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 [26] 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 [20], 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 [20].

Diaz-Prado et al. [27] 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 [28].

There is a contradiction with the passage number at which hAMSCs suffer changes. Some authors [19] 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 [18], Fatimah et al. [25] 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.

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 [30].

The properties which make the amnion successful for transplantation and regenerative medicine are given by the inherent cell characteristics:

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 [34].

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 [31].

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 [21].

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 [16]. 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 [3].

Koizumi et al. [40] 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 [43] 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 [45]. 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 [14]; 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) [18].