Single Nucleotide Polymorphism Typing Going Spatial: In Situ Padlock Probes Targeting mRNA Variants to Identify Haploidentical Cells within the Tissue Environment.

Sister-chromatid exchange and cell replication kinetics in fetal and maternal cells after treatment with chemical teratogens

Long term propagation of human fetal mesenchymal stromal cells (MSC) in vitro has proven elusive due to limited availability of fetal tissue sources and lack of appropriate methodologies. Here, we have demonstrated the presence of fetal and maternal cells within the tips of terminal chorionic villi (TCV) of normal human term placenta, and we have exploited inherent differences in the adhesive and migratory properties of maternal vs. fetal cells, to establish pure MSC cultures of both cell types. The origin and purity of each culture was confirmed by X-Y chromosome-specific fluorescence in situ hybridization (FISH) and short tandem repeat (STR) genotyping. This is the first demonstration of fetal and maternal cells in the TCV of human term placenta and also of deriving pure fetal MSC cultures from them. The concomitant availability of pure cultures of adult and fetal MSC from one tissue provides a good system to compare genetic and epigenetic differences between adult and fetal MSCs; and also to generate new models of cell based therapies in regenerative medicine.

The aim of this study was to investigate the phenotype and function of NKT cells and the effect of NKT cells on the functions of vascular endothelial cells. The study included 30 pregnant women with hypertension and 20 pregnant women with normal delivery. Peripheral blood and placental tissues were collected from all patients and control subjects. Lymphocytes were isolated from peripheral blood and tissues. NKT cell ratio, expression of surface receptors and expression of effector molecules were detected by flow cytometry. Culture supernatant of NKT cells was collected and co-cultured with human umbilical vein endothelial cells (HUVECs). Proliferation was determined by CCK-8 assay, and migration, by transwell assay. The results showed elevated ratios of NKT and NK cells in peripheral blood and placental tissues from women with pregnancy-induced hypertension (PIH) compared with women with normal pregnancies. The expression of NKp30, NKG2D and NKG2A receptors on the surface of NKT cells from women with PIH was altered: the ratio of CD4+ NKT cells was increased but the ratio of CD8+ NKT cells was decreased. The ratios of GranB+ NKT cells in peripheral blood, IFN-γ-positive T cells in placental tissues and IL-17-positive NKT cells in both peripheral blood and placental tissues were elevated in PIH patients. The present study demonstrated that the ratio and activity of NKT cells from peripheral blood and placental tissues of women with PIH were elevated. In addition, NKT cells from peripheral blood of PIH patients inhibited the proliferation and migration of vascular endothelial cells by secreting IL-17.

Prepared by a working party of the BCSH Blood Transfusion (Chairman P. Kelsey) and General Haematology (Chairman J. T. Reilly) Task Forces Working party: J. F. Chapman, B. J. Bain, S. C. Bates, S. M. Knowles, K. H. Shwe, J. Parker-Williams, L. Robson and S. C. Robson Blood Transfusion Task Force: M. Bruce, J. F. Chapman, J. Duguid, S. M. Knowles, M. F. Murphy, L. Williamson, I. Cavill and J. K. Wood Haematology Task Force: R. J. Amos, B. J. Bain, C. Chapman, K. Hyde, E. Matutes, J. Parker-Williams, I. Cavill and J. K. Wood

