**1.3. Regenerative medicine and placenta**

both maternal and fetal cells coexist. Placenta performs functions of metabolic exchange and endocrine regulation between two genetically distinct individuals, the mother and the fetus,

The term placenta derives from the latin and means "flat cake" because of its discoid shape. At the end of pregnancy, it is about 15–20 cm in diameter, 2–3 cm thick, and 500 g in weight,

The placenta is constituted by structures of fetal origin, such as, the placental disk, the fetal membranes, divided in amniotic and chorionic membranes, and the umbilical cord. The placenta is also composed by a membrane of maternal origin termed the decidua that originates from the endometrium. The functional unit of the placenta is the chorionic villosity that forms

Placenta development is a continuous process that starts during early embryological stages, even before gastrulation occurs. Four to five days after fecundation, the morula (solid mass of cells called blastomers) has reached the uterus. The appearance of a fluid-filled inner cavity marks the transition from morula to blastocyst and is accompanied by cellular differentiation: the surface cells become the trophoblast (giving rise to extraembryonic structures, including the placenta and the umbilical cord) and the inner cell mass gives rise to the embryo [3]. Just before the implantation into the endometrium, the internal cell mass or embryoblast, goes through important changes such as cellular reorganization that gives place to a top layer, the epiblast and a bottom layer named hypoblast or primitive endoderm. Some extraembryonic tissues such as the amnion derive from the epiblasts that delimit the amniotic cavity that hosts the embryo during pregnancy. Because of the increase in production of amniotic liquid during gestation, the amnion will expand, and merge with the trophoblast to give rise to the amnionchorionic membrane. Another of the earliest differentiation events in human embryogenesis takes place in the trophoblast with the development of the external syncytiotrophoblast

**Figure 1.** First stage in the interaction between fetal and maternal blood circulation. The syncytiotrophoblast erodes

while maintaining immunological tolerance between them [1, 2].

228 Stromal Cells - Structure, Function, and Therapeutic Implications

the border between maternal and fetal blood during pregnancy (**Figure 1**).

that is, 1/6 of the fetal weight.

**1.2. Placenta development**

maternal vessels.

Regenerative medicine is an interdisciplinary field within translational medicine whose purpose is to heal or replace damaged tissues or organs as a result of age, illness or trauma. It may involve the transplantation of stem cells that will repair the damaged tissue, stimulate the body's own repair processes or serve as delivery-vehicles for therapeutic agents such as genes, cytokines, or therapeutic drugs.

Stem cells are unspecialized cells that have the capacity to renew themselves or differentiate toward more specialized cells. The proliferation of stem cells is indispensable for the maintenance of the stemness niche. The differentiation is the process by which, under certain physiological or experimental conditions, unspecialized cells are induced to become tissue- or organ-specific cells. The differentiation potential of stem cells is essential during the development of the embryo. In the adult, the main function of stem cells is the maintenance of the tissue homeostasis acting as an internal repair system.

Both embryonic and adult tissues are sources of stem cells with therapeutic potential. However, embryonic stem cells have some limitations in clinical practice, such as ethical concerns, difficulty in obtaining, and tumorigenicity. Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, adipose tissue, skeletal muscle, skin, teeth, heart, gut, liver, and placenta. Though the number of stem cells is very small in many adult tissues, their isolation involves several risks and, once removed from the body, the cells have a limited capacity of proliferation and differentiation, making the generation of large quantities of stem cells difficult.

mobilization of placental HSC to the fetal liver and other developing hematopoietic organs

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The three layers of the placenta, such as the amnion, the chorion, and the decidua, are sources of stem cells. The amniotic layer is composed of a single-cell epithelial layer and a deeper mesodermal layer derived from the epiblast and hypoblast, respectively [23]. The chorion sheet is composed of the inner chorionic mesoderm similar to the mesenchymal region of the amnion and an outer layer of trophoblastic origin. The decidua, the uterine component of the

Amniotic epithelial cells (AEC) are very valuable stem cells for regenerative medicine. They have stem cell molecular markers such as OCT-4, Nanog, SOX-2, and Rex-1 (23). AEC do not have telomerase reverse transcriptase, show a stable karyotype, and do not originate tumors when injected. Amnion does not express MHC class II antigens, so AEC can elude the immune system. AEC can also modulate the immune system through an inhibition of the proliferation

