Effect of preeclampsia on umbilical cord blood hematopoietic progenitor-stem cells

Transcription

1 Effect of preeclampsia on umbilical cord blood hematopoietic progenitor-stem cells Daniel V. Surbek, MD, a Enrico Danzer, a Christian Steinmann, MD, a André Tichelli, MD, b Aleksandra Wodnar-Filipowicz, PhD, c Sinuhe Hahn, PhD, a and Wolfgang Holzgreve, MS, MD a Basel, Switzerland OBJECTIVE: The aim of the present study was to determine the influence of preeclampsia on cord blood hematopoietic progenitor-stem cells obtained at delivery because cord blood is increasingly used clinically for stem cell retrieval as an alternative to bone marrow. STUDY DESIGN: Umbilical cord blood was collected from patients fulfilling the criteria for preeclampsia and from gestational age and birth weight matched control subjects at delivery (patient/control subjects ratio, 1:2). Cord blood volume and nucleated cell content were measured, and the number of hematopoietic progenitor-stem cells was determined by means of fluorescence-activated cell sorting with the CD34 + epitope and by means of colony assays with different hematopoietic growth factors. In addition, the expression of adhesion molecules by CD34 + progenitor-stem cells was examined. RESULTS: In pregnancies affected by preeclampsia, volume and nucleated cell and total CD34 + cell contents in the collected cord blood were significantly smaller compared with those of control subjects. Furthermore, there was a trend toward a smaller relative number of CD34 + cells and colony-forming units per nucleated cell in cord blood samples from preeclamptic patients. No difference in the expression of the cell-adhesion molecules leukocyte function associated antigen 1, very late activation antigen 4, and L- selectin by CD34 + cells could be found. CONCLUSION: This study shows that preeclampsia affects umbilical cord blood volume and nucleated cell and progenitor-stem cell numbers obtained at birth. ( 2001;185:725-9.) Key words: Umbilical cord blood, hematopoietic stem cells, preeclampsia Umbilical cord blood (also referred to as residual placental blood) is increasingly used as a source of hematopoietic stem cells for related 1 and unrelated 2 transplantation. Cord blood stem-progenitor cells have been shown to have increased proliferative capacity compared with that of adult bone marrow. 3 Furthermore, recipients of stem cell transplants from cord blood are less likely to have severe graft-versus-host disease. 4 One major disadvantage, however, is the limited amount of cells obtainable after a full-term delivery, and many samples do not contain enough cells for successful marrow reconstitution in adults. 5 This is of particular importance because the outcome of hematopoietic stem cell transplantation From the Department of Obstetrics and Gynecology, a and the Laboratory of Hematology b and the Department of Research, c Division of Experimental Hematology, University of Basel. Supported by a research grant from the Basel Cord Blood Project Foundation. Received for publication January 3, 2001; revised April 27, 2001; accepted May 18, Reprint requests: Daniel V. Surbek, MD, Department of Obstetrics and Gynecology, University Hospital, Schanzenstrasse 46, 4031 Basel, Switzerland. Copyright 2001 by Mosby, Inc /2001 $ /1/ doi: /mob with cord blood has been shown to correlate with the number of nucleated and hematopoietic progenitor-stem cells transplanted in relation to the recipient s weight. 2 The yield of nucleated cells and hematopoietic progenitor-stem cells collected from cord blood is therefore important, especially if the cells are collected for transplantation in a sibling affected by a genetic or malignant disorder. We and others have previously shown that gestational and obstetric factors, including gestational age, 6 length of labor, 7 stress during labor, 8 mode of delivery, 9 cord blood collection technique, 10, 11 and positioning of the newborn after delivery, 12 can have an influence on the amount of cord blood cells obtained after delivery. However, little information about the effect of common disorders complicating pregnancy is available. In particular, it is not known whether preeclampsia affects cord blood hematopoietic cells. Because previous studies have suggested that fetuses from preeclamptic mothers show reduced hepatic hematopoiesis, 13 the amount of circulating progenitorstem cells in cord blood might be altered. The aim of our study was therefore to determine whether there is a difference between number of cord blood hematopoietic cells obtained from preeclamptic versus nonpreeclamptic patients. In addition to obtaining new knowledge about the 725

