Amniotic Fluid and Amniotic Membrane Stem Cells: Marker Discovery

Amniotic fluid (AF) and amniotic membrane (AM) have been recently characterized as promising sources of stem or progenitor cells. Both not only contain subpopulations with stem cell characteristics resembling to adult stem cells, such as mesenchymal stem cells, but also exhibit some embryonic stem cell properties like (i) expression of pluripotency markers, (ii) high expansion in vitro, or (iii) multilineage differentiation capacity. Recent efforts have been focused on the isolation and the detailed characterization of these stem cell types. However, variations in their phenotype, their heterogeneity described by different groups, and the absence of a single marker expressed only in these cells may prevent the isolation of a pure homogeneous stem cell population from these sources and their potential use of these cells in therapeutic applications. In this paper, we aim to summarize the recent progress in marker discovery for stem cells derived from fetal sources such as AF and AM, using novel methodologies based on transcriptomics, proteomics, or secretome analyses.


Introduction
Both amniotic fluid (AF) and amniotic membrane (AM) represent rich sources of stem cells that can be used in the future for clinical therapeutic applications. Ethical concerns regarding the isolation of stem cells from these sources are minimized [1][2][3], in contrary to the issues emerging from human embryonic stem cell (ESC) research [4][5][6]. AF is collected during scheduled amniocenteses between 15th and 19th week of gestation for prenatal diagnosis and the excess of sample can be used for cell sourcing [2,[4][5][6][7][8][9], whereas AM is usually collected during the caesarean sections of term pregnancies [10,11]. Given the heterogeneity of the stem cell populations derived from these sources, the isolation of specific cell types is difficult and requires a detailed phenotypic and molecular characterization of the respective cells. Studies that include omics approaches are fundamental in better understanding the mechanisms of molecular expression of these cells and defining the correct methodologies for their isolation, prior to their use in therapeutic approaches. This paper aims to present the main biological and molecular characteristics of AF-and AM-derived stem cells and also to highlight the recent advances in marker discovery using global methodologies, such as transcriptomics, proteomics, or secretome analyses.
1.1. Amniotic Fluid. AF serves as a protective liquid for the developing embryo, providing mechanical support and the required nutrients during embryogenesis [1,3]. Amniocentesis has been used for many decades as a routine procedure for fetal karyotyping and prenatal diagnosis, allowing the detection of a variety of genetic diseases [1,3,12].
The major component of AF is water; however its overall composition varies throughout pregnancy. At the beginning of pregnancy, the amniotic osmolarity is similar to the fetal plasma. After keratinization of the fetal skin amniotic osmolarity decreases relatively to maternal or fetal plasma, mainly due to the inflow of fetal urine [1]. More interestingly, AF also represents a rich source of a stem cell population deriving from either the fetus or the surrounding 2 Stem Cells International amniotic membrane [1,12]. Additional investigations by several groups have been recently focused on the cellular properties of amniotic derived cells and their potential use in preclinical models [13][14][15][16][17][18] and in transplantation therapies [7,17,[19][20][21][22][23][24].
1.1.1. Amniotic Fluid Stem Cells (AFSCs). The amniotic fluid cells (AFCs) represent a heterogeneous population derived from the three germ layers. These cells share an epithelial origin and are derived from either the developing embryo or the inner surface of the amniotic membrane, which are characterized as amniotic membrane stem cells [12]. The AFCs are mainly composed of three groups of adherent cells, categorized based on their morphological, growth, and biochemical characteristics [12]. Epithelioid (E-type) cell are cuboidal to columnar cells derived from the fetal skin and urine, amniotic fluid (AF-type) cells are originating from fetal membranes, and fibroblastic (F-type) cells are generated mainly from fibrous connective tissue. Both AF-and F-type cells share a fibroblastoid morphology and the dominant cell type appears to be the AF-type, coexpressing keratins and vimentins [1-3, 8, 9, 25-27]. Several studies have documented that human amniotic fluid stem cells (AFSCs) can be easily obtained from a small amount of second trimester AF, collected during routine amniocenteses [2,[4][5][6][7][8][9], a procedure with spontaneous abortion rate ranging from 0.06 to 0.5% [2,28,29]. Up to date, a number of different cultivation protocols have been reported, leading to enriched stem cell populations. The isolation of AFSC and the respective culture protocols were summarized in a recent review by Klemmt et al. [3] and can be categorized as follows: (i) a single step cultivation protocol, where the primary culture was left undisturbed for 7 days or more until the first colonies appear [2,3,[30][31][32], (ii) a two-step cultivation protocol, where amniocytes, not attached after 5 days in culture, were collected and further expanded [3,5,33], (iii) cell surface marker selection for CD117 (c-kit receptor) [3,7,34,35], (iv) mechanical isolation of the initial mesenchymal progenitor cell colonies formed in the initial cultures [9], and (v) short-term cultures to isolate fibroblastoid colonies [36]. The majority of the AFSCs, isolated following these methodologies, shared a multipotent mesenchymal phenotype and exhibited higher proliferation potential and a wider differentiation potential compared to adult MSCs [2, 4-7, 9, 24, 37].