Chorion trophoblastic cells (CTC) represent a mixed and still poorly characterized population of stem cells and there are no reliable methods to isolate them [25], and also, no consistent

Most of stem cells isolated from the placental tissues are cells of mesodermal origin and are named amnion mesenchymal stromal cells (AMSC), chorion mesenchymal stromal cells (CMSC), chorionic villi mesenchymal stromal cells (CV-MSC), and decidua mesenchymal stem cells (DMSC) [9, 27, 28] depending on the layer of origin. Inside the umbilical cord, there is a connective tissue that surrounds the umbilical vein and the two umbilical arteries. This tissue, also known as Wharton's jelly, is a rich source of mesenchymal stromal cells called umbilical cord mesenchymal stem cells (UC-MSC) [29]. They are all considered true mesenchymal stromal cells (MSC), as they meet the three minimal criteria proposed by the International Society for Cellular Therapy [30]. First, placenta-derived MSC exhibit plastic adherence in culture. Second, they express a specific set of cell surface markers, such as CD105, CD73, and CD90, and do not express hematopoietic markers including CD34, CD45 and CD14 or CD11b, CD79a or CD19, and HLA-DR. Third, they have the ability to differentiate in vitro into different mesodermal cell lineages including adipocytes, chondrocytes, and osteoblasts. In addition, AMSC and CMSC are from fetal origin according to the first interna-

Cells with properties of mesenchymal stromal cells have also been isolated from the amniotic fluid (AF) which is used to perform the evaluation of karyotyping and prenatal diagnostic testing. AF is a source of MSC that could be used as autologous cellular therapy for perinatal disorders [32]. These AF-MSC can be easily isolated, have minimal ethical objections, high renewal activity, multiple differentiation capacity, and maintain genetic stability

In this chapter, we will refer to placenta-derived mesenchymal stromal cells as placenta mesenchymal stromal cells (PMSC) regardless of the placenta region where they were isolated.

of T- and B-cells. In addition, AEC inhibit inflammation, as has seen in vitro [24].

within the embryo, such as thymus, spleen, and bone marrow [22].

placenta, is also a source of cells of mesodermal origin.

marking for identifying this population of cells [26].

tional workshop on placenta-derived stem cells [31].

in culture [33].

The placenta is a reservoir of stem cells with several advantages. What makes placenta such an interesting tissue for regenerative medicine? Placenta is spontaneously expulsed at birth, making the use of invasive methods unnecessary as in the case of other sources of adult stem cells. It is considered a medical waste and there are no ethical concerns in its use, unlike using embryonic stem cells [7]. Placenta is a high-yielding source of stem cells compared to other sources such as bone marrow and adipose tissue where the cell recovery decreases with donor age [8]. Versatility and differentiation potential of placental cells is very high probably due to their primitive origin [9]. Furthermore, pregnancy is an example of "tolerated allograft" and placenta is the immunoregulatory organ at the maternal-fetal interface [10]. Placenta is an immunoprivileged organ, and cells isolated from placenta display low immunogenicity in vitro [11] and in vivo [12] when xenotransplanted in immunocompetent animals. The feasibility of placental cells for allogeneic transplantation has been demonstrated [13].

In regenerative medicine, the effects of stem cells are not only restricted to cell or tissue restoration but also to transient paracrine actions. This paracrine action is related to factors produced and secreted by stem cells that will control the injury, modulate the immune responses, and promote self-repair in the surviving injured tissue [14]. Placenta plays a fundamental role in fetomaternal tolerance and this would explain why placenta-derived stem cells have an additional advantage over other stem cells in terms of immunomodulation [15].

Multiple mechanisms underlie maternal tolerance during pregnancy. Fetal and, in particular, placental tissues contribute to its immunoprivileged and immunoregulatory environment. Placental cells are characterized by the absence of MHC class II antigens that normally mediate graft rejection [16]. Placental cells not only express a low level of the highly polymorphic forms of the MHC class I antigens but also express the nonclassical form HLA-G that may play a role in the suppression of immune responses and contribute to maternal-fetal tolerance [17, 18]. Furthermore, through the release of hormones [19], cytokines [20], and soluble forms of MHC antigens, placental cells deviate maternal immune responses toward immune tolerance. Therefore, the cells of the innate immunity of the mother acquire a suppressive profile characterized by a diminished production of pro-inflammatory cytokines. In addition, the B cells and many T cells disappear, leaving the regulatory T cells (Tregs) as the major T-cell subpopulation, with both, immune suppressive and anti-inflammatory characteristics [21].

