Stem Cell Characteristics of Amniotic Epithelial Cells - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

a Departments of Pathology and0 W1 y& }2 c) g; E
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b Cell Biology & Physiology and
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c McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA4 ]1 c& F  V6 B- F, F

Key Words. Placenta ? Amniotic epithelial cell ? Differentiation pluripotent
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Correspondence: Stephen C. Strom, Ph.D., Department of Pathology and McGowan Institute for Regenerative Medicine, University of Pittsburgh, 200 Lothrop St., BST-s450, Pittsburgh, Pennsylvania 15261, USA. Telephone: 412-624-7715; Fax: 412-383-7969; e-mail: strom@pitt.edu
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ABSTRACT

The placenta is comprised of three layers: amnion, chorion, and decidua. Each layer is derived from vastly different sources. Although the decidua is maternally derived, the amnion and chorion are derived from the embryo. Whereas the chorion is derived from the trophoblast layer, the amnion is derived from the epiblast as early as 8 days after fertilization. Thus, the epiblast gives rise to the amnion as wellto as to all of the germ layers of the embryo. Pluripotent embryonal carcinoma cells can only be produced from cells derived before gastrulation , confirming the importance of gastrulation in the differentiation and specification of cell fate. Gastrulation occurs approximately 3 weeks after fertilization, which is nearly 2 weeks after amniotic epithelial (AE) cells are formed from the epiblast. Thus, amnion may retain the pluripotent properties of early epiblast cells.
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The amnion is a thin membrane-lined cavity that fills with fluid and serves, among other things, to cushion the fetus during development and to prevent adhesion of the developing fetus to maternal structures. AE cells have several unique characteristics. Like many immature or stem cells, expression of myosin heavy chain class I antigens is very low on AE cells . Under certain conditions, AE cells have been reported to differentiate to mature neural cells that synthesize and release neurotransmitters, including acetylcholine, norepinephrine, and dopamine . These observations suggest that cells derived from the fetal side of the placenta may retain a multipotent phenotype long after they differentiate from the epiblast. In support of this hypothesis, recent reports have described the identification of pluripotent or multipotent stem cells from human placenta cord blood or amniotic fluid . Pluripotent stem cells were identified in cord blood , whereas multipotent mesenchymal stem cells were detected in various placental tissues . Mesenchymal stem cells have also been isolated from amniotic fluid .

Taken together, these observations suggest that the fetal tissues of the placenta might be a useful source of stem or progenitor cells. We examined the epithelial cell layer of the amnion for cells with stem cell characteristics. The results indicate that the amnion contains cells with significant plasticity and differentiation potential. If methods can be developed for the efficient differentiation of amnion-derived cells to specific cell types, this placental tissue, which is normally discarded, may be a useful source of cells for transplantation and regenerative medicine.7 ]1 z' `+ S5 w9 p* E2 a

MATERIALS AND METHODS, n) ?! a9 F' o/ n) o. c

Stem cell surface markers are present on isolated AE cells. AE cells express SSEA-3 (8.79% ± 2.84%), SSEA-4 (43.94% ± 14.8%), TRA 1-60 (9.82% ± 4.31%), and TRA 1-81 (9.91% ± 4.49%) and do not express SSEA-1 (Fig. 1A). Some AE cells express c-kit, the surface receptor for stem cell factor, and Thy-1 . Although initially low, approximately 50% of the cells express Thy-1 after 6 days of culture (not shown). The cells do not express the hematopoietic stem cell marker CD34. The absence of CD34-positive cells in this population indicates the isolates are not contaminated with hematopoietic stem cells such as umbilical cord blood or embryonic fibroblasts. The results presented are the average values from five different donors.5 L5 P( w. H7 ^* f

Figure 1. Analysis of stem cell markers on AE cells. (A): Data present the average level of cell surface antigen expression of isolated AE cells (n = 5, ± SD) measured by immunofluorescence and flow cytometry. (B): Phase-contrast microscopic images of cultured AE cells with or without EGF (10 ng/ml). Scale bar = 100 μm. (C): Immunohistochemical detection of cytokeratin in cultured AE cells. Insets show a section of amniotic tissue. Mouse IgG1 antibody was used as a negative control. Hematoxylin nuclear counterstaining was performed. (D): Reverse transcription–polymerase chain reaction analysis of AE cells. Stem cell–specific gene primers were used. (E): Expression of Oct-4 and nanog (relative to a beta-actin internal control) in cultured AE cells over the first 18 days after isolation. For this analysis, gene expression in the starting material was set to 1. Spheroid formation was evident in confluent cultures as early as day 6. Abbreviations: AE, amniotic epithelial; ck, cytokeratin; EGF, epidermal growth factor; FGF, fibroblast growth factor; Oct-4, octamer-binding protein 4; TERT, telomerase reverse transcription.
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In the presence of EGF, AE cells proliferate robustly and form a confluent monolayer of cobblestone-shaped epithelial cells (Fig. 1B). Approximately five cell doublings were observed over 8 days, giving these cells an average doubling time of 38.4 hours in the presence of EGF. Without EGF, proliferation ceased and the cells formed multinucleated giant cells reminiscent of trophoblast differentiation of ESCs . Proliferating AE cells showed a normal karyotype (not shown). As shown in Figure 1C, like the single layer of epithelial cells from the amniotic membrane (inset), the cells react with antibodies to pan-cytokeratins, confirming their epithelial nature and the lack of contamination with other cell types, such as mesenchymal fibroblasts.$ s- H" e" G3 X# S, g' M

