Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals.

During pregnancy in placental mammals, small numbers of maternal cells (maternal microchimeric cells, or MMc cells) migrate into the fetus and persist decades, or perhaps for the rest of their lives, and higher frequencies of MMc cells are reported to correlate with variety of phenomena, such as immune tolerance, tissue repair, and autoimmune diseases. While detection of these MMc cells is considered in all pregnancies, their frequency differs largely according to tissue type and disease cases, and it remains unclear whether the number of MMc cells differs significantly among embryos in normal pregnancies. Here, for the first time, we developed a whole embryonic detection method for MMc cells using transgenic mice and counted live MMc cells in each individual embryo. Using this technique, we found that the number of MMc cells was comparable in most of the analyzed embryos; however, around 500 times higher number of MMc cells was detected in one embryo at the latest stage. This result suggests that the number of MMc cells could largely differ in rare cases with unknown underlying mechanisms. Our methodology provides a basis for testing differences in the numbers of MMc cells among individual embryos and for analyzing differences in MMc cell type repertoires in future studies. These data could provide a hint toward understanding the mechanisms underlying the variety of apparently inconsistent MMc-related phenomena.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Chimerism as the basis for organ repair.

Organ and tissue repair are complex processes involving signaling molecules, growth factors, and cell cycle regulators that act in concert to promote cell division and differentiation at sites of injury. In embryonic development, progenitor fetal cells are actively involved in reparative mechanisms and display a biphasic interaction with the mother; and there is constant trafficking of fetal cells into maternal circulation and vice versa. This phenomenon of fetal microchimerism may have significant impact considering the primitive, multilineage nature of these cells. In published work, we have reported that fetal-derived placental cells expressing the homeodomain protein CDX2 retain all "stem" functional proteins of embryonic stem cells yet are endowed with additional functions in areas of growth, survival, homing, and immune modulation. These cells exhibit multipotency in vitro and in vivo, giving rise to spontaneously beating cardiomyocytes and vascular cells. In mouse models, CDX2 cells from female placentas can be administered intravenously to male mice subjected to myocardial infarction with subsequent homing of the CDX2 cells to infarcted areas and evidence of cellular regeneration with enhanced cardiac function. Elucidating the role of microchimeric fetal-derived placental cells may have broader scientific potential, as one can envision allogeneic cell therapy strategies targeted at tissue regeneration for a variety of organ systems.

Male microchimerism in peripheral blood from women with multiple sclerosis in Isfahan Province.

Multiple sclerosis (MS) is referred to as an organ-specific T-cell-mediated autoimmune disease of the central nervous system (CNS). Different genetic and environmental factors increase the risk of developing MS. In recent years, microchimerism (Mc) has been widely studied in autoimmune diseases, although the exact role of this phenomenon in human health is not known well. Microchimerism is the low level presence of DNA or cells from one individual into the tissue or circulation of another individual. In the current study, we evaluated the association of fetal microchimerism (FMc) with MS in Isfahan province. In this study, we enrolled 68 women in four groups. Two groups were MS patients with or without a pregnancy for a son, and the other two groups were MS-negative patients with or without a pregnancy for a son. The presence of the male genome assessed and compared in these groups. Four millilitres of peripheral blood were collected from all subjects in the tube containing EDTA and DNA was extracted. Real-time PCR assay was used for the DAZ (deleted in azoospermia) region Yq 11.23 as a marker for male microchimerism in all subjects. Our results showed that the percentage of DAZ (male genome)-positive women was significantly higher in MS-positive women given birth to a son in comparison with the other three groups. Our results also revealed no significant correlation between the percentage of DAZ-positive women and Expanded Disability Status Scale (EDSS) score and age of onset in the patients' group. For future studies, we suggest enrolling subjects who MS diagnosis occurred before and after pregnancy with a son. Comparing FMc in these two groups might provide a better understanding of the possible role of FMc in later development of MS.

Preliminary evidence of a paternal-maternal genetic conflict on the placenta: Link between imprinting disorder and multi-generational hypertensive disorders.

There has been great research progress on hypertensive disorders in pregnancy (HDP) in the last few decades. Failure of placentation, especially a lack of uterine spiral artery remodeling, is the main pathological finding of HDP. Currently, members of the vascular endothelial growth factor family are used as markers for the early prediction of onset of HDP. Epidemiologic research has also shown that HDP can have effects on the next generation infants, representing a Development Origins of Health and Disease-related disease. However, the precise pathogenic mechanism and the effect of HDP on the offspring remain unclear. The group of strong pro-inflammatory molecules known as "danger signals" have been shown to be released from the placental trophoblast surface and increase in the maternal circulation in HDP, which are then possibly transported into the fetal circulation. These signals, including fatty acids or adipocytokines, may alter the offspring's health in later life. Moreover, a hypoxic condition alters placental methylation, and the change may be passed onto the fetus. Although the genetic origin of the disease is still unknown, a hypothesis has been put forward that a paternal-maternal genetic conflict, mainly at imprinting lesion sites, may be a key factor for disease initiation. In particular, an imbalance in paternal and maternal factors may impede proper placentation, trophoblast invasion, decidualization or immune moderation so as to achieve better nutrition for the fetus (paternal) versus ensuring safe delivery and further pregnancy (maternal). Here, we review this research progress on HDP and focus on this novel genetic conflict concept, which is expected to provide new insight into the cause, pathophysiology, and multi-generational effects of HDP.

