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Center for Women's Health Research, Department of Obstetrics and Gynecology,2 Monash University, Monash Medical Center, Clayton, Victoria 3168, Australia
Monash Institute of Medical Research,3 Monash University, Clayton, Victoria 3168, Australia
Australian Stem Cell Center,4 Clayton, Victoria 3168, Australia
ABSTRACT
The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1; Nanog homeobox; SRY-box 2; and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficiency mice testes. Last, using flow cytometry we have shown that only a very small proportion of primary hAECs contain class IA and class II human leukocyte antigens (HLAs), consistent with a low risk of tissue rejection. However, following differentiation into hepatic and pancreatic lineages, significant proportions of cells contained class IA, but not class II, HLAs. These observations suggest that the term amnion, an abundant and easily accessible tissue, may be a useful source of multipotent stem cells that possess a degree of immune privilege.
amnion, developmental biology, fetal membranes, placenta, stem cells
In pregnancy the human fetus is surrounded by two fetal membranes: the amnion and the chorion. The chorion is the outer of the two membranes and, like the placenta, is derived from the trophectoderm of the implanting blastocyst. The amnion arises prior to gastrulation from embryonic epiblast cells that are also destined to develop into the three primary fetal germ layers: the endoderm, the mesoderm, and the ectoderm. The amnion forms a fluid-filled sac in which the fetus lies. The timing of the derivation of amnion from the epiblast is important, because it has been suggested that tissues that form prior to gastrulation may retain stem cell or stem cell-like capabilities [1].
Indeed, mixed populations of fetal- and amnion-derived cells obtained by amniocentesis in the second trimester of pregnancy express the transcription factors POU domain, class 5, transcription factor 1 (POU5F1) and Nanog homeobox (NANOG) [2], which are necessary for maintaining the undifferentiated state of pluripotent stem cells. Further, the cells obtained at amniocentesis display self-renewal properties forming clonal colonies [2], have decreased telomerase activity [3], resemble mesenchymal stem cells [4] and neural progenitor cells [5], and can differentiate into osteocyte-, adipocyte-, and neural-like cells [2, 6]. While these studies have clearly demonstrated the stem cell-like properties of amniotic fluid cells obtained in the second trimester, amniocentesis is usually undertaken for prenatal genetic diagnosis, and so the majority of retrieved cells are currently used for this purpose. Amniocentesis is also associated with a small but significant excess risk of miscarriage [7]. Thus, if the amnion was to become a useful source of stem cells, a better strategy may be to determine whether cells harvested from fetal membranes that are delivered after birth possess stem cell-like characteristics. This would provide a vast supply of cells from a tissue that is easily accessible and currently discarded after birth. However, to date very few studies have examined whether term amnion cells, as opposed to midtrimester cells, possess stem cell-like properties.
Mixed populations of fetal membrane-derived cells express numerous cell surface and intracellular antigens that are characteristic of mesenchymal stem cells [8], and in a recent study it was reported that passaged term human amniotic epithelial cells (hAECs) can differentiate into cells resembling those derived from neuroectodermal, mesodermal, and endodermal lineages [9].
It has also been shown that native hAECs express the nonpolymorphic, nonclassical human leukocyte antigen G (HLA-G) [10] but lack the polymorphic antigens HLA-A, HLA-B, and HLA-C (class IA) and HLA-DR (class II) on their surfaces [11]. These findings suggest that hAECs may be immunologically inert and would have a reduced risk of rejection upon transplantation. Term amnion is a highly abundant and easily accessible tissue that may potentially be an important source of transplantable stem cells. We investigated whether term hAECs express markers characteristic of stem cells, self-renewal forming clonal colonies, form teratomas, differentiate into cells derived from each of the three primary germ layers, viability of differentiated cells in vitro and whether HLA class IA and class II antigens remain suppressed following differentiation.
Tissue Collection and Processing
The study was approved by the Southern Health Human and Animal Research Ethics Committees of Monash Medical Center. Informed, written consent was obtained from each patient prior to collection. Fetal membranes (n = 21) were retrieved from healthy women delivered at term by elective cesarean delivery. Severe combined immunodeficient (SCID) mice (n = 3) were obtained from Southern Health Animal Facilities, Victoria, Australia. Guidelines relating to the care and use of animals were approved by Southern Health.
