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Volume 53 (3): 337-339, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Trophoblasts Isolated from the Maternal Circulation : In Vitro Expansion and Potential Application in Non-invasive Prenatal Diagnosis
Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel-Hashomer, Israel
Correspondence to: Esther Guetta, Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel-Hashomer, Israel 52621. E-mail: Esther.Guetta{at}sheba.health.gov.il
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Prenatal diagnosis based on rare fetal cells in maternal blood is currently not a feasible option. An effort was made to improve cell yields by targeting trophoblast cells. After sorting, the HLA-G-positive cell fraction was analyzed directly or after culture. In situ hybridization technology was applied to prove fetal cell source in samples from women carrying a male fetus and to predict gender in samples without previous knowledge of fetal sex. In vitro culture led to a significant increase in fetal cells and accurate gender prediction in 93% of these samples. This approach might be useful for non-invasive prenatal diagnosis. (J Histochem Cytochem 53:337–339, 2005)
Key Words: trophoblast cells • prenatal diagnosis • cell sorting • in vitro culture • fluorescence in situ • hybridization
THE CONCEPT OF NON-INVASIVE PRENATAL DIAGNOSIS based on isolation of rare fetal cells from maternal blood has been the source of anticipation and enthusiasm over the past few decades for researchers dedicated to this field. However, the successes reported in the literature have yet to lead to development of a robust protocol that can be applied in the clinical laboratory.
Several groups of target cells have been studied, mainly nucleated red blood cells, progenitor blood cells, and trophoblast cells (Little et al. 1997
; Oudejans et al. 2003; Guetta et al. 2004). Each of these cell types offers advantages. However, none can be considered the optimal target cell. Common to all target cells that have been studied to date is the major disadvantage that they are present in maternal peripheral blood at the frequency of a rare event, one fetal cell in one million to ten million nucleated maternal blood cells (Hahn and Holzgreve 2002).
Trophoblast cells have been isolated from maternal blood by several different methods related to surface antigen expression, e.g., HLA-G, and cell size (van Wijk et al. 2001a; Vona et al. 2002). Proof of fetal origin as well as detection of aneuploidy and polyploidy were accomplished with fluorescence in situ hybridization (FISH) and PCR-based methods (van Wijk et al. 2001a,b; Vona et al. 2002).
Here we revisited trophoblasts as target cells and focused on an aspect of these cells that has not yet been pursued in the context of non-invasive prenatal diagnosis: the potential to grow in vitro under suitable conditions. We hypothesized that trophoblast cells derived from maternal blood can proliferate under in vitro culture conditions, thereby improving fetal cell detection and possibly enabling metaphase chromosome analysis.
To this end, the effect of in vitro culture on fetal cell yields as well as accuracy of gender prediction was studied in the HLA-G-positive fraction of sorted cells.
Peripheral blood samples (20 ml) were collected after obtaining informed consent from women at 17–20 weeks of pregnancy prior to their undergoing amniocentesis at our institute. The study was approved by the institutional Committee on Human Experimentation.
A group of 14 (D1–D14) samples from women carrying a known male fetus was analyzed directly. The samples were separated by single density-gradient centrifugation as previously described (Guetta et al. 2003). After centrifugation, the top cell layer containing mononuclear cells was washed and prepared for magnetic-activated cell sorting (MACS) enrichment. The cells were labeled with a mouse anti-human monoclonal antibody against HLA-G, clone MEM-G/9, at a 1:100 dilution in PBS/0.1% bovine serum albumin (catalog number MCA2044; Serotec, Oxford, UK). The HLA-G-positive fraction of cells was assessed by using an anti-HLA-G FITC-labeled antibody of the same clone used for the MACS enrichment described above (Figure 1A). As negative control, PBS was used instead of antibody. A magnetically labeled secondary antibody, goat anti-mouse IgG microbeads (catalog number 130-048401), was added, and the cells were separated on a MACS minicolumn (both from Miltenyi Biotech; Bergisch Gladbach, Germany) according to the manufacturer's instructions. The entire HLA-G-positive fraction was deposited on glass slides (Biogenex; San Ramon, CA) by centrifugation (Cytospin 3; Shandon, Manchester, UK) and processed for FISH analysis with sex chromosome probes (Guetta et al. 2003). A nucleus was considered to be of male fetal origin when one hybridization signal for each of the X-and Y-chromosome probes was observed. Fetal cells were detected in 11/14 samples (79%) with an average of 1.4 cells/20 ml blood (Figure 1; Table 1).
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A second group of 15 samples was collected without prior knowledge of fetal gender (C1–C15). The sorted HLA-G-positive fraction was seeded in Bio-AMF medium (Biological Industries; Beit Haemek, Israel) on Lab-Tek II-CC2 chamber slides (Nunc; Roskilde, Denmark), harvested after 5–7 days, and subjected to FISH analysis.