Amniotic Membrane (AM).
The amniotic membrane, lacking any vascular tissue, forms most of the inner layer of the fetal membrane [12,38] and is composed of 3 layers: (i) an epithelial monolayer consisting of epithelial cells, (ii) an acellular intermediate basement layer, and (iii) an outer mesenchymal cell layer, rich in mesenchymal stem cells and placed in close proximity to the chorion [12,38]. AM was used in clinic for many decades for wound healing in burns, promoting epithelium formation and protecting against infection [39,40]. Recently, the use of AM has been evaluated as a wound dressing material for surgical defects of the oral mucosa [41], ocular surface reconstruction [40,42], corneal perforations [43,44], and bladder augmentation [45].

Amniotic Membrane Stem Cells (AMSCs).
Amniotic membrane stem cells (AMSCs) include two types, the amniotic epithelial cells (AECs) and the amniotic membrane mesenchymal stem cells (AM-MSCs) derived from the amniotic epithelial and the amniotic mesenchymal layers, respectively [12,46]. Both cell types are originated during the pregastrulation stages of the developing embryo, before the delineation of the three primary germ layers and are mostly of epithelial nature [38,47]. A variety of protocols have been established for AECs and AM-MSCs isolation, primarily based on the mechanical separation of the AM from the chorionic membrane and the subsequent enzymatic digestion [47][48][49][50]. AM-MSCs exhibited plastic adherence and fibroblastoid morphology, while AECs displayed a cobblestone epithelial phenotype. AM-MSCs shared similar phenotypic characteristics with the ones derived from adult sources. More interestingly, AM-MSCs, similarly to AF-MSCs, exhibited a higher proliferation rate compared to MSCs derived from adult sources [12,51] and a multilineage differentiation potential into cells derived from the three germ layers [27].
It was also reported that amniocyte cultures contain a small population of CD117 (a tyrosine kinase specific for stem cell factor present primarily in ESCs and primordial germ cells) positive cells that can be clonally expanded in culture [7]. The differentiation properties of CD117 + AFS were tested for the first time in vivo, proving in this way their stem cell identity [7]. Experimental evidence suggested that AFSCs are derived from spindle-shaped fibroblastoid cells [10].
In an attempt to analyze the AFSCs subpopulations, our group recently identified two morphologically distinct populations of AFSCs of mesenchymal origin, with different proliferation and differentiation properties, termed as spindle shaped (SS) and round shaped (RS) [9]. Both subpopulations were expressing mesenchymal stem cell markers at similar levels. However, it was identified that SS Stem Cells International 3 colonies expressed higher levels of CD90 and CD44 antigens compared to RS colonies [9].
Recent studies provided evidence that a diversity of metabolic enzyme expression in the amnion cells is involved in metabolic and genetic syndromes, and thus, their detection might be important for prenatal diagnosis. A more detailed analysis for determining specific metabolic enzymes present in AFSCs was reported by Oh et al. [59]. Ninety-nine proteins had been identified, such as carbohydrate handing enzymes, amino acid handling enzymes, proteins of purine metabolism, and enzymes of intermediary metabolism [59,60].
A proteomic analysis was also performed on different culture passages of CD117 + AFSCs, exhibiting variations in protein expression that mainly occurred in early passages [35]. Twenty-three proteins were differentially expressed between early and late passages with the most sticking downregulated proteins, the Col1, the Col2, the vinculin (Vcl), the CRABP II, the stathmin (STMN1), and the cofilin-1 (CFL1). In contrast, TAGLN and Col3 are increased during passages [35]. Proteins that showed dysregulated levels along the passages were the 26S protease regulatory subunit 7 (PSMD7), the ubiquitin carboxyl terminal hydrolase isoenzyme L1 (UCH-L1), the heterogeneous nuclear ribonuclear protein H (hnRNP H), and the TAR DNA-binding protein 43 (TDP-43) [35].
In 2007, the proteomic map of human AF-MSCs was constructed and directly compared to the one derived from BM-MSCs [2]. 261 different proteins were identified in AF-MSCs with the majority of the proteins localized in the cytoplasm (41%), whereas others were found in the endoplasmic reticulum (8%), nucleus (13%), mitochondria (12%), ribosomes (1%), cytoskeleton (6%), cytoplasm and the nucleus (5%), and secreted (2%) proteins [2]. AF-MSCs expressed a number of proteins related to proliferation and cell maintenance, such as ubiquilin-1 (UBQLN1), which is known to control cell cycle progression and cell growth, the proliferation associated protein 2G4 (PA2G4), a nucleolar growth-regulating protein, the secreted protein acidic and rich in cysteine (SPARC), which is regulated during embryogenesis and is involved in the control of the cell cycle and cell adhesion, and the enhancer of rudimentary homolog (ERH) that also regulates cell cycle [2]. TAGLN and galectin 1 (Gal 1), both present in stem cells and related to differentiation, were also abundantly expressed in AF-MSCs. Other proteins expressed in high levels in AF-MSCs were related to (i) development, such as Deltex-3-like (DTX3L), and (ii) cytoskeletal organization and movement, such as CFL1, the coactosin-like protein (CLP), and the enabled protein homolog (Enah). As expected, Vim was also expressed in high amounts in AF-MSCs. In this study, a detailed comparison of the common identified proteins in AF cells [58] and AF-MSCs was also described [2].

