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Prenatal Diagnosis of Genetic Abnormalities Using Fetal CD34⁺ Stem Cells in Maternal Circulation and Evidence They Do Not Affect Diagnosis in Later Pregnancies

^a Centre of Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy;
^b Department of Biomedical Sciences and Human Oncology, Division of Clinical Oncology, University of Torino, Torino, Italy;
^c Department of Clinical and Experimental Medicine, Haemathology and Clinical Immunology Section, University of Perugia, Perugia, Italy

Key Words. Fetal stem cells • Maternal blood • Noninvasive prenatal diagnosis • Isolation • Culture

Correspondence: Gian Carlo Di Renzo, Ph.D., Centre of Perinatal and Reproductive Medicine, University of Perugia, Policlinico Monteluce, Via Brunamonti, 06122 Perugia, Italy. Telephone: 39-075-5720563; Fax: 39-075-5729271; e-mail: direnzo{at}unipg.it

	ABSTRACT

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In the present study, we report a new method for enrichment and analysis of fetal CD34⁺ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34⁺ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities.

Peripheral blood samples were obtained from 35 pregnant women, 13 non-pregnant women who had given birth to male offsprings, 12 women who had never been pregnant, and eight pregnant women with male fetuses.

CD34⁺ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood.

Fetal/maternal cells can be isolated and grown "in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34⁺ stem cells.

The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34⁺ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies, it does not seem to affect this new system of enrichment, culture, and FISH analysis of CD34⁺ fetal stem cells.

	INTRODUCTION

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Prenatal diagnosis of genetic abnormalities can be performed by analyzing fetal cells cultured after recovery by amniocentesis or chorionic villus sampling or fetal blood [1–3]. Despite advances in safety and accuracy, these procedures carry a low, but well-documented, risk of fetal loss ranging from 0.5% to 1% [4, 5]. Therefore, they can be offered only to women at risk of bearing children with chromosomal abnormalities or at advanced maternal age (35 years at delivery) [5]. For these reasons, noninvasive methods for prenatal diagnosis are highly desiderable, because about 0.7% of all live-born infants have a congenital abnormality associated with chromosomal defect [6].

Different types of fetal cells are present (erythroblasts, lymphocytes, stem cells) in maternal circulation during pregnancy, thus providing a promising approach for noninvasive prenatal diagnosis of genetic abnormalities [7–10]. Many strategies have been designed to improve the recovery, purity, and yield of these fetal cells from maternal blood, but the success rate varies considerably [11–18]. The major limitations to most of these techniques are the few fetal cells which can be sorted from maternal blood samples [19–22] and the lack of a specific fetal cell marker [23–25]. Furthermore, the hypothesized persistance of fetal cells such as lymphocytes and CD34⁺/CD38⁺ cells in maternal blood for many years after delivery [26] has to date limited the application of these techniques as results in later pregnancies could be misleading. Indeed, fetal cells (such as lymphocytes and CD34⁺/CD38⁺ hematopoietic stem cells) have been claimed to persist in maternal blood after delivery [26] thus providing misleading results in later pregnancies.

To overcome the scarcity of fetal cells in maternal blood, an alternative strategy might be to culture and amplify those fetal cells which proliferate "in vitro." Recent reports have demonstrated maternal and fetal cells, such as erythroid precursors and CD34⁺ hematopoietic stem cells in maternal blood, are clonogenic [27–31].

Several studies have tried to expand "in vitro" fetal erythroblasts and fetal stem cells, isolated from maternal blood [27–31]. Results with erythroblasts, which proliferate to a limited extent, have been disappointing because there is no clear consensus about the degree of amplification of fetal erythroblasts, and because Chen et al. [32] recently reported that culturing fetal/maternal erythroblasts from maternal blood mainly produced erythroid colonies of maternal erythroid progenitor origin. Fetal blood CD34⁺ stem cells, which produce more colony-forming cells [27–30, 33], may theoretically be enough to diagnose genetic abnormalities. After purifying hematopoietic progenitors from umbilical cord blood, studies on proliferation showed the highest proliferative potential of fetal hematopoietic stem cells (hundreds to thousands of progeny). Therefore, stem cells seem to be the best cell type to amplify in cultures.

In the present study we report a new strategy for noninvasive prenatal diagnosis. After puryifing and expanding CD34⁺ stem cells from maternal blood, we used the fluorescence in situ hybridization (FISH) technique to detect genetic abnormalities during pregnancy. Furthermore, we determined whether fetal CD34⁺ stem cells persist in maternal blood after delivery.

