Human Reproduction, Vol. 18, No. 7, 1489-1493,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Oct-4-expressing cells in human amniotic fluid: a new source for stem cell research?
Obstetrics and Gynecology, University of Vienna, Prenatal Diagnosis and Therapy, Währinger Gürtel 18–20, 1090 Vienna, Austria
1 To whom correspondence should be addressed. e-mail: markus.hengstschlaeger{at}akh-wien.ac.at
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Abstract |
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BACKGROUND: It is the hope of investigators and patients alike that in future the isolation of pluripotent human stem cells will allow the establishment of therapeutic concepts for a wide variety of diseases. A major aim in this respect is the identification of new sources for pluripotent stem cells. Oct-4 is a marker for pluripotent human stem cells so far known to be expressed in embryonal carcinoma cells, embryonic stem cells and embryonic germ cells. METHODS: Cells from human amniotic fluid samples were analysed for mRNA expression of Oct-4, stem cell factor, vimentin and alkaline phosphatase via RT–PCR. Oct-4 protein expression was investigated by Western blot analysis and immunocytochemistry. Oct-4-positive cells were also analysed for the expression of cyclin A protein via double immunostaining. RESULTS: Performing RT–PCR, Western blot and immunocytochemical analyses revealed that in human amniotic fluid in the background of Oct-4-negative cells a distinct population of cells can be found, which express Oct-4 in the nucleus. Oct-4-positive amniotic fluid cell samples also express stem cell factor, vimentin and alkaline phosphatase mRNA. The Oct-4-positive amniotic fluid cells are actively dividing, proven by the detection of cyclin A expression. CONCLUSIONS: The results presented here suggest that human amniotic fluid may represent a new source for the isolation of human Oct-4-positive stem cells without raising the ethical concerns associated with human embryonic research.
Key words: human amniotic fluid/Oct-4/pluripotency/stem cells
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Introduction |
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The Oct-4 POU transcription factor is expressed in mouse totipotent embryonic stem and germ cells. The expression of human Oct-4 is comparable with the pattern in the mouse, suggesting that it may have a similar function in preventing human totipotent embryo cells from differentiating. Each type of established mammalian pluripotent stem cell line—embryonal carcinoma (EC) cells, embryonic stem (ES) cells and embryonic germ (EG) cells—express Oct-4, which disappears rapidly when the cells differentiate. Accordingly, Oct-4 is considered to be a marker of pluripotent stem cells (Donovan, 2001
; Pesce and Schöler, 2001).
Human amniotic fluid cells are used for routine prenatal genetic diagnosis of a wide range of fetal abnormalities caused by genetic alterations. Although this routine diagnosis is well established, little is known about the origin and properties of these cells. Cells of different embryonic/fetal origins of all three germ layers have been reported to exist in amniotic fluid, and for specific subsets of amniotic fluid cells the origins still need to be clarified (Milunsky, 1979; Hoehn and Salk, 1982; Gosden, 1983; Prusa and Hengstschläger, 2002).
The purpose of this study was to investigate whether human amniotic fluid contains cells expressing the transcription factor Oct-4.
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Materials and methods |
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Amniotic fluid cells
Amniotic fluid cell samples were obtained from amniocentesis all performed after the 14th week of pregnancy for routine prenatal diagnosis. The indications were advanced maternal age and the cytogenetic analyses revealed normal karyotypes. This project has been reviewed and accepted by the ethics commission of the University of Vienna, Austria (project number: 036/2002) and each patient signed a written informed consent.
RT–PCR
RNA of amniotic fluid cells was prepared using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA). First-strand cDNA synthesis and PCR were performed using the rp-RT Mix (ViennaLab, Austria) following the protocol provided by the manufacturer. Sequences and PCR conditions have been described previously for the first used Oct-4-specific oligonucleotide set (Monk and Holding, 2001; in Figure 1 designated ‘primer set 1’) and for the second set (Xu et al., 2001; in Figure 1 designated ‘primer set 2’). The oligonucleotides specific for stem cell factor have been designed and synthesized by VBC Genomics (Vienna, Austria) according to the sequence published by Martin et al. (1990), and the PCR conditions were the same. For the analysis of vimentin mRNA expression, specific oligonucleotides and PCR conditions were chosen according to Shamblott et al. (2001). For the analysis of alkaline phosphatase mRNA, specific oligonucleotides and PCR conditions were chosen according to Pittenger et al. (1999). As an internal standard, the expression of 18S rRNA was analysed.