The amnion was peeled from the chorion and rinsed in phosphate-buffered saline (PBS). Tissue was digested twice in 0.25% trypsin containing 0.5 mM EDTA in Hanks' Balanced Salt Solution for 20 min at 37°C with agitation. Trypsin was inactivated with fetal calf serum (FCS), and the solution was filtered and centrifuged at 2300 x g for 10 min. After washing cells in M199 medium, contaminating erythrocytes were lysed in hypotonic solution (8% ammonium chloride, 0.84% sodium bicarbonate, and 0.37% EDTA) for 10 min at 37°C with gentle shaking. Cells were washed and resuspended in Dulbecco modified Eagle medium (DMEM)/F12 containing 100 U/ml penicillin/streptomycin solution and 10% FCS. Media and supplements were purchased from Invitrogen (Mount Waverly, Australia).
The purity of the isolates was determined by flow analyses for the epithelial marker cytokeratin 7 (KRT7; Dako, Carpentaria, CA). Briefly, 5 x 105 cells were incubated with permeabilizing reagent (Dako), 10% goat serum, and Iry antibody diluted 1:100 for 30 min at room temperature. After washing, cells were incubated with phycoerythrin-conjugated anti-mouse IgG (1:10; Dako) for 30 min. Isotype-matched mouse IgG at concentrations similar to those of the Iry antibody was used as a negative control. Isolates that were more than 99% positive for KRT7 were used for experiments described below.
Total RNA was isolated from amnion tissue (n = 5) using Trizol (Invitrogen). RNA was also isolated from freshly harvested hAECs (n = 5) and hESC line hES2 using QIAshredder and RNeasy columns (Qiagen, Doncaster, Australia). Following DNase treatment, 1 µg total RNA was reverse transcribed into cDNA using random primers and Superscript III (Invitrogen). The cDNA diluted 1:20 was subjected to 36 cycles of PCR using primers and parameters shown in Table 1. Human ESCs served as a positive control, whereas the negative control consisted of reactions omitting reverse transcriptase enzyme. The resulting products were separated on 1%–2% agarose gels containing ethidium bromide.
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Immunocytochemistry for Stem Cell Markers
Immunocytochemistry was performed on hAECs cultured for 24 h to detect POU5F1, SOX2, stage-specific embryonic antigen-4 (SSEA-4), and germ cell tumor marker 2 (GCTM2; n = 5). SSEA-4 is a glycolipid found on hESCs and cells of the inner cell mass [12, 13]. GCTM2 is a keratin sulphate proteoglycan present in pluripotent embryonal carcinoma and undifferentiated hESCs [14]. Human AECs were fixed in ice-cold ethanol and blocked in 10% human serum for 45 min at room temperature. Primary antibodies against human POU5F1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), SOX2 (1:200; Chemicon, Temecula, CA), SSEA-4 (1:200; Developmental Hybridoma Bank, Iowa City, IA), and GCTM2 (1:2; from Martin Pera, Australian Stem Cell Center, Melbourne, Australia), were applied and incubated overnight at 4°C. Controls were incubated with corresponding concentrations of isotype-matched IgG in lieu of Iry antibodies. These were IgG2b (POU5F1), IgG3 (SSEA-4), IgM (GCTM2), and rabbit IgG (SOX2). Alexa Fluoro dye-conjugated goat anti-mouse or goat anti-rabbit IIry antibodies (1:100; Invitrogen) were applied to hAECs for 1 h at room temperature. Cells were mounted in Vectorshield containing 4',6-diamidino-2-phenylindole nuclear stain (Vector Laboratories, Burlingame, CA). Human AECs in 8–10 randomly selected fields were viewed at 200x magnification, and the percentage (mean ± SEM) of positively staining cells was calculated.