	MATERIALS AND METHODS

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Patients
Peripheral maternal blood samples (20 ml) were obtained in heparinized test tubes from 31 consecutively primigravidae (all over 35 years of age) at 11-16 weeks of gestation. Blood samples were taken before amniocentesis performed in all because of age. Karyotyping confirmed fetal sex.

Immediately after amniocentesis, we also obtained maternal blood samples from two women with Down's syndrome fetuses, one woman with Edwards' syndrome fetus and another with Klinefelter syndrome fetus. Amniocentesis was performed because multiple ultrasound anomalies were observed (such as nuchal thickening and/or other abnormalities) and later confirmed by fetal karyotyping.

Peripheral blood samples from four non-pregnant adult females and three adult males were used as controls as well as three samples of male cord blood. None of the pregnant women and female controls had a history of blood transfusion, miscarriage or abortion before blood sampling.

In order to test the persistance of fetal CD 34⁺ stem cells in maternal blood after birth, 20 ml of peripheral blood were obtained from 13 women who were not pregnant but had given birth to one or two male offspring from 1 year to 21 years before blood sampling. As controls, we recruited 12 young women who had never been pregnant or received blood tranfusions and eight pregnant women carrying male (as visualized by ultrasound) fetuses at 11-16 weeks of gestation. Karyotyping confirmed fetal sex.

The study was approved by the Umbria Ethics Committee, and informed consent was obtained from all pregnant and nonpregnant women enrolled in this study.

Enrichment of Stem Cells (CD34⁺ Stem Cells)
In order to obtain an enriched CD34⁺ stem cell population from the peripheral blood sample, peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque density separation (1.077 g/ml density gradient, Sigma Diagnostic; St. Louis, MO; http://www.sigma-aldrich.com). Isolated and washed PBMCs were split into two equal parts: one was cultured in a semisolid methylcellulose medium and the other underwent CD34⁺ stem cell enrichment as reported previously [31].

Briefly, CD34⁺ stem cells were enriched using MiniMACS magnetically activated cell sorting (Miltenyi Biotech; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com). CD34⁺cells were sorted by positive selection of CD34-expressing cells (clone: QBEND/10, isotype, mouse IgG1) as previously described [31].

Enriched CD34⁺ stem cells were seeded in a semisolid methylcellulose culture medium. In some maternal blood samples, FISH analysis was performed on enriched CD34⁺ stem cells as such and therefore immediately fixed on membrane filters.

Cell Cultures
1 x 10⁵ enriched PBMCs and 1 x 10⁴ CD34⁺ stem cells were cultured in Iscove's modified Dulbecco's medium (Methocult GF H4435, Stem Cell Technology; Vancouver, Canada; http://www.stemcell.com) as previously reported [31]. Colonies were then observed under an inverted microscope and were classified as BFU-E/CFU-E (colony-forming unit-erythroid) and CFU-GM (colony-forming unit-granulocyte, macrophage). The cells were harvested and nucleated cells counted using a Burker hemocytometer and expressed as total number x10⁵ cells [31].

FISH

Sample Analysis   We performed FISH analysis of uncultured CD34⁺ cells and nucleated cells after CD34⁺ cell cultures from normal maternal blood samples. Samples from nucleated cells (obtained after enrichment and culture) were also analyzed from all pregnancies with aneuploid fetuses.

In 5 of 12 blood samples from pregnant women with normal male fetuses and in Klinefelter and Edwards' syndromes, FISH was performed in samples obtained after culture of unenriched PBMC and enriched CD34⁺ stem cells in order to evaluate the efficacy of CD34⁺ enrichment as determined by recovery of fetal stem cells.

Samples from nucleated cells (obtained after enrichment and culture) were analyzed from all pregnancies with aneuploid fetuses.

Preparation of Slides   The noncultured CD34⁺ cells were immediately fixed with a solution of formaldehyde (10%) and incubated for 3 minutes; after centrifuge at 1,200 RPM for 5 minutes, the preparation was filtered through membrane filters (Nucleopore Track-Etch Membrane, Corning Separation Division; New York, NY) and stored frozen at –20°C until use.

Cultured CD34⁺ cells were processed as previously described [31] and seeded directly on a glass slide. The slides were frozen at –20°C and stored until use.

Fluorescent Probes   The target chromosome was identified by fluorescent probes which varied according to the clinical indication of pregnancy.

To distinguish fetal male cells from the maternal cells, X and Y chromosomal analysis was performed using CEP X ( satellite) SpectrumOrange hybridizing centromere of human chromosome X (bands Xp 11.1-q11.1, locus DXZ1) and CEP Y ( satellite) SpectrumGreen hybridizing centromere of human chromosome Y (bands Yq 12, locus DYZ1; Vysis; Downers Grove, IL).