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Western blot analyses
For Western blot analyses, protein extracts were prepared in buffer containing 20 mmol/l HEPES pH 7.9, 0.4 mol/l NaCl, 2.5% glycerol, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 0.5 mmol/l NaF, 0.5 mmol/l Na3VO4, 0.02 µg/ml leupeptin, 0.02 µg/ml aprotinin, 0.003 µg/ml benzamidine chloride, 0.1 µg/ml trypsin inhibitor and 0.5 mmol/l dithiothreitol (DTT). Cells were lysed by freezing and thawing, the extracts were centrifuged and the supernatants were stored at –70°C. Protein concentrations were determined using the Bio-Rad protein assay reagent with bovine serum albumin as the standard. Proteins were run on an SDS–polyacrylamide gel and transferred to nitrocellulose. Blots were stained with Ponceau-S to visualize equal amounts of loaded protein (for the method see also Kubista et al., 2002
). For immunodetection, the anti-Oct-4 antibody C-10 (Santa Cruz, CA, USA) was used. As suggested by the manufacturer of the antibody, the rodent teratocarcinoma cell line F9 has been analysed as an Oct-4-positive control.
Immunocytochemistry
For immunocytochemical analyses of cellular Oct-4 expression, cells were fixed in cold methanol/acetone (1:1) and incubated with anti-Oct-4 antibody C-10 (Santa Cruz) overnight at 4°C. Thereafter, cells were washed, incubated with a biotinylated secondary antibody (B7401, Sigma), washed again, and incubated with ExtrAvidin-Cy3 conjugate (red) (E4142, Sigma). For double staining, a second incubation with the cyclin A-specific antibody H-432 (Santa Cruz) was performed. This antibody was detected directly with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (green) (F9887, Sigma). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue).
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Results |
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RT–PCR was performed with 11 independent amniocentesis samples. In five samples, Oct-4 mRNA expression was detectable. In Figure 1 representative samples are presented. To obtain more information regarding the nature of these cells and their potential, we investigated stem cell factor, vimentin and alkaline phosphatase mRNA expression. All three of these molecules are markers for pluripotent stem cells (for a detailed description of such markers see http://www.nih.gov/news/stemcell/scireport.htm). Stem cell factor (Martin et al., 1990
) is known to be expressed in ES cells, EC cells, haematopoietic stem cells and mesenchymal stem cells. Vimentin is known to be expressed in ectodermal, neural and pancreatic progenitor cells (compare also Shamblott et al., 2001). Alkaline phosphatase (compare Pittenger et al., 1999) expression can be found in ES cells and in EC cells. By analysing six independent amniotic fluid cell samples, we found that all the three Oct-4-positive samples also expressed these three markers, whereas the three Oct-4-negative cell samples did not (Figure 1).
Western blot analyses demonstrated Oct-4 protein expression in samples with positive RT–PCR results (Figure 2). Immunocytochemical analyses of positive samples revealed that 
0.1–0.5% of amniotic fluid cells expressed Oct-4 protein (Figures 3 and 4). The low number of Oct-4-expressing cells can be assumed to be the explanation for the low expression levels in the amniotic fluid samples detected by Western blot analyses (Figure 2). In agreement with the known localization of this POU transcription factor (Pesce and Schöler, 2001), Oct-4 signals were detected in the nucleus (Figures 3 and 4).
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To investigate further the question of whether the Oct-4-positive amniotic fluid cells are actively dividing, we performed double immunostaining experiments with anti-cyclin A antibody. The cell cycle regulator cyclin A is only expressed in proliferating cells (for a review see Hengstschläger et al., 1999
). Our finding that cyclin A protein is expressed in Oct-4-positive amniotic fluid cells demonstrates that these cells proliferate (Figure 4). |
Discussion |
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A major aim in stem cell research is the identification of new sources of stem cells with as yet unknown properties. There is no doubt as to the importance of Oct-4 for the maintenance of stem cells. Each established mammalian pluripotent stem cell line expresses Oct-4, which rapidly disappears when the cells differentiate (Pesce and Schöler, 2001
). ‘‘If you wanted a marker of pluripotent stem cells, you wouldn’t go far wrong if you picked the POU-domain transcription factor, Oct-4.’’ (Donovan, 2001). Accordingly, we have analysed human amniotic fluid cell samples for the expression of this marker. For RT–PCR detection of Oct-4 mRNA, two independent sets of oligonucleotides have been used, which previously were shown to be specific for the human sequence of this transcription factor (Monk and Holding, 2001; Xu et al., 2001). In the RT–PCR-positive samples, we detected the Oct-4 protein of the correct size using a specific antibody in Western blot analyses. To check further whether the amniotic fluid cells exhibit the correct intracellular localization of this transcription factor, we performed immunocytochemical investigations. The observed nuclear localization again confirms that the expression of the correct molecule has been analysed in our study. In addition, this finding furthers suggest that Oct-4 expressed in amniotic fluid cells is functional.