Clonal Culture and Cloning Efficiency
To test for self-renewal properties through clonogenicity, hAECs from five fetal membranes were seeded at a clonal density of 50 cells/cm2 in 60-mm diameter Petri dishes (Becton Dickinson Biosciences, Bedford, MA). Control cultures were maintained in DMEM/F12 with 10% FCS. Cells were also grown in serum-free DMEM/F12 supplemented with 10 ng/ml epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF), a combination of EGF + FGF (10 ng/ml each), or activin A (10 ng/ml). EGF and bFGF were purchased from Sigma-Aldrich (St. Louis, MO), and activin A was purchased from R&D Systems (Minneapolis, MN). Cultures were maintained for 21 days, with media replaced thrice weekly. Cell colonies were fixed in 10% formalin for 10 min, immunostained for KRT7 (1:100; Dako), and visualized with 3,3'-diaminobenzidine (DAB) chromogen. A cluster was considered to be a clonal colony when the cell number exceeded 50 [15]. Cell number and cloning efficiency percentage were calculated as described previously [15]. Briefly, numbers of cells within each colony were estimated using a 10 x 10 grid mounted on an eyepiece graticule. The cloning efficiency percentage was calculated using the formula cloning efficiency (%) = (number of colonies/number of cells seeded) x 100.
Transplantation of hAECs into Murine Testes
Freshly isolated hAECs (5 x 105 viable cells) were inoculated into one testis each of three adult SCID mice, whereas saline was injected into the other testis. Ten weeks after inoculation, animals were killed, and testes were excised and fixed in 4% paraformaldehyde. To identify hAECs, immunohistochemistry for human KRT7 (1:100) was performed on 5-µm sections. Endothelial cells lining murine blood and lymph vessels were identified using rabbit anti-mouse PECAM1 (1:100; Dako). Immunostaining was visualized using DAB chromogen.
Differentiation of hAECs into Multiple Lineages and Their Characterization
Freshly isolated hAECs (5 x 104 viable cells/well) were plated onto collagen IV-coated coverslips seated in 24-well plates in standard medium consisting of DMEM/F12 supplemented with 10% FCS. Plated cells were divided into three groups. In group 1, cultures were maintained in standard medium for 24 h only, whereas cells in group 2 were cultured for 4 wk. In group 3, media was removed after 24 h of plating, and fresh DMEM/F12 with 10% FCS and the supplements shown in Table 2 was added to induce mesodermal (adipocytic, osteocytic, myocytic, and cardiomyocytic) and endodermal (pancreatic and hepatic) differentiation.
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Neuroectodermal differentiation was induced by plating cells on poly-D-lysine/laminin-coated coverslips and grown in neural basal A medium (Invitrogen) containing supplements (Table 2). Cultures treated with supplements were also maintained for 4 wk. These experiments were performed in quadruplicate wells from hAECs isolated from five fetal membranes. The percentage of viable cells was determined by the trypan blue dye exclusion test.
For evidence of adipogenic differentiation, cells were tested for lipid granules using Oil Red O stain. Briefly, cells were fixed in 10% neutral-buffered formalin vapor at room temperature for 15 min and rinsed in 60% isopropanol. The Oil Red O solution was applied for 20 min at room temperature. Cells were rinsed in 60% isopropanol, then in distilled water, and were mounted in aqueous mounting medium.
Osteoblastic differentiation was assessed by identifying calcium accumulation using Von Kossa stain. Cells fixed as described above were washed in distilled water and incubated in 5% silver nitrate solution for 1 h under ultraviolet light. Cells were washed in distilled water, dehydrated, and mounted using DPX mounting medium.
Immunocytochemistry was also carried out to identify antigens associated with differentiated cell phenotypes. These included glucagon (GCG; pancreatic cells); albumin (ALB) and hepatocyte growth factor (HGF; hepatocytes); troponin T (TNNT; cardiomyocytes) [16]; smooth muscle alpha-actin (ACTA2; myocytes); neural progenitor marker nestin (NES); neuronal-specific marker; microtubule-associated protein 2 (MAP2); and astroglial marker glial fibrillary acidic protein (GFAP).