2 726 Surbek et al September 2001 pathophysiology of preeclampsia, we wanted to find out for practical reasons whether cord blood from preeclamptic women is suitable for stem cell retrieval. Patients and methods This prospective case-control study was approved by the institutional review board of the University Hospital of Basel. We consecutively included patients with preeclampsia fulfilling the following criteria: (1) minimum gestational age of 24 weeks, as confirmed by firsttrimester ultrasonography; (2) blood pressure above 140/90 mm Hg on 2 or more occasions and proteinuria of at least 0.3 g/l; and (3) willingness to participate in the study with written informed consent. The control group consisted of gestational age and estimated fetal weight matched pregnant women delivered of their infants preterm or at term without signs of preeclampsia. Matching for gestational age and birth weight was performed to exclude bias from these factors, which have been shown to affect cord blood hematopoietic cells. 6, 14 For every preeclamptic patient, control subjects were matched according to gestational age and birth weight at a patient/control subject ratio of 1:2. Cord blood collection. At delivery, cord blood was collected from the umbilical vein with the placenta in situ by using a closed collection system containing citratephosphate-dextrose. Briefly, the cord was clamped and dissected within 30 seconds after delivery of the newborn, according to the routine management protocol of our obstetric service. Modification of cord clamping during the study was not allowed. Initially, an umbilical cord fragment of 3 to 5 cm in length was obtained to assess arterial and venous cord blood acid-base status. Thereafter, the umbilical vein was punctured, and cord blood was collected by means of passive drainage into a sterile, closed blood-collection system containing citrate-phosphatedextrose. If collection of cord blood could not be performed before placenta expulsion for any reason, it was done after expulsion in the same fashion. In case of cesarean section, the same procedure was performed, as described elsewhere. 11 The identical collection technique was used for both the preeclamptic and control groups. Cord blood analysis. The volume of collected cord blood was determined by subtracting the volume of citrate-phosphate-dextrose in the bag before collection from the total measured volume in the bag. Nucleated cell content per milliliter of cord blood was assessed with a Technicon H*3 RTC (Bayer Technicon), and the total amount of nucleated cells per cord blood sample was calculated by multiplication with the volume contained in the bag. The fraction of CD34 + cells within the leukocyte population was determined by means of dual-color flow cytometry (fluorescence-activated cell sorting) with FACScan (Becton-Dickinson). Briefly, red blood cell lysis was performed with Ortho-mune lysing reagent (Ortho Pharmaceuticals). For staining of progenitor-stem cells, phycoerythrin cyanid labeled anti-cd45 (Immunotech) and phycoerythrin-labeled anti-cd34 (Becton-Dickinson) were used, with CD34 being a surrogate marker for hematopoietic stem cells. 15 The gating strategy consisted of a first gate (G1) of CD45 + intermediate-to-high forward-scatter events (leukocyte gate) and a second gate (G2) of CD34 + high-low side-scatter events (CD34 + gate). The proportion of CD34 + cells among the leukocytes (nucleated cells) was calculated as the proportion of G1 within G2; a minimum of 75,000 events were acquired and analyzed with Cellquest 3.1f software (Becton-Dickinson). The total amount of CD34 + cells per cord blood sample was calculated by multiplication of the relative CD34 + cell count (percentage) with the total amount of nucleated cells. We also compared the profile of β 2 -integrin leukocyte function associated antigen (LFA-1; CD11a/CD18), the β 1 -integrin very late activation antigen 4 (VLA-4; CD49d/CD29), and L-selectin (CD62L) expression on CD34 + cells. These adhesion molecules were chosen because of previous reports about their role in the migration (mobilization and homing) of hematopoietic stem cells. 16 Fluorescein isothiocyanate labeled CD11a, CD49d, and CD62L antibodies (all from Immunotech) were applied for cell adhesion molecule staining. Three-color flow cytometry (FACScan, Becton-Dickinson) was used for cell analysis. The CD34 hi population was gated versus low side scatter. At least 1000 events were obtained within the CD34 gate. The proportion of CD34 + cells coexpressing the respective adhesion molecule was calculated. A 2-week assay of colony-forming units (CFUs) was used to assess the proliferative capacity of hematopoietic progenitors in vitro. Mononuclear cells were isolated by means of Ficoll density centrifugation, resuspended in Iscove s modified Dulbecco s medium, and allowed to adhere overnight. Nonadherent mononuclear cells were plated into 1% methylcellulose culture medium containing fetal calf serum, bovine serum albumin, and transferrin supplemented with either erythropoietin alone or in combination with granulocyte colony-stimulating factor, granulocyte monocyte colony-stimulating factor, interleukin 3, and stem cell factor. After 14 days, the number of CFUs per 10 5 plated mononuclear cells was counted. Statistical analysis. The number of nucleated cells per cord blood sample was chosen as the primary outcome parameter because of the known relation to outcome after transplantation. 2 The sample size had a power of 80% to detect a clinically significant difference of 40% in the cord blood nucleated cell number at a significance level of.05, assuming a standard deviation of ± nucleated cells, as observed in previous studies. 11 Continuous nonparametric data of preeclamptic and control pregnancies were compared with the Mann-Whitney U