The incidence and potentially different biological impact of naturally acquired maternal and fetal cells were enthusiastically and extensively discussed when basic scientists and clinicians met at the Medical University of Graz in Austria for an International Symposium on Chimerism (Fig. 1). The two day program featured presentations by speakers from 4 different continents, intermingled with short presentations focusing on technical aspects of chimerism analysis by representatives from the companies who sponsored the meeting. Details on the program and speakers list are available at the meeting’s website (http://chimerism.medunigraz.at/cms/website.php). As also exemplified by the various different types of studies published in the recently released journal Chimerism, this field is slowly but steadily expanding as new research teams working outside and within the transplantation field have become interested in the biologically intriguing phenomena of chimerism and microchimerism (Mc). The highlights of the meeting are summarized in this meeting report. Figure 1. The Symposium on Chimerism. Photo courtesy of Harry Schiffer, Copyright Graz Tourism. Technical Aspects of Chimerism Analysis Since the 1979 paper by Herzenberg and colleagues reporting enrichment of cells from pregnant women presumed to be fetal using flow-sorting techniques,1 many different studies have been published describing fetal or maternal cells in diverse hematopoietic cell types or tissues and organs. During the past decade, major technical improvements regarding the detection of these generally rare cells have been made. This has resulted in a shift from the more traditional XY fluorescent in situ hybridization (FISH) staining technique applied to tissue sections to more refined technologies such as real-time PCR for the detection of male-specific DNA sequences in genomic DNA extracted from peripheral blood cells or processed tissues. Real-time PCR reactions for mismatched HLA alleles as well as for other genetic markers (insertion/deletion) have been developed in the laboratories of meeting participants Dr Nelson (Fred Hutchinson Cancer Research Center, Seattle, USA), Dr Lambert (INSERM UMRs 1097, Marseille, France) and Dr van Halteren (Leiden University Medical Center, Leiden, the Netherlands). Unfortunately, many researchers are faced by the problem that this approach requires the availability of DNA from mother or offspring in order to find informative genetic markers. Of note, the newly developed QuantiChimera platform established at the laboratory of Dr Lambert can be contacted for technical support regarding state-of-the art PCR technology for Mc detection. Particularly relevant for investigators who are restricted to using FISH techniques for the identification of rare maternal or fetal chimeric cells is the collective observation that this technique does not always give consistent staining results. This may be explained by the fact that archived tissue samples are often subjected to different fixation protocols. As discussed by Dr Sedlmayr and Dr Kroneis (Medical University, Graz, Austria), FISH by itself is a less than optimal technique for the analysis of rare microchimeric cells given the frequency of false positive cells. This issue can in part be addressed by using two reverse sets of differently labeled X and Y probes. Also biochemical markers such as unique cell surface molecules or markers that can be used to distinguish fetal cells from adult cells are not perfectly reliable in the setting of analysis of extremely rare cells. Unambiguous identification of individual microchimeric cells can be done by combining either FISH analysis or analysis of biochemical markers with DNA genotyping of candidate cells isolated by laser microdissection.2 The introduction of a whole genome amplification step subsequently even allows combining DNA genotyping of single microchimeric cells with sequencing or comparative genome hybridization.3This exciting new approach is expected to further boost the chimerism field.

Hypermethylation of RASSF1A in Human and Rhesus Placentas

Production of interferon was studied in fibroblasts cultured from human fetal, neonatal, and maternal tissue. Human fetal and maternal cells were paired to diminish genetic variability. Fetal cells displayed an increased response to two inducers of interferon, virus and synthetic double-stranded ribopolynucleotide. Fetal cells released 300-fold more interferon than maternal cells on exposure to poly rI:rC. This enhanced capacity for interferon production was consistent in cultures developed from fetal skin obtained between the 10th and 20th gestational week. The response was relatively stable, persisting in cells cultured for 18 generations (about 14 weeks). On infection with Newcastle disease virus, fetal cells produced, on the average, 4 to 6.5 times more interferon than maternal or neonatal cells. The virus was adsorbed with equal efficiency by each type of cell. Increased production is apparently independent of the rates of overall protein synthesis, since fetal and maternal cells have very similar rates of total protein synthesis.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Fetal nucleated cells circulating in maternal peripheral blood are a noninvasive source of fetal DNA for prenatal genetic diagnoses. The successful isolation of fetal cells from maternal blood depends upon identification of differences between fetal and maternal cell surface antigen expression. To our best knowledge, a monoclonal antibody that binds only fetal blood cells has not yet been identified. We studied antigens recognized by six different monoclonal antibodies for their biologic expression on fetal blood cells as a function of gestational age, and compared their ability to bind fetal but not maternal cells. The results suggest a relationship between gestational age and nucleated cell surface antigen expression. The monoclonal antibodies FB3-2, H3-3, CD71 and 2-6B/6 are suitable reagents for first or early second trimester fetal cell isolation, although FB3-2 and H3-3 are more specific for fetal cells due to significantly lower expression of these antigens on maternal mononuclear cells. The observation that samples from fetuses with chromosome abnormalities or multiple structural anomalies express higher levels of these antigens indicates that these reagents will potentiate the detection of abnormal fetal cells in maternal blood samples.