Cells fixed in ethanol were blocked for 45 min at room temperature with 10% human serum in PBS (NES, GFAP, and MAP2) or 10% donkey serum and 1% bovine serum albumin in PBS (GCG, HGF, ALB, ACTA2, and TNNT). Primary antibodies were applied at the following dilutions: ACTA2 (1:100; Dako); TNNT, GCG, HGF, and ALB (1:10; R&D Systems); NES (1:200; Chemicon); GFAP (1:400; Chemicon); and MAP2 (1:100; Lab Vision Corp., Fremont, CA). Corresponding concentrations of mouse IgG1 (ACTA2, TNNT, NES, MAP2, and GFAP), mouse IgG2a (GCG and ALB), or goat IgG (HGF) in 1% BSA in PBS were applied to controls. Cells were incubated overnight at 4°C. After several rinses, horseradish peroxidase-labeled biotin-strepavidin (LSAB kit; Dako) was applied at room temperature for 30 min, followed by DAB to detect ACTA2, GCG, HGF, ALB, and TNNT. To detect NES, MAP2, and GFAP, 1:1000 diluted Alexa Fluor goat anti-mouse IIry antibody (Invitrogen) was applied for 1 h, and cells were washed and mounted in Vectorshield. Cells in 8–10 randomly selected fields viewed at 200x magnification showing positive staining/phenotypic changes characteristic of differentiated cells were expressed as a percentage (mean ± SEM).
Insulin concentrations were measured in media conditioned by cells grown in pancreatic differentiation media using a commercial assay (Access/DXI Ultra Sensitive Insulin Assay; Beckman Coulter, Gladesville, Australia).
Human AECs differentiating into hepatic, pancreatic, and cardiomyocytic cells were further characterized through RT-PCR and/or flow cytometric analyses and transmission electron microscopy (TEM).
Total RNA isolated from control cultures and differentiated cells was analyzed (n = 4). For cardiomyocytic cells, mRNA expression of GATA-binding protein 4 (GATA4); atrial natriurertic peptide (ANP); calcium channel, voltage-dependant, L type, alpha 1C subunit (CACNA1C); potassium voltage-gated channel, Shal-related subfamily, member 3 (KCND3); and myosin light chain 7 (MYL7) was analyzed, and for pancreatic cells, amylase, alpha 2B (AMY2B) was analyzed. RNA isolation and cDNA synthesis were carried out as described earlier. The cDNA was amplified for 36 cycles using PCR primers and conditions described previously [16, 17]. GATA4 is a marker of precardiac cells [16], ANP is a vasorelaxant hormone secreted by cardiomyocytes [18], and MYL7 is important in the development of cardiac myofibrillogenesis and myocardial contraction [19]. CACNA1C and KCND3 are cardiac-specific L-type calcium channel and transient outward potassium channel proteins, respectively [20, 21]. AMY2B is an enzyme produced by pancreatic exocrine cells [22].
For flow analyses, cells were incubated with antibodies against ALB, HGF, GCG, and TNNT for 45 min at room temperature at concentrations used for the immunocytochemistry described above. After several washes, 1:100 diluted allophycocyanin (APC)-conjugated (Becton Dickinson Biosciences) goat anti-mouse (for ALB, GCG, and TNNT) and donkey anti-goat (for HGF) IIry antibodies (Becton Dickinson Biosciences) were applied for 30 min. Cells were washed and analyzed by flow cytometry.
For TEM, cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at room temperature and then left at 4°C overnight. Cells were postfixed in 1% osmium tetroxide and dehydrated in graded acetone, infiltrated, and embedded in Spurrs resin at 60°C for 24 h. Ultrathin sections (80 nm) stained with 3% uranyl acetate and Renoylds stain were examined.
Flow Cytometric Analyses for HLA Class IA and II Antigens
Freshly isolated hAECs, control cultures grown in standard media, and cells maintained in media inducing hepatic, pancreatic, and cardiomyocytic differentiation for 4 wk were analyzed (n = 4). Cells (5 x 105 cell/ml) were incubated with 10 µl APC-conjugated mouse anti-human HLA-A, HLA-B, or HLA-C (Becton Dickinson Biosciences) or 10 µg/ml mouse anti-human HLA-DP, HLA-DQ, or HLA-DR (Becton Dickinson Biosciences) for 45 min in PBS containing 1% BSA. Corresponding concentrations of APC-conjugated IgG1
and IgG2a (Becton Dickinson Biosciences) in 1% BSA was applied to the negative controls. For cells incubated with class II antibody, after several washes, 1:100 diluted APC-conjugated goat anti-mouse IIry antibody (Becton Dickinson Biosciences) was applied for 30 min. Cells were washed and analyzed by flow cytometry.
Quantified data are presented as the mean ± SEM. The data were analyzed by the Kruskal-Wallis one-way analysis of variance followed by Dunn's posthoc test using GraphPad Prism software (version 4.03). Significance was accorded at P < 0.05.