### **1.4. Placenta-derived stem cells**

Different populations of cells with features of stem/progenitor cells have been isolated from placenta: hematopoietic, epithelial, trophoblasts, and mesenchymal cells.

Placenta is a hematopoietic organ since it harbors a large pool of hematopoietic stem cells (HSC) that possess functional properties of true HSC. Placenta-derived HSC can differentiate into all types of mature blood cells and are able to sustain the hematopoiesis during the life of the embryo. Placental HSC activity declines toward the end of gestation, possibly reflecting mobilization of placental HSC to the fetal liver and other developing hematopoietic organs within the embryo, such as thymus, spleen, and bone marrow [22].

is very small in many adult tissues, their isolation involves several risks and, once removed from the body, the cells have a limited capacity of proliferation and differentiation, making

The placenta is a reservoir of stem cells with several advantages. What makes placenta such an interesting tissue for regenerative medicine? Placenta is spontaneously expulsed at birth, making the use of invasive methods unnecessary as in the case of other sources of adult stem cells. It is considered a medical waste and there are no ethical concerns in its use, unlike using embryonic stem cells [7]. Placenta is a high-yielding source of stem cells compared to other sources such as bone marrow and adipose tissue where the cell recovery decreases with donor age [8]. Versatility and differentiation potential of placental cells is very high probably due to their primitive origin [9]. Furthermore, pregnancy is an example of "tolerated allograft" and placenta is the immunoregulatory organ at the maternal-fetal interface [10]. Placenta is an immunoprivileged organ, and cells isolated from placenta display low immunogenicity in vitro [11] and in vivo [12] when xenotransplanted in immunocompetent animals. The feasi-

bility of placental cells for allogeneic transplantation has been demonstrated [13].

additional advantage over other stem cells in terms of immunomodulation [15].

In regenerative medicine, the effects of stem cells are not only restricted to cell or tissue restoration but also to transient paracrine actions. This paracrine action is related to factors produced and secreted by stem cells that will control the injury, modulate the immune responses, and promote self-repair in the surviving injured tissue [14]. Placenta plays a fundamental role in fetomaternal tolerance and this would explain why placenta-derived stem cells have an

Multiple mechanisms underlie maternal tolerance during pregnancy. Fetal and, in particular, placental tissues contribute to its immunoprivileged and immunoregulatory environment. Placental cells are characterized by the absence of MHC class II antigens that normally mediate graft rejection [16]. Placental cells not only express a low level of the highly polymorphic forms of the MHC class I antigens but also express the nonclassical form HLA-G that may play a role in the suppression of immune responses and contribute to maternal-fetal tolerance [17, 18]. Furthermore, through the release of hormones [19], cytokines [20], and soluble forms of MHC antigens, placental cells deviate maternal immune responses toward immune tolerance. Therefore, the cells of the innate immunity of the mother acquire a suppressive profile characterized by a diminished production of pro-inflammatory cytokines. In addition, the B cells and many T cells disappear, leaving the regulatory T cells (Tregs) as the major T-cell subpopulation, with both, immune suppressive and anti-inflammatory characteristics [21].

Different populations of cells with features of stem/progenitor cells have been isolated from

Placenta is a hematopoietic organ since it harbors a large pool of hematopoietic stem cells (HSC) that possess functional properties of true HSC. Placenta-derived HSC can differentiate into all types of mature blood cells and are able to sustain the hematopoiesis during the life of the embryo. Placental HSC activity declines toward the end of gestation, possibly reflecting

placenta: hematopoietic, epithelial, trophoblasts, and mesenchymal cells.

the generation of large quantities of stem cells difficult.

230 Stromal Cells - Structure, Function, and Therapeutic Implications

**1.4. Placenta-derived stem cells**

The three layers of the placenta, such as the amnion, the chorion, and the decidua, are sources of stem cells. The amniotic layer is composed of a single-cell epithelial layer and a deeper mesodermal layer derived from the epiblast and hypoblast, respectively [23]. The chorion sheet is composed of the inner chorionic mesoderm similar to the mesenchymal region of the amnion and an outer layer of trophoblastic origin. The decidua, the uterine component of the placenta, is also a source of cells of mesodermal origin.