During pregnancy, fetal CD34+ cells enter the maternal circulation, persist for decades, and create a state of physiologic microchimerism. Many studies have confirmed the residual presence of fetal cells in maternal blood and tissues following pregnancy. Fetal cells may respond to maternal injury by developing multilineage capacity in maternal organs. To verify that fetal microchimeric cells express markers of epithelial, leukocyte, and hepatocyte differentiation within maternal organs. Archived paraffin-embedded tissue section specimens from 10 women who had male offspring and were previously found to have high numbers of microchimeric cells, and 11 control women who had no prior male pregnancies. Male cells were identified by fluorescence in situ hybridization, using X and Y chromosome-specific probes, followed by histologic and immunochemical studies using anticytokeratin (AE1/AE3) as a marker of epithelial cells, anti-CD45 as a leukocyte marker, and heppar-1 as a hepatocyte marker. Percentage of microchimeric cells expressing nonhematopoietic markers. A total of 701 male (XY+) microchimeric cells were identified (mean [SD], 227 [128] XY+ cells per million maternal cells). In maternal epithelial tissues (thyroid, cervix, intestine, and gallbladder), 14% to 60% of XY+ cells expressed cytokeratin. Conversely, in hematopoietic tissues, such as lymph nodes and spleen, 90% of XY+ cells expressed CD45. In 1 liver sample, 4% of XY+ cells expressed heppar-1. Histologic and immunochemical evidence of differentiation, as assessed by independent observers, was highly concordant (kappa = 0.72). The detection of microchimeric male cells, bearing epithelial, leukocyte, or hepatocyte markers, in a variety of maternal tissue specimens suggests the presence of fetal cells that may have multilineage capacity.

Inflammation and oxidative stress at the maternal-fetal interface characterize the placental dysfunction that underlies the pregnancy disorder preeclampsia. Specialized fetal trophoblasts directly interact with leukocytes at both sites of the maternal-fetal interface; the uterine wall decidua; and the placenta. TLR3 has been implicated in the harmful inflammation at the maternal-fetal interface in preeclampsia, but the cellular involvement in the decidua and placenta has not been determined. This study aimed to characterize and quantify cell-specific TLR3 expression and function at the maternal-fetal interface in normal and preeclamptic pregnancies. TLR3 expression was assessed by immunohistochemistry and quantified by a novel image-based and cell-specific quantitation method. TLR3 was expressed at the maternal-fetal interface by all decidual and placental trophoblast types and by maternal and fetal leukocytes. Placental, but not decidual, TLR3 expression was significantly higher in preeclampsia compared to normal pregnancies. This increase was attributed to placental intravillous tissue and associated with both moderate and severe placental dysfunction. TLR3 pathway functionality in the decidua and placenta was confirmed by TLR3 ligand-induced cytokine response, but the TLR3 expression levels did not correlate between the two sites. In conclusion, functional TLR3 was broadly expressed by maternal and fetal cells at both sites of the maternal-fetal interface and the placental intravillous expression was increased in preeclampsia. This suggests TLR3-mediated inflammatory involvement with local regulation at both sites of the maternal-fetal interface in normal and preeclamptic pregnancies.