In addition to the SSEA and TRA surface markers, there is consensus agreement that human embryonic stem cell (hESC) lines express telomerase, Oct-4, SOX-2, FGF-4 , and Rex-1. Freshly isolated AE cells were examined for these markers. The human hepatoblastoma cell line HepG2 cells served as the control. With the exception of telomerase, all other stem cell markers were expressed on freshly isolated AE cells (Fig. 1D). Telomerase RT expression was detected in HepG2 but not in AE cells. Neither was telomerase activity detectable in AE cells by the TRAP assay (Trapez telomerase detection kit; InterGen, Burlington, MA, http://www.intergen.com, data not shown).

Isolated AE cells express Oct-4 and nanog, two genes known to be required for self-renewal and pluripotency . The expression of nanog was confirmed by the use of three different primer sets and sequencing the amplified product. Both genes were readily detected in AE cells at the time of isolation. When AE cells were kept in high-density culture, the expression of Oct-4 and nanog increased over the first 12–15 days as AE cells formed spheroid structures above the basal layer (Fig. 1E). Suspecting that the stem cell markers may be derived from the cells in the spheroids, the expression of the stem cell markers was examined in these structures. As shown in Figure 2A, metabolism of a fluorescent substrate by alkaline phosphatase was restricted to the cells within the spheroids. Immunofluorescent staining and confocal microscopy revealed that the stem cell surface antigens SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were also localized to the spheroids (Fig. 2B), whereas 98% of the basal layer cells under the spheroid structure did not react. Nearly 100% of the cells in spheroids reacted with antibodies to SSEA-4, whereas 5%–15% of the cells in spheroids were positive for SSEA-3, TRA1-60, or TRA1-81. Nuclear localization of Oct-4 was evident within the sections of the spheroids visualized by confocal microscopy, confirming the RNA data (Fig. 2C). Cells at the middle level of the spheroid showed both nuclear and cytoplasmic staining. These data suggest that some cells in the spheroid structures retain their initial stem cell characteristics and that stem cell markers are reduced when the cells attach to culture dishes.: i- L+ e; K% _' [# b

Figure 2. Examination of stem cell markers on spheroids. Scale bar = 100 μm. (A): Phase-contrast and fluorescent microscopic view of a spheroid and basal layer cells observed after incubation with vector red, a fluorescent alkaline phosphatase (ALP) substrate. (B): Cells in the spheroids were incubated with antibodies to stem cell surface markers, and confocal images were taken at sections through the spheroid indicated in the side and bottom views. Stem cell markers SSEA-3, SSEA-4, TRA1-60, and TRA 1-81 were all visualized with a fluorescein isothiocyanate–conjugated secondary antibody (green), f-actin was stained with rhodamine phalloidin (red) to visualize the spheroid structure, and nuclei were counterstained with DRAQ5 (blue). TRA 1-60–positive and TRA 1-81–positive cells are indicated with a yellow arrow. (C): Confocal microscopic images of a spheroid after incubation with an antibody to Oct-4 (green). Cells at the middle level of the spheroid showed both nuclear and cytoplasmic staining. Structural and nuclear counterstaining are the same as in (B).3 A0 G  K* b, ]
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After 5 days in culture, the cells floating in the media (Sup) were removed by aspiration of the media. Clusters of cells attaching loosely over the adherent cells (Mid) and those cells attached to the culture dish (adherent fractions) were collected by differential trypsinization (Fig. 3A). Some of the collected cells were stained for stem cell surface markers, and the remainder were used for RT-PCR analysis. The expressions of Oct-4 and nanog were examined by quantitative real-time RT-PCR. The results clearly indicate that the nanog and Oct-4 expression is higher in the cells in the middle layer of the cultures than in the adherent fraction (Fig. 3B). The middle cell fraction also contained more cells that express the stem cell surface markers SSEA-4, TRA1-60, and TRA 1-81 than the cells adherent to the culture dish. There was no significant difference in the expression of SSEA-3 (Fig. 3C). These data suggest that cells remaining in culture over the basal layer of adherent cells contain more cells with stem cell characteristics than the adherent fraction.7 n) H( ^3 T. O2 {! |9 S+ z: ^* G: _