3 Volume 185, Number 3 Surbek et al 727 Table I. Maternal characteristics and perinatal data Preeclamptic group (n = 11) Control group (n = 22) P value* Maternal age (y) 29.4 ± ± Gestational age (wk) 37.3 ± ± BP (systolic; mm Hg) 174 ± ± BP (diastolic; mm Hg) 100 ± 3 73 ± Proteinuria (>0.3 g/l) 11 (100%) 3 (14%).001 Vaginal delivery 7 (64%) 10 (46%).465 Birth weight (g) 2910 ± ± Placental weight (g) 496 ± ± Fetal sex male 7 (64%) 11 (50%).712 Apgar score <8 at 5 min 0 0 Umbilical artery ph 7.25 ± ± Umbilical vein ph 7.32 ± ± Values are means ± SEM where indicated. BP, Maximum blood pressure. *Mann-Whitney U test (continuous data) or Fisher exact test (categoric data) comparing preeclamptic group versus control group. Table II. Cord blood data Preeclamptic group (n = 11) Control group (n = 22) P value* Volume (ml) 37 ± 6 67 ± Nucleated cells ( 10 8 ) 4.6 ± ± CD34 + cells ( 10 5 ) 11.3 ± ± CD34 + cells (%) 0.22 ± ± CFUs (EPO) 56 ± 9 64 ± CFUs (EPO/G-CSF/GM-CSF/IL-3/SCF) 135 ± ± CD11a + cells (%) 49.7 ± ± CD49d + cells (%) 92.1 ± ± CD62L + cells (%) 76.4 ± ± Values are means ± SEM where indicated. EPO, Erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte monocyte colony-stimulating factor; IL-3, interleukin 3; SCF, stem cell factor. *Mann-Whitney U test comparing preeclamptic versus control group. Numbers of CFUs in methylcellulose cultures with erythropoietin alone or in combination with granulocyte colony-stimulating factor, granulocyte monocyte colony-stimulating factor, interleukin 3, and stem cell factor are given per nonadherent mononuclear cells. Proportion of CD34 + cells coexpressing the adhesion molecules CD11a, CD49d or CD62L, respectively. test, and the Fisher exact test was used for categoric variables. A 2-tailed P value of less than.05 was considered significant. The SPSS for Windows software package was used for calculations. Results The results of the comparison of maternal characteristics and perinatal data between the preeclamptic and control groups are summarized in Table I. Although the difference in maximum systolic and diastolic blood pressure and presence of proteinuria was pronounced (as expected), there was no difference regarding perinatal parameters, including gestational age, neonatal and placental weight, and short-term neonatal outcome. No statistically significant difference between the groups could be found regarding intrapartum fetal distress (as defined by nonreassuring fetal heart rate tracings) or obstructed labor as an indication for cesarean section. Intravenous β-mimetics were administered to 18% and 23% in the preeclampsia and control groups, respectively (P = not significant), and 27% and 23%, respectively, received 1 or 2 courses of betamethasone for induction of lung maturation (P = not significant). In the preeclamptic group 73% were given intravenous magnesium sulfate for prevention of eclamptic seizures. We found, however, a statistically significant and large difference in cord blood volume and total nucleated cell number, being almost twice as low in the preeclamptic group compared with the control group (Table II). There was also a lower relative number of CD34 + cells in cord blood from preeclamptic patients (0.22% vs 0.35%), although this difference did not reach statistical significance. Functional progenitor cell assessment showed only a very limited and nonsignificant difference in the number of CFUs among nucleated cells; samples from preeclamptic patients produced less CFUs in erythropoietin- and mixed growth factor supplemented cultures. Nevertheless, in conjunction with the smaller number of nucleated cells, a significantly lower absolute CD34 + cell count per cord blood sample was seen in the

4 728 Surbek et al September 2001 preeclamptic group. In contrast, no clear difference in expression of the adhesion molecules LFA-1, VLA-4, and L-selectin could be identified between the cord blood from preeclamptic patients and that from control subjects. The comparison between patients with mild-tomoderate preeclampsia (n = 6) and those with severe preeclampsia (n = 5) did not show any difference in cord blood values. Comment In this study we found that cord blood volume and nucleated cell and CD34 + cell counts were smaller in pregnant patients affected by preeclampsia compared with gestational age and birth weight matched control subjects. These differences are not due to lower placental weight or intrapartum factors (eg, fetal distress or prolonged labor) because there was no significant variation between the groups regarding these factors. Accordingly, the numbers of patients receiving β-mimetics and betamethasone were similar, and magnesium sulfate (which was used in most patients with preeclampsia) is not known to have a significant influence on hematopoietic cells or fetoplacental blood volume. Although other studies have examined the influence of gestational and perinatal factors on umbilical cord blood hematopoietic cells, 7-9, 14 this is the first published study addressing the question of effects of preeclampsia on cord blood hematopoietic cells. Hiett et al 14 have previously studied cord blood obtained from small-for-gestational-age neonates. They found a significantly smaller number of progenitors compared with numbers found in gestational age matched neonates of appropriate weight. Although some fetuses from mothers affected by pregnancy-induced hypertension or preeclampsia were included in this series, this subgroup was not identified separately from other growthrestricted fetuses. Recently, a histomorphometric study found a decreased number of erythroid precursors and early granulopoietic cells in the liver of fetuses affected by preeclampsia. 