Human placentation entails the remarkable integration of fetal and maternal cells into a single functional unit. In the basal plate region (the maternal-fetal interface) of the placenta, fetal cytotrophoblasts from the placenta invade the uterus and remodel the resident vasculature and avoid maternal immune rejection. Knowing the molecular bases for these unique cell-cell interactions is important for understanding how this specialized region functions during normal pregnancy with implications for tumor biology and transplantation immunology. Therefore, we undertook a global analysis of the gene expression profiles at the maternal-fetal interface. Basal plate biopsy specimens were obtained from 36 placentas (14-40 wk) at the conclusion of normal pregnancies. RNA was isolated, processed, and hybridized to HG-U133A&B Affymetrix GeneChips. Surprisingly, there was little change in gene expression during the 14- to 24-wk interval. In contrast, 418 genes were differentially expressed at term (37-40 wk) as compared with midgestation (14-24 wk). Subsequent analyses using quantitative PCR and immunolocalization approaches validated a portion of these results. Many of the differentially expressed genes are known in other contexts to be involved in differentiation, motility, transcription, immunity, angiogenesis, extracellular matrix dissolution, or lipid metabolism. One sixth were nonannotated or encoded hypothetical proteins. Modeling based on structural homology revealed potential functions for 31 of these proteins. These data provide a reference set for understanding the molecular components of the dialogue taking place between maternal and fetal cells in the basal plate as well as for future comparisons of alterations in this region that occur in obstetric complications.

Cidial, an organophosphorous insecticide (also known as phenthoate), was tested for its genotoxic effect on both maternal and fetal cells. Cidial was administered at three different dose levels (53.5, 106.9, and 171 mg/kg) to pregnant mice on day 16 of gestation. Maternal bone marrow and embryonic liver cells were examined for chromosomal aberrations and cellular proliferation. Cidial was found to increase the percentage of cells with chromosomal aberrations in both mothers and fetuses. It also significantly inhibited the rate of mitotic activity of both maternal and fetal cells, with the inhibitory effect being more appreciable in fetal cells than in maternal cells. The data indicate that cidial, which is widely used in rural areas, is hazardous to both mothers and their transplacentally exposed babies.

Cidial, an organophosphorous insecticide (also known as phenthoate), was tested for its genotoxic effect on both maternal and fetal cells. Cidial was administered at three different dose levels (53.5, 106.9, and 171 mg/kg) to pregnant mice on day 16 of gestation. Maternal bone marrow and embryonic liver cells were examined for chromosomal aberrations and cellular proliferation. Cidial was found to increase the percentage of cells with chromosomal aberrations in both mothers and fetuses. It also significantly inhibited the rate of mitotic activity of both maternal and fetal cells, with the inhibitory effect being more appreciable in fetal cells than in maternal cells. The data indicate that cidial, which is widely used in rural areas, is hazardous to both mothers and their transplacentally exposed babies. Environ. Mol. Mutagen. 29:53–57, 1997 © 1997 Wiley-Liss, Inc.

Frequencies of baseline and cyclophosphamide-induced sister chromatid exchanges (SCE) were measured in mouse maternal and fetal cells between days 11 and 19 of gestation. Baseline levels of SCE did not vary as a function of gestational age in either the mother or fetus. Cyclophosphamide-induced SCE frequencies remained constant in maternal cells but declined dramatically in the fetus throughout the latter half of development. Because cyclophosphamide is a metabolically activated mutagen, a direct-acting drug, mitomycin C, was given on days 11 and 15 to determine if the decline in induced SCE levels seen with gestational results from alterations in activating enzymes. A similar decline in mitomycin C-induced SCE levels was noted in fetal tissues as a function of gestational age. Dose-response curves to cyclophosphamide performed on day 13 of gestation showed increases in SCE as a function of cyclophosphamide concentration in both the mother and the fetus. When mutagen-induced SCE levels were compared in different fetal organs, the direct-acting drugs (mitomycin C and daunomycin) were found to induce similar levels in all tissues. Cyclophosphamide, which is metabolically activated, induced higher SCE levels in fetal liver than in lung or gut. Whereas cyclophosphamide induced similar SCE levels in fetal and maternal cells on day 13 of gestation, daunomycin produced fetal SCE levels that were approximately 50% of maternal levels. Simultaneous measurement of the distribution of [14C]cyclophosphamide and [3H]daunomycin in maternal and fetal cells revealed that the lower SCE induction by daunomycin was probably due to decreased ability to cross the placental barrier.