Cross-section of the amniotic sac stained with hematoxylin–eosin. On the left hand, subdivisions of amniotic sac: amnion, intermediate layer, and chorion (magnification ×40). On the right hand, subsections of amnion: epithelium, basement membrane, and mesenchymal layer (magnification ×200)
Amnion comprises three layers (Fig. 17.1): uniformly organized, a single layer of epithelium derived from ectoderm , the basement membrane and a mesenchymal layer. The mesenchymal layer also can be subdivided into the compact layer, the fibroblast layer, and an intermediate layer (zona spongiosa) .
The HAM protects against infections, traumas, and toxins. It acts as a filter and a preventive shock  because of their mechanical properties. It has no nerves, muscles or lymph vessels  and is strong, elastic, transparent, and semipermeable. It contains different types of collagens (I, III, IV, V, and VI), which provide resistance to rupture  and, also contains fibronectin, nidogen, laminin, proteoglycans and hyaluronan, as well as growth factors [3, 7, 8].
HAM could to be suitable for allotransplantation and regenerative medicine. There are several advantages which make it suitable: procurement of a high cell number, easily to obtain because HAM is usually discarded after parturition, and their use is within the legal and ethical framework. Also, HAM reduces pain and inflammation, inhibits scarring and shows little or no immunogenicity. It seems to be immune-privileged, and possess antimicrobial, antitumorigenic, antifibrotic and antiangiogenic characteristics. It does not represent transplantation risk, it enhances wound healing and epithelialization and acts as an anatomical and functional barrier [9–14].
It has been suggested that the amniotic membrane may retain a reservoir of stem cells throughout pregnancy coming from embryonic epiblast cells, prior to gastrulation .
HAM contains two different cell types (Fig. 17.2): human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs), from different embryological origins [16, 17]. Both populations show similar cell surface receptor expression pattern but many differences with regard to cell shape (Fig. 17.2c, d) and cell localization (Fig. 17.1) [18, 19]. hAECs are located in the epithelium layer (Fig. 17.1, empty arrowhead) and hAMSCs in the mesenchymal layer (Fig. 17.1, arrowhead) .
Identity and morphology of HAM-isolated cells. (a) Human amniotic mesenchymal stem cells (hAMSCs) were negative for cytokeratin immunostaining. (b) hAECs showed positivity for cytokeratin immunostaining (magnification ×400). (c) Morphology of cultured hAMSCs isolated from amniotic stroma. (d) Morphology of cultured hAECs isolated from amniotic epithelium (magnification ×100)
Using immunohistochemical stains we can distinguish the identity of both the cell types (Fig. 17.2a, b), because cytokeratins are epithelial markers only expressed on cultured hAECs but not on mesenchymal ones . hAECs forms a continuous monolayer of epithelium derived from embryonic ectoderm, in contact with the amniotic fluid (Fig. 17.1) . hAMSCs, which underlies amnion epithelium , are cells sparsely distributed, derived from embryonic mesoderm (Fig. 17.1) .
2 Isolation and Culture of Mesenchymal Stem Cells from Human Amniotic Membrane
Both hAECs and hAMSCs can be isolated easily from the epithelial and mesenchymal regions of the amnion. Different methods to isolate HAM-derived cells have been published [14, 16, 20, 23–25].
First of all, the amniotic sac is obtained from scheduled cesareans, avoiding contamination through the birth canal. Then, the amniotic membrane is separated mechanically from the chorion, through the spongy layer  and washed to remove the red blood cells. All protocols are based only in the use of digestive enzymes to separate the two cell types [14, 16, 23–25], except the Barbati et al.’s protocol , which introduce a second mechanical separation (epithelium from mesenchymal layer) to avoid the cross-contamination between cells. Some of these authors [14, 16, 25] suggest a first trypsin digestion to release hAECs from the HAM and a subsequent incubation to obtain the hAMSCs, using other digestive enzymes such as dispase, trypsin and/or collagenase, alone or combined with DNAse. However, Bacenkova et al. and Soncini et al. [23, 24] isolated first the hAMSCs with those digestive enzymes and then isolated hAECs through a trypsin digestion (Fig. 17.3). These protocols avoid the reduction of hAMSCs in the quantitative cellular yield at isolation and the contamination of hAECs with hAMSCs .