Messenger RNA Expression of Stem Cell Markers
Term amnion and freshly isolated hAECs expressed POU5F1, SOX2, CFC1, NANOG, DPPA3, PROM1, and PAX6. The mRNA of the pluripotent markers FOXD3 and GDF3 was not detected. Undifferentiated hESCs expressed all pluripotent and multipotent markers tested. Amplified cDNA was absent in negative control reactions that lacked the reverse transcriptase enzyme (Fig. 1A).
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Localization of Stem Cell Markers
The cell surface antigens SSEA-4 and GCTM2 localized to the majority of isolated cells (mean ± SEM percentages: 96 ± 1.2 and 98 ± 1.5, respectively; n = 5). The transcription factors POU5F1 and SOX2 were also detected in hAECs cultured overnight, with nuclear staining restricted to a minority of cells. The percentages of cells showing nuclear staining for POU5F1 and SOX2 were 4.8 ± 1.2 and 6.3 ± 1.8, respectively. Control cultures incubated with isotype-matched IgG lacked immunostaining (Fig. 1B).
Following overnight incubation, cells seeded at a clonal density of 50/cm2 had adhered onto the culture dishes and were sparsely distributed. Colony formation was not observed in control cultures grown in DMEM/F12 with 10% FCS for up to 21 days. Human AECs were also grown in serum-free DMEM/F12 media supplemented with EGF, bFGF, and activin A. Clonal colony formation was not supported by activin A (data not shown). EGF and bFGF alone and in combination promoted colony growth, with small clusters of cells visible by Day 7 and large, flattened, undifferentiated colonies containing numerous cells by Day 21. Colonies were successfully subcloned and expanded in the presence of EGF and bFGF. Cells within the colonies stained positively for KRT7, a cytoskeletal protein present in hAECs but not mesenchymal cells and fibroblasts. The cloning efficiency percentage was calculated on Day 14, when discrete colonies containing more than 50 cells were clearly visible. Cloning efficiency was significantly higher in the presence of EGF and bFGF compared with EGF or bFGF alone (P < 0.01). The effect of EGF and bFGF on clonal colony formation and cloning efficiency is shown in Figure 2.
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Transplantation of hAECs into Mouse Testes
Following transplantation of freshly isolated hAECs into the testes of SCID mice, no palpable tumors were detected after 10 wk after injection. However, examination of testes tissue sections showed monolayers of cells that were positive for human KRT7 surrounding some blood/lymph vessels distant to the sites of injection. Murine testicular cells were negative for KRT7. Murine blood and lymph vessels were identified by positive immunostaining for murine PECAM1, an antigen found on endothelial cells lining the blood/lymph vessels (Fig. 3).
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Although hAECs did not form teratomas in vivo, we tested their ability to differentiate into cells derived from primary germ layers using supplements given in Table 2.
Freshly harvested hAECs showed strong immunostaining for the early neural lineage marker NES, the neuronal marker MAP2, and astrocytic protein GFAP (mean ± SEM percentages 93.7 ± 3.2, 96.4 ± 2.9, and 94.8 ± 3.3, respectively). Upon culturing hAECs in neural differentiating medium, cell numbers declined sharply. After 4 wk, a small percentage of NES-positive cells and MAP2-positive neurons with large central bodies and thin, elongated processes were observed. The majority of cells remaining after 4 wk produced GFAP (Table 2). These cells had large cell bodies and thin processes characteristic of astrocytes. The neural lineage markers present in hAECs soon after isolation and following culture with supplements is shown in Figure 4.
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Freshly isolated hAECs and cells grown in standard media for 4 wk lacked GCG, ALB, and HGF. Cells grown with hepatocyte-inducing supplements were large, with the majority demonstrating dense, granule-rich cytoplasm and binucleated cells characteristic of human hepatocytes [23]. The majority of differentiated cells produced ALB and HGF (Fig. 5 and Table 2). Ultrastructural analyses revealed marked differences between control and differentiated cells. The double-layered nuclear membrane, extensive rough endoplasmic reticulum surrounding the nucleus, and vesicles resembling bile canaliculi characteristic of human hepatocytes were present in differentiated cells but absent in hAECs grown in standard media (Fig. 5, E–H).