Amniotic Membrane Stem Cells.
A detailed approach for studying human AM proteins was described by Hopkinson et al. [61]. In this study, the authors performed a proteomic analysis of AM samples that were prepared for human transplantation, by using 2-DE gels. The wash media from the AM samples were also examined and the secreted proteins were identified. Proteins detected in both AM and the wash media suggested that partial protein release had occurred. These proteins were mostly soluble cytoplasmic proteins and were categorized according to their subcellular localization and function [61]. One example of the most abundant and consistent proteins in AM is THBS1 which is reported to play role in wound repair, inflammatory response, and angiogenesis [62,63]. Mimecan (also named osteoglycin/OGN) is another protein detected in AM that represents a small leucine-rich proteoglycan, found in the ECM of connective tissue. Mimecan is reported to maintain the tensile strength and hydration of the tissue [61,[64][65][66]. In addition, the larger form of mimecan was expressed in AM cells and was susceptible to proteolytic cleavage [65]. TGF-β-induced protein ig-h3 (βIG-H3), an ECM adhesive molecule acting as a membrane-associated growth factor during cell differentiation and wound healing, and intergrin α6 (CD49f), a component of α6β4 integrin, were also present in significant amounts in AM cells [61,67,68]. It is well known that α6β4-βIG-H3 interaction plays an important role in mediating cell adhesion and wound repair signaling pathways [69].
Another important study by Baharvand et al. [70] was focused on the analysis of epithelium-denuded human AM showing both quantitative and qualitative differences compared to nontreated AM [61]. They investigated the proteome of the human AM epithelium, which was used as a limbal stem cell niche for treating ocular surface reconstruction [71,72]. 515 spots were detected in all the 2-DE gels and 43 proteins were identified using MALDI TOF/TOF MS in AM. The most abundant proteins were different isoforms of lumican (LUM) and OGN, both members of the proteoglycan (PG) family. In particular, OGN might play role in many biological processes including cell growth, angiogenesis, and inflammation [66]. Other proteins detected included collagen VI α-1/α-2 (Col6a1/Col6a2), fibrinogen beta chain (FGB), transglutaminase 2 isoform A (TGM2A), b-actin variant (ACTB), 70 kD heat shock protein 5 (HSPA5), nidogen 2 (NID2), CD49f, βIG-H3, and tubulointerstitial nephritis (TIN) [70]. Some of the proteins identified in this study were also related to extracellular matrix (ECM). Among the detected ones, fibronectin (FN), laminins, and collagen IV (Col4) and VII were reported to promote epithelial adhesion and migration [73,74].

Secretome
Recently, significant progress has been made regarding the analysis of the secreted proteins from AFSCs. It has been documented that AFSC secretome was responsible for enhancing vasculogenesis and was capable of evoking a strong angiogenic response in murine recipients [75]. According to this study, a detailed analysis of the AFSC-conditioned media revealed the presence of known proangiogenic and antiangiogenic factors using Luminex's MAP Technology. Vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and two angiogenesis inhibitors, interferon-gamma (IFNγ) and interferon gamma-induced protein 10 (IP-10), were identified as secreted proteins [75][76][77]. It was also demonstrated that a relative small number of AFSC was enough to secrete a detectable amount of proangiogenic growth factors and cytokines. The secretion of these can be regulated in a dose-dependent manner according to the initial cell number of the cells used [24,75].
A systematic study on AFSC-secreted proteins led to the conclusion that proangiogenic soluble factors from AFSCs can mediate the recruitment of endothelial progenitors in an ischemic rat model [78]. In particular, conditioned medium derived from AFSCs could topically deliver angiogenic growth factors and cytokines into the skin flap of the ischemic rat model and was responsible for triggering the endogenous repair by recruiting endothelial progenitor cells [78].

Summary
The current data so far suggest that amniotic fluid and amniotic membrane may represent promising sources for stem cells of mesenchymal origin. Indeed, MSCs are more abundant and a wide range of protocols has been described for their isolation. However, it is reported that different culture conditions of the same type of cells may affect their differential gene expression pattern, which represents a limitation for their isolation and expansion in vitro. Studies including phenotypic analysis, using methodologies such as flow cytometry and immunohistochemistry, as well as transcriptomics, proteomics, and secretome analyses approaches, aim to determine the protein profile of these cells (Figure 1). Data generated by such studies are expected to clarify their differential repertoire and validate the molecular profile of these stem cells. However, the main issue urged to be addressed is the isolation of a homogenous population that may facilitate systematic studies for the elucidation of the function of these multipotent cells.
Such approaches may lead to the identification of key antigens that mirror the phenotype of these cells and explain their distinct features properties. This type of studies will open the way for a systematic and efficient isolation of these cells prior to their use at the clinical setting.
Since there is no common marker available for AFSC and AMSC, a wider panel of markers needs to be employed. This also urges the conduction of further detailed array and functional analyses in order to define the most appropriate markers for AFSC and AMSC characterization.