Figure 3. Feeder cell–like function of basal layer amniotic epithelial (AE) cells. (A): Side-view image scheme of AE cell primary culture. After 5 days in culture, AE cells could be divided into three groups, a basal layer of cells that attached to the plastic dish (Adh), cells in an intermediate layer weakly adherent over the basal layer (Mid), and cells floating freely in the media (Sup). Cells in the intermediate layer were rounded-up single or small clusters of cells, many of which eventually form spheroid structures. (B): Semiquantitative mRNA expression analysis. The value was normalized by ?-actin expression of each sample and shown with gene expression in the middle layer set to 100% (n = 3). The cell group (Mid) that attached on the basal layer cells expressed higher levels of the stem cell–specific marker genes Oct-4 and Nanog than the other two populations. (C): Flow cytometric cell surface antigen analysis. Cells were stained with the antibodies that recognize stem cell surface antigens SSEA-1, SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, and Thy-1 (CD90) and analyzed by fluorescence-activated cell sorter. Values are mean ± SD (n = 4).9 w( `4 y4 t% K3 J4 F* V5 M

Because undifferentiated ESCs induce tumor formation upon transplantation, we examined the tumorigenicity of AE cells by injection of 1 million cells per site into the rear leg muscles (n = 3) and/or the testis (n = 2) of SCID/beige mice and into the liver (n = 10) and/or the interscapular fat pad (n = 5) of Rag-2–/– mice in 100 μl of PBS using a 30-gauge needle. Although AE cells showed some stem cell characteristics, when a total of 23 million cells (1 million cells per site) were injected into immunodeficient SCID/beige or Rag2–/– mice, none of the recipients developed tumors.

Animals were observed for up to 7 months with no evidence of tumor formation, whereas tumors were observed in animals transplanted with the transformed cell line (HepG2) in approximately 2–3 weeks.: }1 {6 G( m* Z4 D. R

Because AE cells do not form teratocarcinomas, the examination of the ability of AE cells to differentiate to cells from all three germ layers was conducted in vitro. We focused our studies on four cell types that are among those most useful for cell therapy. Endodermal (pancreatic) lineage differentiation of AE cells was examined. As shown by RT-PCR analysis (Fig. 4A), freshly isolated AE cells express pancreas duodenum homeobox-1 and the mRNA expression is maintained when AE cells are cultured in the presence of nicotinamide . The expression of the downstream transcription factors paired box homeotic gene 6, the NK2 transcription factor–related locus 2 (Nkx 2.2), and the mature hormones insulin and glucagon was induced when AE cells were cultured with nicotinamide. Immunostaining for glucagon was also observed (Fig. 4A), suggesting that culture treatments enhance pancreatic differentiation of AE cells. These data indicate that under specific conditions, AE cells differentiate to endodermal cells. Under the same conditions, immunoreactivity to proinsulin or c-peptide could not be demonstrated.9 K! M7 f1 B" B  O7 Q  q4 p
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Figure 4. Pancreatic, neural, and cardiac in vitro differentiation of AE cells. (A): Pancreatic differentiation of AE cells. One-step RT-PCR was conducted with the indicated primers on total RNA extracted from cells cultured for 14 days with media supplemented with nicotinamide (10 mM). The expression of the early pancreatic transcription factor PDX-1 and the downstream transcription factors Pax-6 and Nkx 2.2 and the mature hormones insulin and glucagon (Cy3, red) were identified. The photograph shows immunolocalization of glucagon expression with DAPI nuclear counterstaining in blue. Scale bar = 100 μm. (B): Neural differentiation of AE cells. Neural-specific gene expression was examined by one-step RT-PCR. GFAP immunostaining: more than 90% of the cells are GFAP-positive (Cy3, red). Approximately 5%–10% of cells are positive for CNP (fluorescein isothiocyanate, green). DAPI nuclear counterstaining (blue). Scale bars = 100 μm. (C): Cardiomyocyte differentiation of AE cells. One-step RT-PCR for cardiomyocyte-specific genes from AE cells cultured for 14 days in basal media supplemented with ascorbic acid 2-phosphate. Immunofluorescent image with an anti–alpha-actinin antibody (Cy-3, red) and DAPI nuclear counterstaining (blue). Scale bar = 50 μm. Abbreviations: AE, amniotic epithelial; CNP, cyclic nucleotide phosphodiesterase; DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Nkx 2.2, NK2 transcription factor–related locus 2; Pax-6, paired box homeotic gene 6; PDX-1, pancreas duodenum homeobox-1; RT-PCR, reverse transcription–polymerase chain reaction.+ }7 R2 D8 g/ G3 v% D5 D+ s& }
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Next, neural differentiation was examined. AE cells were cultured in media supplemented with all-trans retinoic acid and FGF-4 for 7 days to induce neural-specific gene expression (ectodermal lineage) . Like hESCs, freshly isolated AE cells express many markers of neural differentiation (Fig. 4B). The expression of nestin and glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA biosynthesis, increased over 7 days (Fig. 4B). As opposed to the epithelial morphology shown in Figure 1B, cells induced toward neural differentiation take on an elongated, neuronal morphology and react with antibodies to GFAP (glial cell lineage) and CNP (Fig. 4B, oligodendrocyte lineage). Nearly 90% of AE-derived cells were positive for GFAP, and 5% of cells were positive for CNP. These data indicate that under these culture conditions, AE cells are induced to differentiate to glial and neuronal cells.