13 The authors of this report suggested that these alterations in fetal hematopoiesis are a consequence of preeclampsia itself rather than growth restriction alone. Our data support these findings. The smaller amount of hematopoietic cells in the fetal liver and in fetal blood might be the result of an unspecific suppression of hematopoietic activity in the fetus. The exact pathophysiologic mechanism leading to the reduced number of hematopoietic progenitors in cord blood from fetuses affected by preeclampsia, however, is unknown. One possible explanation might be the influence of cytokine and growth factor patterns in fetuses affected by preeclampsia. The regulation of hematopoiesis in the fetus, including the physiologic change of the predominant site of hematopoiesis from the fetal liver to the fetal bone marrow toward the end of gestation, depends on complex interactions between hematopoietic cells, hematopoietic stroma (microenvironment), and circulating cytokine-growth factors, leading to ontogenetically predetermined patterns of self-renewal, proliferation, apoptosis, lineage-specific differentiation, and migration of hematopoietic progenitor cells and stem cells. 17, 18 Several studies have shown that the level of cytokines and growth factors in fetal blood are altered in preeclampsia. 13, 19 Although the addition of granulocyte colonystimulating factor in escalating doses in vitro apparently does not affect the short-term proliferative capacity of progenitors, 20 a long-term alteration of the pattern of hematopoietic growth factors and cytokines in the fetus induced by preeclampsia might have an influence on hematopoietic activity in the progenitor cell population. In this study we further determined whether the expression of LFA-1, VLA-4, and L-selectin on hematopoietic progenitors differs in preeclampsia. These cell-adhesion molecules are involved in molecular interactions between hematopoietic and stromal cells, which are important determinants of the process of migration, mobilization, and homing of hematopoietic progenitor-stem cells. 21 We and others 22, 23 have described development stage-specific differences of expression patterns in fetal hematopoietic cells, suggesting that these patterns are likely to be involved in the ontogenesis of the predominant site of hematopoiesis. In the present study we did not find a difference in the expression of LFA-1, VLA-4, and L-selectin in preeclampsia. From this finding, we conclude that it does not seem likely that the lower number of hematopoietic cells in the fetal peripheral blood is due to increased homing in the bone marrow niches mediated by increased adhesion molecule expression. Regarding the question of whether cord blood from preeclamptic patients can be used for stem cell retrieval, it must be realized that the difference in the relative progenitor-stem cell content among nucleated cells was rather limited and statistically nonsignificant in our study. However, the difference in absolute numbers of CD34 + cells is clinically significant in conjunction with the decreased cord blood volume, leading to a clearly lower yield of hematopoietic cells per cord blood sample. Accordingly, the content of CFUs per cord blood sample is also lower (data not shown). The large SD of the relative content of these cells might be the cause for the lack of statistical significance in our limited sample size. This large variation in relative hematopoietic cell content has to be taken into account if only the nucleated cell number is used to determine whether a cord blood sample is appropriate for transplantation. From our findings, we conclude that patients with preeclampsia should be excluded a priori from unre-

5 Volume 185, Number 3 Surbek et al 729 lated cord blood banking. In contrast, if targeted cord blood collection and banking for stem cell transplantation in a family member is planned and preeclampsia occurs, the collection of cord blood is still justified. This is because despite the lower cord blood progenitor-stem cell yield in preeclampsia, many patients might still be adequate donors of cord blood for transplantation, especially if the recipient is small. However, on the basis of our results, the use of cord blood from preeclamptic patients should be considered on a case-by-case basis. Most importantly, modified cord blood collection techniques aiming to increase cord blood yield, such as very early cord clamping, should not be applied in preterm infants born to mothers with preeclampsia because of the increased risk for severe anemia of prematurity necessitating blood transfusion. 24 Furthermore, because transplacental cell traffic has been shown to be increased in preeclampsia, 25 a potentially increased contamination of maternal cells in the cord blood graft is of some concern and has to be taken into account. In summary, we have shown that umbilical cord blood collected after delivery contains fewer nucleated cells and hematopoietic progenitor-stem cells if the pregnancy is affected by preeclampsia. REFERENCES 1. Wagner JE, Kernan NA, Steinbruch M, Broxmeyer HE, Gluckman E. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. 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