To detect fetal cells with trisomy 21, slides were processed with TriGen LSI 21 SpectrumOrange (21q22.13-q22.2) and CEP X SpectrumGreen/CEP Y-alpha SpectrumOrange DNA FISH Panel (Vysis).

To detect fetal cells with trisomy 18, slides were processed using CEP 18 ( satellite) SpectrumOrange hybridizing centromere of human chromosome 18 (18p11.1-q11.1; Vysis). The sample slides were processed by FISH procedure as described above for CEP XY.

Hybridation was performed according to the manufacturers' instructions [31].

Detection of Fetal Cells   Slides were analyzed by a fluorescence microscopy (Olympus BH-2, Tokyo, Japan) equipped with a triple bandpass filter set (DAPI/Green/Orange, Vysis) designed to visualize the SpectrumGreen and SpectrumOrange. The fluorescence microscope is equipped with a PM20 automatic photomicrographic system (Olympus).

Male (fetal) cells had one red and one green fluorescent signal; female (maternal) cells had two red fluorescent signals. The fetal cells were scored and the percentage was calculated.

Trisomic fetal cells had three red chromosome 21 fluorescence signals. The maternal cell population had two. Fetal trisomic cells were scored and the percentage was calculated.

Trisomic fetal cells had three red chromosome 18 fluorescent signals. The maternal cell population had two. Fetal trisomic cells were scored and the percentage was calculated.

Polymerase Chain Reaction (PCR) Analysis on Sorted CD34⁺ Stem Cells   The persistence of male DNA in sorted CD34⁺ cells was evaluated after MiniMACS enrichment (as described above) in blood samples from women who were carrying male fetuses and in the never- and non-pregnant women.

Genomic DNA was extracted by boiling the CD34⁺ sorted cells for 5 minutes. To detect Y-positive cells, a nested PCR protocol with primers Y1.5, Y1.6, Y1.7, Y1.8 (GeneAmp, Perkin Elmer Cetus; Norwalk, CT) already described by Lo et al. [34] was used. In dilution experiments this system showed a very high sensitivity with 1 positive cell in 10⁵ to 10⁶ negative cells. Extreme caution was taken when preparing sorted cells for PCR amplification to avoid false-positive results and PCR carryover as previously suggested [35]. All laboratory technicians were women. Correct diluted positive and negative internal controls were mandatory preliminary criteria in the validation of each PCR amplification experiment. After PCR amplification one-tenth of the PCR product was separated by 2% agarose gel and evaluated for the presence of the 198 bp positive product.

In five cases with PCR positivity for Y-chromosome (two patients who gave birth to boys, one patient who had never been pregnant and two pregnant woman) and two cases with PCR negativity for Y-chromosome (two pregnant women), we used FISH analysis on CD34⁺ preparation without culture and on cultured nucleated cells as a second check for the Y-chromosome.

Statistical Analysis   Intergroup differences were determined using the ANOVA-test. For all statistical comparisons p values less than 0.05 were considered statistically significant. Results are expressed as means ± standard deviation (SD).

FISH sensitivity was calculated as (true-positive/true-positive + false-negative) x100; true-positive is the male fetus correctly identified and false-negative represents the identification of female fetus in presence of male fetal sex.

The purity of fetal cells was calculated as a percentage:

	RESULTS

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In peripheral blood samples from normal and abnormal pregnant women, a mean of 14.7± 4.6 x 10⁶ (range, 4.9-21.2 x 10⁶) of PBMCs was isolated after gradient density centrifugation. The number of CD34⁺ stem cells was a mean of 72.6 ± 71.4 x 10³ (range, 4.5-28.0 x 10³) cells after MiniMACS separation. The recovery of CD34⁺ stem cells was 88.5 ± 28.7% with purity reaching 55.1 ± 19.3%.

After the culture period of 14 days in a semisolid culture medium, we obtained a total number of nucleated cells of 18.1 ± 5.7 x 10⁵.

FISH Analysis of CD34⁺ Stem Cells
The hybridation specificity of FISH was 95%, 88.9% and 96.3% when 100% pure female, male, and male cord mononuclear cells were respectively analyzed. No false-positives detecting XY or XX signals in female or male preparations, respectively, were detected.

Table 1 shows the results of FISH analysis of enriched uncultured CD34⁺ stem cells. In eight cases of samples from women carrying male fetuses (Pt. Nos. 1-8) we detected fetal CD34⁺ stem cells in only two cases and their sex chromosomes were correctly identified by FISH. We found a percentage gender prediction of 25%.