The immunocytochemical analyses were also of importance in answering the question of exactly how many cells within one positive amniotic fluid cell sample express Oct-4. It has been reported previously that some adult human tissues express low levels of Oct-4 as analysed by RT–PCR. Whether Oct-4 protein expression is detectable in these tissues remained elusive (Takeda et al., 1992). In Western blot analyses, we found that specific human tissues samples, others than amniotic fluid cells, were positive for Oct-4 expression in RT–PCR but did not express Oct-4 protein (data not shown). This finding provides evidence for post-transcriptional control of Oct-4 expression in specific human cells. In the study presented here, we observed that a subpopulation within amniotic fluid cell samples can be found to be Oct-4 positive. The fact that only 0.1–0.5% of the cells within such a positive sample express this transcription factor, together with the finding that only about half of the analysed amniotic fluid samples contained Oct-4-positive cells, suggests that (i) the Oct-4-positive signals are specific for a distinct subpopulation within an amniotic fluid cell sample and are not due to a low background of Oct-4 expression in all different cell types existing in such a sample; and (ii) Oct-4-positive cells cannot be detected in every analysed sample. The reasons for this observation still need to be clarified.
When ES cells are triggered to differentiate, Oct-4 is down-regulated. Loss of Oct-4 expression at the blastocyst stage causes the cells of the inner cell mass to differentiate into trophoectoderm cells (Pesce and Schöler, 2001). Experiments in mouse models indicate that an Oct-4 expression level of 50–150% of the endogenous amount in ES cells is permissive for self-renewal and maintenance of totipotency. Up-regulation above these levels causes stem cells to express genes involved in the lineage differentiation of primitive endoderm (Niwa et al., 2000). At present, this model has not been proven in humans. However, it could be considered of interest to compare the level of expression of Oct-4-positive amniotic fluid cells with that in different stem cell types and lines to obtain further insights into what type of stem cells are detected in amniotic fluid cell samples. Experiments with human ES cells or stem cell lines are forbidden in Austria.
To obtain further information about amniotic fluid cells in connection with stem cell research, we sought to answer two important questions. (i) Can other markers, known to be expressed in pluripotent human stem cells, be found in amniotic fluid samples? (ii) Are the Oct-4-positive amniotic fluid cells actively dividing? RT–PCR analyses revealed that in amniotic fluid cell samples containing Oct-4 positive cells, the mRNAs of three genes, known to be expressed in pluripotent human stem cells, can be detected: stem cell factor, vimentin and alkaline phosphatase. Double staining experiments further demonstrated that Oct-4-positive amniotic fluid cells can actively divide, which was proven by the detection of cyclin A expression. However, final proof for a putative pluripotency of such Oct-4-positive amniotic fluid cells would include experiments to isolate them and to differentiate them into different lineages. Such experiments are planned in our laboratory. We believe this new source of human stem cells to be interesting, even if these cells could be proven to harbour the potency to differentiate only in a specific subset of lineages. In this respect, it will also be of interest to try to elucidate the origin of the Oct-4-positive cells in the human amniotic fluid. There exist a lot of different possibilities, including, for example, aberrantly migrating primordial germ cells.
Another important marker for human pluripotent stem cells is telomerase activity (for a detailed description of such markers see http://www.nih.gov/news/stemcell/scireport.htm). All ES cell lines express high levels of telomerase, the enzyme that helps to maintain telomeres which protect the ends of chromosomes. Telomerase activity and long telomeres are characteristic of proliferating cells in embryonic tissues and of germ cells. Human non-transformed somatic cells, however, do not show telomerase activity and their telomeres are considerably shorter (Amit et al., 2000). Interestingly, telomerase activity can be detected in human amniotic fluid cell samples (Mosquera et al., 1999). This observation is in agreement with the assumption that such samples can contain stem cells of high potency, as suggested by our data.
We believe the findings reported here, together with the recent demonstration of the successful usage of amniotic fluid cells for tissue engineering approaches (Kaviani et al., 2001), to be encouraging for the further investigation of human amniotic fluid as a putative new source of stem cells with high potency.
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Submitted on October 2, 2002; resubmitted on February 13, 2003; accepted on March 18, 2003.
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