Obstetrics and Gynecology, University of Vienna, Prenatal Diagnosis and Therapy, Währinger Gürtel 1820, 1090 Vienna, Austria
1 To whom correspondence should be addressed. e-mail: markus.hengstschlaeger{at}akh-wien.ac.at
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: human amniotic fluid/Oct-4/pluripotency/stem cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RTPCR
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.
|
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).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blot analyses demonstrated Oct-4 protein expression in samples with positive RTPCR results (Figure 2). Immunocytochemical analyses of positive samples revealed that 0.10.5% 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).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 RTPCR. 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 RTPCR 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
0.10.5% 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
50150% 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? RTPCR 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.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Donovan, P.J. (2001) High Oct-ane fuel powers the stem cell. Nature Genet., 29, 246247.[CrossRef][ISI][Medline]
Gosden, C.M. (1983) Amniotic fluid cell types and culture. Br. Med. Bull., 39, 348354.[ISI][Medline]
Hengstschläger, M., Braun, K., Soucek, T., Miloloza, A. and Hengstschläger-Ottnad, E. (1999) Cyclin-dependent kinases at the G1S transition of the mammalian cell cycle. Mutat. Res., 436, 19.[CrossRef][ISI][Medline]
Hoehn, H. and Salk, D. (1982) Morphological and biochemical heterogeneity of amniotic fluid cells in culture. Methods Cell Biol., 26, 1134.[ISI][Medline]
Kaviani, A., Perry, T.E., Dzakovic, A., Jennings, R.W., Ziegler, M.M. and Fauza, D.O. (2001) The amniotic fluid as a source of cells for fetal tissue engineering. J. Pediatr. Surg., 36, 16621665.[CrossRef][ISI][Medline]
Kubista, M., Rosner, M., Kubista, E., Bernaschek, G. and Hengstschläger, M. (2002) Brca1 regulates in vitro differentiation of mammary epithelial cells. Oncogene, 21, 47474756.[CrossRef][ISI][Medline]
Martin, F.H., Suggs, S.V., Langley, K.E., Lu, H.S., Ting, J., Okino, K.H., Morris, C.F., McNiece, I.K., Jacobsen, F.W., Mendiaz, E.A., et al. (1990) Primary structure and functional expression of rat and human stem cell factor DNAs. Cell, 63, 203211.[ISI][Medline]
Milunsky, A. (1979) Amniotic fluid cell culture. In Milunsky, A. (ed.) Genetic Disorder and the Fetus. Plenum Press, New York, pp. 7584.
Monk, M. and Holding, C. (2001) Human embryonic genes re-expressed in cancer cells. Oncogene, 20, 80858091.[CrossRef][ISI][Medline]
Mosquera, A., Fernandez, J.L., Campos, A., Goyanes, V.J., Ramiro-Diaz, J.R. and Gosalvez, J. (1999) Simultaneous decrease of telomere length and telomerase activity with ageing of human amniotic fluid cells. J. Med. Genet., 36, 494496.
Niwa, H., Miyazaki, J-i. and Smith, A.G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self renewal of ES cells. Nature Genet., 24, 372376.[CrossRef][ISI][Medline]
Pesce, M. and Schöler, H.R. (2001) Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells, 19, 271278.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143147.
Prusa, A.R. and Hengstschläger, M. (2002) Amniotic fluid cells and human stem cell research: a new connection. Med. Sci. Monitor, 8, 253257.
Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, P.D., Huggins, G.R., Cui, Y., Cheng, L. and Gearhart, J.D. (2001) Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc. Natl Acad. Sci. USA, 98, 113118.
Takeda, J., Seino, S. and Bell, G.I. (1992) Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic Acids Res., 20, 46134620.[Abstract]
Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D. and Carpenter, M.K. (2001) Feeder-free growth of undifferentiated human embryonic cells. Nature Biotechnol., 19, 971974.[CrossRef][ISI][Medline]
Submitted on October 2, 2002; resubmitted on February 13, 2003; accepted on March 18, 2003.