Table 1 also shows the results of FISH analysis on enriched, cultured CD34⁺ stem cells in normal and abnormal pregnancies. In normal pregnancies (Nos. 15-26), the range of visualized fetal cells was between 0 to 11 cells on a range of scored maternal cells between 100 to 2,499 (mean ± SD, 711 ± 603). The percentage of gender prediction accuracy was 83.3% (10/12). In the five cases of pregnancy with female fetuses, we did not observe any cell with XY fluorescent signals in cultured CD34⁺ stem cells. Results were confirmed by chromosome analysis.

FISH Analysis of CD34⁺ Stem Cells on Trisomic Fetuses (Nos. 32-35)
We detected trisomic fetal cells after CD34⁺ cell cultures in all the maternal samples. When we analyzed samples from pregnancies involving 21-trisomic fetuses, we scored 19 and 8 trisomic fetal cells in 659 and 100 maternal cells, respectively. The ratio of fetal to maternal cells was 1/34 and 1/12 with a fetal purity cell percentage of 2.9% and 8.0%.

When we analyzed a sample from one pregnancy involving an 18-trisomic fetus, we found seven trisomic fetal cells out of 125 scored maternal cells. The ratio of fetal/maternal cells was 1/17 with a fetal purity cell percentage of 5.6%.

Table 2 shows the efficacy of CD34⁺ stem cell enrichment in pregnancies with normal (Nos. 15-19) and abnormal fetuses (Nos. 32-35), as determined by recovery of fetal stem cells. After PBMC culture, the number of XY fetal cells detected in normal maternal blood ranged from 0 to 5 (on 648 ± 362 scored maternal cells). After CD34⁺ stem cell enrichment and culture, it ranged from 4 to 11 (on 663 ± 562, scored maternal cells). Interestingly, in sample No. 16, we did not observe fetal XY cells after PBMC culture, but we detected male fetal cells after enrichment and culture of CD34⁺ stem cells.

After culture of PBMCs from a pregnancy with an 18-trisomic fetus (No. 35), we found three trisomic fetal cells in 127 scored maternal cells with a ratio of 1/42 and a fetal cell purity percentage of 2.3%. We observed an increase in the number of fetal cells after enrichment and CD34⁺ cell culture (Table 2). Interestingly, in this pregnancy, we found one trisomic metaphase, confirming that fetal cells can divide actively "in vitro" (Fig. 1).

View larger version (69K): [in this window] [in a new window]   Figure 1. Metaphase of cultured CD34+ stem cells from pregnancy with fetus with trisomy 18. Fetal methaphase shows three 18 orange spots and a maternal nuclei with two 18 orange spots.

In the 13 women who had given birth to one or more sons years earlier (mean ± SD, 10 ± 7.4; range, 1-21 years), we detected male DNA in three (23%) (Table 3).

There was no evidence of false-positive amplification in reagent controls and in the preparation of the CD34⁺ stem cells.

In three women (two women who had given birth to sons and one woman who had never been pregnant) with positivity to male DNA (samples with asterisk on Table 3), a mean of 721.0 ± 333.8 CD34⁺ stem cells without culture (range: 326-1,139) and another aliquot of CD34⁺ cells after culture (mean 972.2 ± 205.9, range 839-1,278, NS), were analyzed by FISH to check for male fetal cells. We did not find cells with male XY chromosomes in any of these three women. We also analyzed consecutively two pregnancies with positivity to male DNA and two pregnancies who were negative male DNA (samples with asterisk on Table 3). A mean of 780 ± 104.8 CD34⁺ stem cells without culture (range: 695-865) and after culture (mean 860 ± 120.6, range 775-945) was analyzed by FISH to check for male fetal cells. After enrichment of CD34⁺ stem cells, we observed male fetal cells in one of four samples (patient No. 26, Table 3); while after culture we found male cells in all samples (mean 3 ± 1.6, range 1-5).

	DISCUSSION

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Studies on human maternal blood for the presence of cells bearing the Y chromosome demonstrated that fetal cell migration in maternal blood occurs during pregnancy [36]. Recently, fetal clonogenic cells such as hematopoietic progenitors and stem cells have been found in maternal circulation from early gestation onwards [27–31]. Although to date some success has been reported in using them for prenatal diagnosis [27–31], the degree of their expansion is insufficient against the higher number of background hemopoietic maternal cells.