Fetal gender predicted by scoring the X and Y signals in the nuclei was compared with fetal sex determined by karyotype analysis of amniocytes. Gender prediction was successful in 14/15 samples (93%) (Table 1). The result from one sample (C-14) was false-positive, as it was obtained from a woman carrying a female fetus and one XY nucleus was detected. However, it should be noted that in all the true male samples, a minimum of two XY cells was detected. In the remaining six samples from women carrying a female fetus, fetal gender was predicted correctly. Fetal male cells were detected in 100% of the samples (8/8) from women carrying a male fetus compared with 79% of the samples analyzed without previous culture.
An average of 6.9 XY cells/20 ml blood was counted in the samples originating from women carrying a male fetus, representing a significant fivefold increase in the yield of fetal cells compared with samples directly analyzed (p<0.01).
A model system in which XY hybridization signals served as proof of fetal origin and fetal gender prediction was applied in this study. The results indicate that targeting trophoblast cells and exposing them to in vitro culture results in improved yields of fetal cells. A larger group of samples should be processed to confirm the findings of this preliminary study. Moreover, adjustments in the protocol, such as manipulation of the culture medium components, might further improve fetal cell yields.
A common problem exists with all the cell types investigated in the context of non-invasive prenatal diagnosis and remains beyond the scope of the current study: it is still not possible to define fetal origin based on sorting according to a surface antigen, and much improvement is needed to render this process more specific. Future advances in this area will be applied toward improving the specificity of the in vitro protocol.
It can be argued that the increase in fetal cells after culture represents clonal expansion of the rare cells detected in the direct analysis. In this case, the additional cells retrieved as a result of culture might be redundant and cannot be treated as individual clones. To circumvent this risk, the protocol could be adapted by seeding single cells in each well of a multi-well plate. Each arising colony could then be considered an individual clone. On the other hand, it is possible that the culture step actually represents an enrichment phase that enables the analysis of additional clones that are not detected in the direct method. Nevertheless, even if each cell does not represent a unique clone, the information derived would still be useful, especially if it will be possible to capture cells at the metaphase stage.
We are now investigating the possibility of combining the in vitro culture protocol described here with appropriate methods for metaphase arrest and processing to carry out chromosome analysis.
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Footnotes |
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Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich Schiller University, Jena, Germany, April 17–18, 2004.
Received for publication May 27, 2004; accepted September 2, 2004
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Literature Cited |
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TopSummaryLiterature Cited |
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Guetta E, Gordon D, Simchen MJ, Goldman B, Barkai G (2003) Hematopoietic progenitor cells as targets for non-invasive prenatal diagnosis: detection of fetal CD34+ cells and assessment of post-delivery persistence in the maternal circulation. Blood Cells Mol Dis 30:13–21[CrossRef][Medline]
Guetta E, Simchen MJ, Mammon-Daviko K, Gordon D, Aviram-Goldring A, Rauchbach N, Barkai G (2004) Analysis of fetal blood cells in the maternal circulation: challenges, ongoing efforts, and potential solutions. Stem Cells Dev 13:93–99[Medline]
Hahn S, Holzgreve W (2002) Prenatal diagnosis using fetal cells and cell-free fetal DNA in maternal blood: what is currently feasible? Clin Obstet Gynecol 45:649–656[CrossRef][Medline]
Little MT, Langlois S, Wilson RD, Lansdorp PM (1997) Frequency of fetal cells in sorted subpopulations of nucleated erythroid and CD34+ hematopoietic progenitor cells from maternal peripheral blood. Blood 89:2347–2358.
Oudejans CB, Tjoa ML, Westerman BA, Mulders MA, van Wijk IJ, van Vugt JM (2003) Circulating trophoblast in maternal blood. Prenat Diagn 23:111–116[CrossRef][Medline]
van Wijk IJ, de Hoon AC, Griffioen S, Mulders MA, Tjoa ML, van Vugt JM, Oudejans CB (2001b) Identification of triploid trophoblast cells in peripheral blood of a woman with a partial hydatidiform molar pregnancy. Prenat Diagn 21:1142–1145[Medline]
van Wijk IJ, Griffioen S, Tjoa ML, Mulders MA, van Vugt JM, Loke YW, Oudejans CB (2001a) HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy. Am J Obstet Gynecol 184:991–997[CrossRef][Medline]
Vona G, Beroud C, Benachi A, Quenette A, Bonnefont JP, Romana S, Dumez Y, et al. (2002) Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood. Am J Pathol 160:51–58
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