Using culture conditions reported to induce cardiac differentiation of ESCs  (mesodermal lineage), we examined car-diomyocyte-related gene expression in AE cells. Results shown in Figure 3C indicate that cardiac-specific genes atrial and ventricular myosin light chain 2 (MLC-2A and MLC-2V) and the transcription factors GATA-4 and Nkx 2.5 are expressed or are induced in cultured AE cells over 14 days in media supplemented with ascorbic acid. Immunohistochemical analysis of alpha-actinin expression is presented in Figure 4C. Although the staining pattern does not indicate the functional localization of -actinin seen in mature cardiomyocytes, this staining pattern is very similar to that reported by Cheng et al.  with hESC-derived cardiomyocytes.

We also investigated hepatic (endodermal lineage) differentiation of AE cells by mRNA expression, protein production, and functional activity. mRNA expression of characteristic hepatocyte genes albumin and A1AT  was examined by real-time quantitative PCR over time in culture (Fig. 5A). Steady and time-dependent increases in the expression of these genes were observed when cells were cultured in EGF and dexamethasone. Immunolocalization of albumin and hepatocyte nuclear factor 4-alpha (HNF-4) revealed that up to 33% of cells were positive for albumin or HNF-4 (Fig. 5B). Some albumin-positive cells were binucleated and resembled normal human hepatocytes. Cells maintained in culture longer contained small cells with refractive cell junctions and characteristic hepatocyte morphology (Fig. 5B, lower right). Although it could be argued that immunoreactivity with albumin could be the result of cross-reaction with bovine serum albumin taken up from the media, the immunohistochemical data are consistent with the expression of human albumin at the RNA level, and the antibody used for localization studies was made up in a solution containing 1% bovine serum albumin.

Figure 5. Hepatic differentiation of amniotic epithelial (AE) cells. (A): Induction of hepatocyte-specific mRNA, albumin (n = 4), and 1-antitrypsin (A1AT, n = 3) over 3 weeks. Real-time quantitative polymerase chain reaction data are plotted relative to the starting material. (B): Immunohistochemistry with anti-human albumin (upper panel) on AE cells cultured for 14 days. Original magnification x 100 (upper left) and x 400 (upper right). Lower left, HNF4 staining. Mixture of cytoplasm-positive cells, nuclear-positive cells, and dead cells is observed. Lower right, a phase-contrast image of AE-derived hepatocyte-like cells after long-term culture (28 days). Scale bar = 100 μm. (C): Functional assay of drug metabolism by CYP1A, ethoxyresorufin-o-deethylase (EROD), conducted on naive AE cells, hepatocyte-like cells produced from AE cells, and freshly isolated human hepatocytes. CYP1A activity was induced by exposure to ?-naphthoflavone (10 μM) for 48 hours before analysis.( a& |& T7 g) `, m: G7 P- B
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An important liver function is drug metabolism. Mature liver expresses basal and inducible CYP450 genes, which encode the drug metabolizing enzymes. Data presented in Figure 5C show the results of an assay for enzymatic activity for CYP1A conducted on AE cells and authentic human hepatocytes. After culture of AE cells with dexamethasone, hepatocyte-like cells express very high levels of the drug metabolizing enzyme CYP1A, with relative levels equal to 60% of that found in normal human liver by the EROD assay  (Fig. 5C). The expression of CYP450 enzymes is considered a relatively late event in liver differentiation and demonstrates that AE cells can differentiate to cells that display mature liver functions.

DISCUSSION

We thank Gerald Schatten and S.P.S. Monga for critical review of this manuscript and acknowledge support from the Alpha-1 Foundation and NIH/DK 92310, Liver Tissue Procurement and Distribution System.: ~6 G. O/ ?# |

DISCLOSURES" S# E$ H" d( B* V2 E

T.M. owns stock in Stemnion and within the past 2 years has acted as a consultant for Stemnion.
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