Amniotic epithelial cells (AEC) are very valuable stem cells for regenerative medicine. They have stem cell molecular markers such as OCT-4, Nanog, SOX-2, and Rex-1 (23). AEC do not have telomerase reverse transcriptase, show a stable karyotype, and do not originate tumors when injected. Amnion does not express MHC class II antigens, so AEC can elude the immune system. AEC can also modulate the immune system through an inhibition of the proliferation of T- and B-cells. In addition, AEC inhibit inflammation, as has seen in vitro [24].

Chorion trophoblastic cells (CTC) represent a mixed and still poorly characterized population of stem cells and there are no reliable methods to isolate them [25], and also, no consistent marking for identifying this population of cells [26].

Most of stem cells isolated from the placental tissues are cells of mesodermal origin and are named amnion mesenchymal stromal cells (AMSC), chorion mesenchymal stromal cells (CMSC), chorionic villi mesenchymal stromal cells (CV-MSC), and decidua mesenchymal stem cells (DMSC) [9, 27, 28] depending on the layer of origin. Inside the umbilical cord, there is a connective tissue that surrounds the umbilical vein and the two umbilical arteries. This tissue, also known as Wharton's jelly, is a rich source of mesenchymal stromal cells called umbilical cord mesenchymal stem cells (UC-MSC) [29]. They are all considered true mesenchymal stromal cells (MSC), as they meet the three minimal criteria proposed by the International Society for Cellular Therapy [30]. First, placenta-derived MSC exhibit plastic adherence in culture. Second, they express a specific set of cell surface markers, such as CD105, CD73, and CD90, and do not express hematopoietic markers including CD34, CD45 and CD14 or CD11b, CD79a or CD19, and HLA-DR. Third, they have the ability to differentiate in vitro into different mesodermal cell lineages including adipocytes, chondrocytes, and osteoblasts. In addition, AMSC and CMSC are from fetal origin according to the first international workshop on placenta-derived stem cells [31].

Cells with properties of mesenchymal stromal cells have also been isolated from the amniotic fluid (AF) which is used to perform the evaluation of karyotyping and prenatal diagnostic testing. AF is a source of MSC that could be used as autologous cellular therapy for perinatal disorders [32]. These AF-MSC can be easily isolated, have minimal ethical objections, high renewal activity, multiple differentiation capacity, and maintain genetic stability in culture [33].

In this chapter, we will refer to placenta-derived mesenchymal stromal cells as placenta mesenchymal stromal cells (PMSC) regardless of the placenta region where they were isolated.

### **1.5. Placenta-derived mesenchymal stromal cells**

Mesenchymal stromal cells (MSC) can be isolated from virtually all adult tissues in the body, although not always in large quantities. They are thought to be a precursor cell population capable of reconstituting all the cellular elements that comprise the supportive stromal tissue in each organ [34]. First described in bone marrow as a subset of non-hematopoietic cells [35], they have become the paradigm cell in regenerative medicine. MSC are the most widely studied cell type in both preclinical and clinical trials. The advantages of MSC include ease of isolation and subsequent maintenance in culture, high expansion capacity, high plasticity, and tissue repair activity. The restorative activity of MSC is not necessarily by the replacement of dead or damaged cells, but also, by paracrine actions that mediate immune-regulation and promote cell growth and/or differentiation (**Figure 2**). Besides, MSC do not form teratomas after transplantation, ensuring safety to the host and, their low immunogenicity makes them suitable for allogeneic transplantation. Furthermore, these cells have the ability to migrate to inflammatory microenvironments [36] and tumors [12, 37], where they play an active role inducing many processes, such as angiogenesis and wound healing, mainly in a paracrine manner [38]. This feature provides an important therapeutic advantage to MSC since they can be injected via systemic infusion and can be used as vehicles for the delivery of drugs such as anticancer agents to the tumor site.