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Cells grown in media promoting pancreatic differentiation formed masses of cells with a cystlike appearance known to be characteristic of pancreatic cells [24]. These cells expressed the pancreatic exocrine cell marker AMY2B and produced the hormone GCG (Fig. 6 and Table 2). However, insulin was not detected in the conditioned media. Differentiated cells showed ultrastructural features characteristic of acinar beta exocrine pancreatic cells, including bodies resembling beta and zymogen granules, condensing vacuoles, and distended Golgi saccules (Fig. 6).
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Freshly isolated hAECs and cells cultured in standard DMEM/F12 for 4 wk lacked immunostaining for the myocytic marker ACTA2 or for mineralized bone or lipid deposits characteristic of osteocytes and adipocytes, respectively. However, hAECs cultured with myocytic-promoting supplements formed elongated, striated cells that were positive for ACTA2.
Compared with controls, under conditions inducing osteocytic differentiation, cells enlarged two to three times, and many were binucleated and contained mineralized bone deposits. Cells grown in adipocytic media also enlarged three to four times, with numerous multinucleated cells containing lipid droplets scattered throughout the cytoplasm. Control cultures and those grown with supplements inducing differentiation into mesodermal lineages are shown in Figure 7.
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Freshly isolated, control, and cells cultured in cardiomyocytic-promoting medium expressed the cardiomyocytic precursor marker GATA4 and differentiated cell markers ANP, MYL7, CACNA1C, and KCND3. TTNT was absent in freshly isolated cells and control cultures. However, TTNT was abundant in differentiated cells (Fig. 8 and Table 2). Ultrastructural analyses showed that compared with control cultures, differentiated cells contained T tubules, numerous myofilaments and myofibrils, and H bands characteristic of relatively mature cardiomyocytes (Figs. 5E and 8, D–F).
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Viability and Transformation Efficiency
Cells showing the differentiated phenotypes and markers described above were evident within 2 wk of culture. Trypan blue dye exclusion tests showed that more than 96% of cells were viable after a further 2 wk in culture in media containing supplements. However, the efficiency of transformation as assessed by the percentage of cells displaying the markers and phenotypic appearances of differentiated cells varied considerably among the mesodermal- and endodermal-derived lineages. The transformation efficiency into cells resembling myoctyes, cardiomyocytes, hepatocytes, and pancreatic cells was high compared with adipocytes (Table 2). Further, more than one distinct phenotype was observed in cells grown with supplements inducing hepatic, pancreatic, and cardiomyocytic differentiation.
Cells grown in pancreatic, cardiomyocytic, and hepatic differentiation media were selected for HLA studies on the basis of high transformation efficiency into these lineages and potential therapeutic use in clinically important diseases, such as diabetes, cardiomyopathies, and hepatic disorders. The mean ± SEM percentage of freshly isolated cells with HLA-A, HLA-B, and HLA-C (class IA) was 2.73 ± 0.05, and HLA-DR, HLA-DP, and HLA-DQ (class II) was 0.83 ± 0.16 (n = 4). The percentage of cells with class IA and II antigens after 4 wk in standard medium was similar to that of freshly isolated cells. Upon differentiation into pancreatic and hepatic cells there was a significant increase in the percentage of cells containing HLA class IA antigens (P < 0.0001 compared to controls). However, there were no significant changes in the percentage of cells expressing HLA class II antigens following differentiation into hepatic and cardiomyocytic cells compared with controls. Representative flow analysis diagrams and the percentage of cells expressing HLA antigens are shown in Figure 9.
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In this study we have demonstrated that hAECs obtained from normal term pregnancies 1) express markers usually ascribed to stem cells, 2) are clonogenic, and 3) can be differentiated in vitro into endodermal-, mesodermal-, and ectodermal-derived lineages. We have also demonstrated that hESCs do not form teratomas in vivo. Taken together, these observations provide important and novel evidence for the multipotentiality of hAECs. We have also shown that undifferentiated cells are likely to possess a degree of immune privilege but that this may alter following differentiation into some lineages.
Although hAECs express mRNAs and/or proteins present in hESCs and multipotent stem cells, the epithelial cells lining the amnion do not transdifferentiate in vivo. It is possible that there are endogenous substances and factors in the amniotic fluid that suppress differentiation, and it may be worthwhile identifying such factors. This could also assist in understanding the pathways governing differentiation of adult and embryonic stem cells.
