracrine capacity and secrete growth factors, cytokines, and other bioactive molecules, all of which result in immune modulation (Crivelli et al., 2017;Özen et al., 2012;Trohatou & Roubelakis, 2017). It is known that MSCs can regulate the expression of anti-inflammatory molecules and growth factors related to angiogenesis and reduction of apoptosis; they can negatively regulate pro-inflammatory cytokines .
The secreted factors and their quality depend on the physiology of the donor; however, these factors are mainly observed in fetal tissue-derived cells. To our knowledge, no study has assessed the immunomodulation capacity of MSCs derived from the YS; however, it is known that MSCs can inhibit the proliferation of T lymphocytes, as well as downregulate the expression of cytokines IL-2, IL-12, TNF-α, and IFN-γ and increase the release of IL-10, IL-4, IL-6, and IL-17 from stem cells derived from the human placenta (Abumaree et al., 2017). MSCs derived from adipose tissue and bone marrow have also been demonstrated to perform immunomodulation similar to cells derived from the placenta; however, cells in fetal membranes exhibit higher levels of cytokines IL-2, IL-4, and IL-13 (Lee et al., 2012).
In addition, MSCs derived from the canine YS were recently used in cell therapy in dogs with hip dysplasia, where they ameliorated the symptoms (Moyle et al., 2019). This model exhibits remarkable similarities with humans compared to other traditional models, being predominantly used for research in cell transplantation because it exhibits stem cell kinetics similar to those observed in humans (Bentin-Ley et al., 1994).

Conclusion and future directions
Although few therapeutic studies have been conducted to date with stem cells derived from placental attachments, MSCs from dogs and cats are commonly used as models for the treatment of several pathologies, such as osteoarthritis (Guercio et al., 2012), spinal cord injury Penha et al., 2012), autoimmune diseases (Tyndall & Uccelli, 2009), renal dysfunction (Quimby et al., 2016;Vidane et al., 2017), Duchenne muscular dystrophy (Hyzewicz et al., 2017), and heart disease (Kim et al., 2010). Thus, stem cells derived from the YS can serve as an important source for cell therapy in different species.

Ethics statement
Not applicable (review article).

Conflict of interests
No conflict of interest.

Authors' contributions
PAFP, VMP, LCBM, MFA, TGS, VCO, LCM -Writing, Review and Editing manuscript. CEA -Acquisition of the financial support for the project leading to this publication, Review manuscript.

Availability of complementary results
The work was carried out at Laboratório de cultivo de células tronco e terapia gênica, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.