reducing the risk of ex vivo senescence influencing gene expression and resulting in aging phenotype [41, 42]. The senescent state needs to be taken into account for quality control of PMSC in cellular therapy. In addition, the clinical efficacy and safety of PMSC could be higher, compared to other sources of MSC, since PMSC are younger cells that have been exposed less time to harmful agents, such as reactive oxygen species (ROS), chemical and biological agents, and physical stressors [43]. Also, PMSC have a limited capacity to grow in culture related to low telomerase activity, which is also lost during proliferation, making them a safe product to be used in regenerative medicine [9]. Moreover, PMSC could be advantageous with respect to migratory properties and homing capacities into damaged tissues. Homing of MSC is basically dependent on the release of chemoattractants by the injured tissue and the expression of chemokine receptors on the MSC membrane. For extravasation into tissue, MSC have to attach to and migrate through the endothelium. Several integrins and other adhesion molecules are known to be expressed on MSC. Dependence on the VLA-4/VCAM-1 (very late antigen-4/vascular cell adhesion molecule-1) axis for MSC adherence to endothelial cells has been demonstrated [44]. PMSC have a higher expression of VLA-4 compared to bone marrow MSC suggesting that PMSC may have enhanced prop-

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**2. Therapeutic applications of placenta mesenchymal stromal cells** 

among others, have been widely evaluated at the preclinical level [9, 46, 47].

**2.1. Use of placental mesenchymal stem/stromal cells in cardiovascular diseases**

Stem cell therapies are expected to provide substantial benefits to patients suffering a wide range of pathologies. The plasticity and pleiotropic properties of PMSC that include immunomodulation and inflammation control, angiogenesis, neuroprotection, and antiapoptosis,

Myocardial infarction (MI) is a major cause of death and disability worldwide. MI occurs when there is an interruption in blood flow to the heart muscle followed by heart ischemia. Since regeneration of heart muscle is virtually absent, damaged myocardium after infarct is replaced by scar tissue leading to reduced cardiac function. PMSC transplantation is a promising strategy to restore cardiac function and reduce myocardial fibrosis in MI due to their

PMSC have the potential to differentiate into cardiomyocytes, and exhibit spontaneous beating under in vitro conditions suggesting that they can therapeutically act in the cardiac repair process [9, 48, 49]. Several groups have investigated the effects of PMSC when transplanted in animal models of MI. PMSC injected into rat hearts after the induction of a MI showed integration into cardiac tissues and in vivo transdifferentiation into cardiomyocytes [48]. The CXCR4 chemokine receptor and its ligand, stromal cell-derived factor (SDF-1)

erties for homing to damaged tissue [45].

**(PMSC) in preclinical models**

angiogenic and immunosuppressive properties.

*2.1.1. Myocardial infarction*

The use of placenta as a source of MSC has several advantages with respect to other adult MSC. Besides the ease of extraction of MSC from the placenta without invasive methods, the isolated MSC represent a more homogeneous and primitive population [9, 39]. The last feature is associated with a higher proliferative rate in culture compared to bone marrow MSC [40]. This fact makes it possible to achieve a greater number of cells in fewer passages

**Figure 2.** PMSC mechanisms of action. PMSC can migrate, home, and differentiate into tissue specific cells to repair injured tissue, transport restorative genes and used as a cellular vehicles of therapeutic agents. PMSC also exert their actions through paracrine effects and have immunomodulatory properties.

reducing the risk of ex vivo senescence influencing gene expression and resulting in aging phenotype [41, 42]. The senescent state needs to be taken into account for quality control of PMSC in cellular therapy. In addition, the clinical efficacy and safety of PMSC could be higher, compared to other sources of MSC, since PMSC are younger cells that have been exposed less time to harmful agents, such as reactive oxygen species (ROS), chemical and biological agents, and physical stressors [43]. Also, PMSC have a limited capacity to grow in culture related to low telomerase activity, which is also lost during proliferation, making them a safe product to be used in regenerative medicine [9]. Moreover, PMSC could be advantageous with respect to migratory properties and homing capacities into damaged tissues. Homing of MSC is basically dependent on the release of chemoattractants by the injured tissue and the expression of chemokine receptors on the MSC membrane. For extravasation into tissue, MSC have to attach to and migrate through the endothelium. Several integrins and other adhesion molecules are known to be expressed on MSC. Dependence on the VLA-4/VCAM-1 (very late antigen-4/vascular cell adhesion molecule-1) axis for MSC adherence to endothelial cells has been demonstrated [44]. PMSC have a higher expression of VLA-4 compared to bone marrow MSC suggesting that PMSC may have enhanced properties for homing to damaged tissue [45].