Stem cells are characterized by the expression of the CD34⁺ cell-surface antigen [37–39]. Some studies have demonstrated the fetal blood presents a higher frequency of hematopoietic progenitors than adult peripheral blood [39]. In particular, fetal blood from an early second-trimester fetus has an elevated number of stem cells compared with term umbilical cord blood, adult peripheral blood and bone marrow [40]. Furthermore, fetal blood at 16 weeks' gestation has a frequency of BFU-E that is 35 times higher than in adult peripheral blood [39]. All these data suggest that early fetal blood (<24 weeks' gestation) has a great frequency of hematopoietic progenitors/stem cells and a powerful clonogenic potential, and that these cells commonly cross the placental barrier into maternal circulation. So, fetal stem cells appear to be the best cell type to isolate from maternal circulation and to culture.

Our results demonstrate fetal/maternal cells can be isolated and grown "in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than results obtained from uncultured CD34⁺ stem cells. We also proved that during the culture phase, clonal amplification of fetal cells occurred. Indeed, we observed two fetal metaphases (one normal and one with 18 trisomy), providing evidence that fetal cells can actively divide "in vitro." One further point is worth noting: as we cultured only blood samples from primigravidae, we are sure the clonal amplification occurred on fetal cells of these ongoing pregnancies.

We also evaluated the potential loss of fetal CD34⁺ stem cells during cellular enrichment and found fetal cell recovery was improved after CD34⁺ cell enrichment and the fetal/maternal cell ratio was higher. The same results were obtained from maternal samples with aneuploid fetuses. Consequently, our data demonstrate that enrichment procedures for CD34⁺ stem cells do not cause fetal cell loss [29]. Although our methodology increases the number of post-culture fetal cells, it does not entirely overcome the problem of the maternal cell background. Further studies will focus on maternal cell suppression and selective "in vitro" culture of fetal cells. Investigations into the best culture time for fetal CD34⁺ cell growth [29, 38], supplements for culture media [41, 42], and biological differences between fetal and maternal cells appear to be the most promising approaches [18, 43].

Like other studies [44–47], we detected many more fetal cells in aneuploid pregnancies than in normal pregnancies. As we sampled maternal blood immediately after amniocentesis in these cases, an objection might be that the invasive procedure could have affected our results. However, a recent study reported amniocentesis does not significantly influence the passage of fetal cells into maternal blood, so the number of fetal aneuploid cells we observed is probably reliable [44].

The mechanisms that lead to an increase in fetal aneuploid cells in maternal blood are still unclear, but there is evidence to support the hypothesis that trisomy-related abnormalities in the placenta barrier may increase the trafficking of fetal cells into maternal circulation [48, 49].

A second goal of our study was to determine persistence of fetal stem cells from previous pregnancies in maternal circulation and the role of such cells in our FISH-based prenatal diagnostic system. Fetal stem cells have been postulated to persist in maternal blood and be detected by PCR amplification of Y chromosome-specific genomic sequences for as long as 27 years postpartum [26] suggesting that a status of long-term cellular microchimerism is induced in maternal circulation [50]. The obvious consequence is that results of prenatal diagnostic tests in subsequent pregnancies can be altered by persistence in maternal blood of these progenitor cells. An alternative explanation is that positive cells may have paternal cells since fallopian tubes are open to the peritoneal cavity. Sexual activity may continue into pregnancy [51] and cellular microchimerism may be induced [52]. Fetal cells in maternal blood have been demonstrated to have a mean half-life of 16.3 minutes and therefore, there is a rapid clearance of fetal cells from maternal circulation [53, 54].

Using a well-described PCR amplification protocol [34], we identified Y positivity in samples from 3/13 mothers who gave birth to male offspring, 2/8 women who were pregnant with male fetuses and 1/12 women who had never been pregnant. The karyotype of this woman was a normal 46, XX. Two other pregnant women with negative PCR analysis had previously given birth to male children. The somewhat higher percentage of false-negative women in our experience could be due to patient characteristics, such as the number of previous pregnancies with male fetuses or different subsets of fetal cells analyzed. We used our FISH-based method to test two of three positive PCR samples obtained from mothers who gave birth to male children and the one positive sample from a woman who had never been pregnant. CD34⁺ stem cells were enriched and analyzed as such and after culture. FISH analysis did not detect any male fetal cells in any sample. Therefore, even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies, that does not seem to affect at all the FISH analysis system we describe in the present sudy.

Overall, the fetal cell purity we achieved is indicative of ongoing fetal development rather than replication of fetal cells from previous pregnancies. Furthermore, the results of FISH analysis of samples with a positive PCR confirm this hypothesis.

	ACKNOWLEDGMENT

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The authors would like to thank Dr. Geraldine A. Boyd for reviewing the manuscript. This work was in part supported by a grant from CNR and Fondazione Cassa di Risparmio di Perugia and by Associazione Italiana Ricerca sul Cancro (AIRC).

Profs. Antonio Tabilio and Gian Carlo Di Renzo contributed equally to this work.

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