Our finding that hAECs are clonogenic, forming large, flattened, undifferentiated colonies containing several hundred cells within a few weeks of culture is both novel and important. Cells within the colonies stained positively for KRT7, an epithelial cytoskeletal protein found in hAECs, indicating that colonies did not arise from contaminating fibroblast or mesenchymal cells [25]. Clonogenicity, the ability of a single cell to form a clonal colony, is a key defining function that demonstrates the self-renewal properties of stem cells [26]. That hAECs are clonogenic supports our stem cell-like marker findings. Indeed, the cloning efficiency of the hAECs was comparable to some hESC lines [27] and higher than some adult epithelial stem cells, such as those derived from the human endometrium [15]. However, while we have shown that hAECs are clonogenic, we have yet to establish their long-term self-renewal capabilities. This will be important to do in future studies.
Teratoma formation is an important feature ascribed to pluripotent hESCs. While the marker expression and clonogenecity suggest some similarity to hESCs, hAECs did not form teratomas upon transplantation into the testes of SCID mice. This confirms previous reports [9, 28] and studies using either amniotic membranes to repair damaged ocular surfaces [29–31] or amnion cells to improve congenital lysosomal storage diseases [32] that also found that these cells did not form tumors. Taken together, the current and previous studies would suggest that amnion cells are not pluripotent-like hESCs. On first inspection this may seem to be a limitation of hAECs but, if proven, the absence of tumor formation following transplantation could actually be an advantage in future clinical applications. Indeed, an understanding of the mechanisms whereby hAECs do not form tumors, despite possessing similar molecular machinery to hESCs, may be useful in directing lineage-specific and tumor-suppressing differentiation of hESCs. It is possible that the lack of telomerase activity in hAECs [3] may contribute to tumor suppression in vivo. However, equally importantly, the absence of teratoma formation may indicate that hAECs are unable to differentiate at all in vivo. It is therefore important to determine their capacity to differentiate in vivo using appropriate animal models of disease—the focus of our ongoing studies.
Nonetheless, in this study we investigated whether hAECs could respond to environmental cues and differentiate into cells derived from the three germ layers in vitro. We thought that this was likely, as it has been reported that neural progenitor, neuronal, and glial cell markers are present in the amniotic epithelium [33, 34]. Consistent with these reports, we found NES, GFAP, and the neuronal-specific marker MAP2 to be abundant in freshly isolated hAECs. However, under conditions known to stimulate neural differentiation of hESCs, the survival and differentiation of hAECs into cells resembling neurons was poor. Interestingly, in vivo studies in which hAECs have been injected into injured rodent brain or bonnet monkeys with spinal cord injury did not show conclusive evidence of differentiation, but that immunosuppressive and/or neurotrophic factors produced by the cells seem to aid repair [35–38]. The roles played by neurotrophic factors and neurotransmitters catecholamines acetylcholine, norepinephrine, and dopamine [39] produced by hAECs during gestation in fetoplacental growth and function are not widely understood. However, it is possible that they may hinder differentiation along neural lineages, at least in vitro.
On the other hand, this is the first report that native hAECs can differentiate into cells with a phenotype and markers characteristic of mesodermal-derived myocytes, osteocytes, and adipocytes. Human AECs also formed elongated TNNT-positive cells resembling cardiomyocytes, consistent with a previous report describing 
-actin staining cardiomyocyticlike cells derived from passaged term hAECs [9]. Interestingly, freshly isolated cells and those grown under standard conditions also expressed the mRNAs of genes that are important for cardiomyoctyic lineage specification, such as GATA4 and function, including ANP, MYL7, CACNA1C, and KCND3. However, ultrastructrual analysis revealed that features consistent with relatively mature cardiomyocytes, such as myofilaments, myofibrils, H bands and T tubules [40], were present only in differentiated cells. These findings demonstrate that hAECs can attain subcellular specialization in response to environmental cues. However, differentiated cells did not contract spontaneously under the culture conditions used. This may be due to the lack of myofibrillar bundle organization into densely packed sarcomeres, as is seen in terminally differentiated cardiomyocytes [40]. It would be worthwhile investigating whether hAECs can attain terminal differentiation and their electrophysiologic properties.