Diaz-Prado et al.  compared two previously published protocols [16, 23] for the isolation of hAMSCs, with different digestive enzymes and digestion in different periods of time. This quantitative study showed that with Soncini’s protocol it was obtained an increase in the hAMSCs isolation yield with regard to Alviano’s protocol. Also, the former protocol allowed the isolation and expansion in a very short time of a larger number of cells. Procurement of cells in a ready and rapid availability is one requirement of a source of MSCs for it to be considered for cell transplantation.
HAM isolated cells can be grown in Dulbecco’s modified Eagle’s media (DMEM) or a similar culture medium (e.g., DMEM:F12), supplemented with fetal bovine serum (FBS) or fetal calf serum (FCS), glutamine and antibiotic–antimycotic, and seeded into culture flasks. Both populations should be expanded at 37 °C, in a humidified 5 % CO2 atmosphere. Although these culture mediums are widely used in basic research, for clinical applications it is necessary to have the culture and expansion under xenobiotic-free conditions for a good manufacturing practice, which could lead to differences in expression markers, capacity to differentiate and other changes in their characteristics .
There is a contradiction with the passage number at which hAMSCs suffer changes. Some authors  founded that their proliferation stops beyond passage 5, others could expanded them in vitro at least 15 passages [16, 23]. Although phenotype of hAMSCs seems to be maintained from passage 0 to passage 9 , Fatimah et al.  showed that after serial passages, stemness gene expression decreases. However, Bailo et al. [17, 29] agreed that hAMSC’s immune-inhibitory properties were not affected by serial passaging.
3 Characterization of Mesenchymal Stem Cells from Human Amniotic Membrane
The International Society for Cellular Therapy proposed three criteria to define all types of stem cells: self-renewal, multipotency, and the ability to reconstitute in vivo a tissue. Because the absence of specific markers for MSCs, there are some additional requirements for their identification, which include: adherence to culture flask plastic, fibroblast-like morphology, prolonged capacity for proliferation, the capacity to differentiate in vitro into cells of mesodermal lineage, expression of CD90, CD73, CD105 and do not express CD45, CD34, CD14 or HLA-DR .
The properties which make the amnion successful for transplantation and regenerative medicine are given by the inherent cell characteristics:
Amnion cells seem to be immune-privileged [12, 14]. They express at a very low level HLA A-B-C, corresponding to class I of the major histocompatibility complex (MHC), and do not express the HLA-DR and HLA-DQ, corresponding to class II of the MHC [3, 15, 17, 21, 26, 31]. Furthermore, amnion cells secrete HLA-G (class I MHC) and Fas ligand [26, 32], immunosuppressive factors. They have also been shown to inhibit the lymphocyte proliferation response [17, 29].
HAM-isolated cells secrete interleukin (IL)-1 receptor antagonist, activin A, tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) and IL-10, antiangiogenic and anti-inflammatory proteins which are deposited within the amniotic membrane stroma .
hAMSCs show plastic adherence and fibroblast-like growth usually observed in MSCs from bone marrow. It is possible to obtain a population of adherent mesenchymal cells with a fibroblastic-like morphology, after 3–4 weeks of culture (Fig. 17.2). Meanwhile, hAECs are small-size cells which exhibit cuboid morphology (typical epithelial) and grow in culture into a tightly packed monolayer .
Studies of transmission electron microscopy showed that hAMSCs present dispersed mitochondria, glycogen lakes, and stacks of rough endoplasmic reticulum cisternae. Characteristics of higher specialization were absent, such as the presence of assembled contractile filaments, prominence of endocytotic traffic, and junctional communications .