We were also able to differentiate hAECs into endodermal-derived hepatocytes and pancreatic cells, confirming previous reports [9, 41]. We have extended those reports by showing that the differentiated cells have high long-term survival rates in culture. We have also further characterized the cells, showing that the hepatic cells produce HGF, a key growth factor, and that at the ultrastructural level they had the key morphologic features of normal adult hepatocytes. Similarly, while we could demonstrate that the hAEC-derived pancreatic cells produced GCG, we could not coax them to secrete insulin. Interestingly, at the ultrastructural level the pancreatic cells looked like exocrine beta-acinar cells consistent with an inability to secrete insulin. It is also important to note that while a high proportion of cells displayed markers characteristic of specialized cells, we observed secondary phenotypes among some of these lineages. It would be important to characterize these secondary phenotypes. However, given these promising preliminary findings, in vivo animal studies investigating migration, engraftment, and tissue-specific differentiation of transplanted hAECs would seem warranted. Indeed, mixed populations of membrane-derived cells have been shown to synthesize migration and adhesion molecules and proteases that could facilitate their transmigration and homing to injured sites in vivo [8].
Native hAECs are thought to have low levels of polymorphic HLA-A, HLA-B, and HLA-C and negligible levels of HLA-DR mRNA expression [42]. This is important in terms of potential clinical applications, as native hAECs would be expected to have a reduced risk of rejection upon allotransplantation. Indeed, mixed populations of fetal membrane-derived cells have been found to suppress mixed allogeneic lymphocyte reactions [8]. Our findings show that only a very small fraction of native cells or those maintained long term under control conditions contain these antigens. Importantly, however, HLA-A, HLA-B, and HLA-C antigens are clearly present in significant proportions of hAEC-derived hepatocytes and pancreatic cells but not cardiomyoctyes. Understanding the factors stimulating/suppressing HLA antigenicity would be important if these cells were to be useful clinically. Human AECs also secrete immunosuppressive factors [37, 43] that could facilitate successful engraftment and secrete growth factors and cytokines that may stimulate the differentiation of endogenous stem cell populations and/or assist in wound healing and repair in vivo [43–48]. Given the abundance of human term amnion tissue, minimal ethical and legal issues associated with its usage and our findings that term hAECs possess stem cell-like characteristics and have considerable multilineage differentiation potential warrants further investigation into their differentiation and functional potential in vivo.
ACKNOWLEDGMENTS
We thank the patients and staff of Monash Medical Center, Nicki Sam for tissue collection, Budi Marjono and Claire Walker for their assistance in maintaining cultures, Paul Hutchinson and James Ngui for their assistance with the flow analyses, and Anna Friedhuber for the TEM and ultrastructural analyses.
Correspondence: 1Ursula Manuelpillai, Department of Obstetrics and Gynecology, Monash University, Monash Medical Center, 246 Clayton Rd., Clayton, Victoria 3168, Australia. FAX: 61 3 9594 6389; e-mail: ursula.manuelpillai{at}med.monash.edu.au
Received: 2 July 2006.
First decision: 21 August 2006.
Accepted: 30 April 2007.
REFERENCES
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  C.-P. Chen, S.-H. Liu, J.-P. Huang, J. D. Aplin, Y.-H. Wu, P.-C. Chen, C.-S. Hu, C.-C. Ko, M.-Y. Lee, and C.-Y. Chen Engraftment potential of human placenta-derived mesenchymal stem cells after in utero transplantation in rats Hum. Reprod., January 1, 2009; 24(1): 154 - 165. [Abstract] [Full Text] [PDF]
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 M Mimeault and S K Batra Recent progress on normal and malignant pancreatic stem/progenitor cell research: therapeutic implications for the treatment of type 1 or 2 diabetes mellitus and aggressive pancreatic cancer Gut, October 1, 2008; 57(10): 1456 - 1468. [Abstract] [Full Text] [PDF]
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 O. Parolini, F. Alviano, G. P. Bagnara, G. Bilic, H.-J. Buhring, M. Evangelista, S. Hennerbichler, B. Liu, M. Magatti, N. Mao, et al. Concise Review: Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells Stem Cells, February 1, 2008; 26(2): 300 - 311. [Abstract] [Full Text] [PDF]
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