Immunophenotypic characterization of hAMSCs demonstrates the presence of the common and well-defined human MSC markers (Fig. 17.4) (CD90, CD44, CD73, CD166, CD105, and CD29), described for bone marrow. It also demonstrates the absence of the hematopoietic markers (CD34 and CD45) and monocyte (CD114), macrophage (CD11) and fibroblast markers [26, 35, 36]. Other markers present in hAMSCs are CD13, CD10, CD49c, CD49d, CD49e, CD54, CD271 low, CD349, CD140, CD324, and E-cadherin .
Graphic representation of hAMSCs surface markers expression. It is represented typical (CD29, CD44, CD73, CD90, CD105, CD166, CD117) and non-typical (CD34) markers of MSCs, and pluripotent markers (STRO-1 and SSEA-4)
hAMSCs express specific transcription factors for pluripotential stem cells such as Oct4 (octamer binding protein 4), NANOG, SOX2 (SRY-related HMG-box gene 2) and REX-1 [21, 37–39]. They may be considered as superior in their differentiation and proliferation capacity to adult MSCs due to their higher OCT4 mRNA levels . These cells are also positive for embryonic stem cell markers as stage-specific embryonic antigen 3 and 4 (SSEA-3, SSEA-4), tumor rejection antigen (TRA)-1-60 and TRA-1-80 .
4 Multilineage Differentiation Potential of Mesenchymal Stem Cells from Human Amniotic Membrane
Koizumi et al.  determined that hAMSCs are capable of secreting several growth factors that support angiogenesis and tissue remodeling, and decrease inflammation. These growth factors include: epidermal growth factor (EGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and basic fibroblastic growth factor (bFGF). The expression of growth factors related to different cell types gives us an idea of their pluripotency. Different authors demonstrated that hAMSCs can be induced to differentiate, in vitro, into tissues from all three germ layers: endoderm, mesoderm, and ectoderm [15, 16, 41].
hAMSCs grown in a specific-cell differentiation medium were capable of expressing specific cell markers. It was found that hAMSCs expressed typical neuronal markers (ectodermal lineage), Nestin, Musashi-1, neuron-specific enolase, neurofilament medium, microtubule-associated protein [MAP]-2 and Neu-N, and the typical glial marker GFAP (glial fibrillary acidic protein) [22, 42–44].
Alviano et al. and Fatimah et al. [16, 25] found that these cells expressed angiogenic and endothelial markers as FLT-1 (receptor of the vascular endothelial growth factor 1), KDR (receptor of the vascular endothelial growth factor 2), I-CAM-1/CD54, CD34, von Willebrand factor (vWF), PECAM-1/CD31 (platelet-endothelial adhesion molecule-1), and endothelial nitric oxide synthase (eNOS). It was found that these cells were capable of myogenic differentiation  due to the expression of MyoD and myogenin. Also, hAMSCs grown in a differentiation-specific medium were capable to express the cardiac-specific factor GATA4, cardiac-specific genes atrial myosin light chain (MLC)-2a, ventricular MLC-2v, and the cardiac troponins cTnl and cTnT . Moreover, the expression of bone morphogenetic proteins (BMPs) and their receptors BMPR-IA and BMPR-IB indicates the capacity to induce the differentiation of mesenchymal cells into osteoblast and chondroblast lineages. The expression of oligomeric matrix protein (COMP), SRY-related HMG box family (SOX) as SOX5, SOX6, SOX9, and the presence of collagen type II and aggrecan (Fig. 17.5) in the extracellular matrix of differentiated cells indicates the capacity to differentiate into a chondrocyte-like phenotype ; presence of calcium deposits (Fig. 17.5) and expression of osteopontin (OP) and alkaline phosphatase (ALP) indicates the capacity to differentiate into osteoblast-like cells. hAMSCs differentiation to another mesodermal lineage, adipocytes, was also detected by the presence of lipid drops (Fig. 17.5) and the expression of adiponectin (APM1), fatty acid binding protein (FABP4) and lipoprotein lipase (LPL) .