Monitoring of Venus transgenic cell migration during pregnancy in non-transgenic rabbits.

Cell transfer between mother and fetus were demonstrated previously in several species which possess haemochorial placenta (e.g. in humans, mice, rats, etc.). Here we report the assessment of fetal and maternal microchimerism in non-transgenic (non-TG) New Zealand white rabbits which were pregnant with transgenic (TG) fetuses and in non-TG newborns of TG does. The TG construct, including the Venus fluorophore cDNA driven by a ubiquitous cytomegalovirus enhancer, chicken ß-actin promoter (CAGGS), was previously integrated into the rabbit genome by Sleeping Beauty transposon system. Three different methods [fluorescence microscopy, flow cytometry and quantitative polymerase chain reaction (QPCR)] were employed to search for TG cells and gene products in blood and other tissues of non-TG rabbits. Venus positive peripheral blood mononuclear cells (PBMCs) were not detected in the blood of non-TG littermates or non-TG does by flow cytometry. Tissue samples (liver, kidney, skeletal and heart muscle) also proved to be Venus negative examined with fluorescence microscopy, while histology sections and PBMCs of TG rabbits showed robust Venus protein expression. In case of genomic DNA (gDNA) sourced from tissue samples of non-TG rabbits, CAGGS promoter-specific fragments could not be amplified by QPCR. Our data showed the lack of detectable cell transfer between TG and non-TG rabbits during gestation.

Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy.

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals.

Monozygotic Monochorionic Twins Discordant for Trisomy 21: A Reason to Evaluate Both Fetuses: A Case Report.

BACKGROUND: The occurrence of a discordant chromosomal abnormality in monozygotic twins is an extremely rare condition. CASE: We report the prenatal sonographic findings and cytogenetic studies in a monochorionic twin pregnancy discordant for severe fetal anomalies. Amniocentesis was normal for both twins. The pregnancy was managed conservatively, resulting in the delivery of discordant twins at 28 weeks. Cytogenetic analysis performed on cultured lymphocytes from peripheral blood revealed a mosaic 47XY+21 (in 2% of the cells)/46XY (in 98%) in the structurally normal twin, and a mosaic 47XY+21 (4%)/46XY (96%) for the abnormal twin. The abnormal neonate died shortly after delivery. The structurally normal twin survived without sequelae and had a normal karyotype 2 years later. CONCLUSION: This report adds to the literature a case of a monochorionic twin pregnancy with a mosaic fetus who gives his co-twin trisomic cells through placental vascular anastomoses, this twin being a chimera, highlighting the necessity of performing molecular genetics with polymorphic DNA markers to differentiate chimerism from mosaicism and define the origin of cell lines.

Maternal microchimerism in health and disease.

Circulating maternal cells transfer to the fetus during pregnancy, where they may integrate with the fetal immune and organ systems, creating a state of maternal microchimerism (MMc). MMc can persist throughout the child's life, and it has been implicated in the triggering or perpetuation of chronic inflammatory autoimmune diseases, in the context of specific major histocompatibility genes. Correlative data in humans have now been tested in animal model systems. Results suggest that maternal-fetal tolerance may have health implications far beyond the time of pregnancy and into the child's life.

Fetal microchimerism and maternal health: a review and evolutionary analysis of cooperation and conflict beyond the womb.

The presence of fetal cells has been associated with both positive and negative effects on maternal health. These paradoxical effects may be due to the fact that maternal and offspring fitness interests are aligned in certain domains and conflicting in others, which may have led to the evolution of fetal microchimeric phenotypes that can manipulate maternal tissues. We use cooperation and conflict theory to generate testable predictions about domains in which fetal microchimerism may enhance maternal health and those in which it may be detrimental. This framework suggests that fetal cells may function both to contribute to maternal somatic maintenance (e.g. wound healing) and to manipulate maternal physiology to enhance resource transmission to offspring (e.g. enhancing milk production). In this review, we use an evolutionary framework to make testable predictions about the role of fetal microchimerism in lactation, thyroid function, autoimmune disease, cancer and maternal emotional, and psychological health. Also watch the Video Abstract.

Fetal cell microchimerism: a protective role in autoimmune thyroid diseases.

OBJECTIVE: The physiological persistence of fetal cells in the circulation and tissue of a previously pregnant woman is called fetal cell microchimerism (FCM). It has been hypothesized to play a role in systemic autoimmune disease; however, only limited data are available regarding its role in autoimmune thyroid disease (AITD). DESIGN: Circulating FCM was analyzed in a large series of previously pregnant women with Graves' disease (GD), Hashimoto's thyroiditis (HT), or no disease (healthy controls (HCs)). To exclude the possible bias related to placental factors, the polymorphic pattern of human leukocyte antigen-G (HLA-G) gene, which is known to be involved in the tolerance of fetal cells by the maternal immune system, was investigated. METHODS: FCM was evaluated by PCR in the peripheral blood, and the Y chromosome was identified by fluorescence in situ hybridization in some GD tissues. HLA-G polymorphism typing was assessed by real-time PCR. RESULTS: FCM was significantly more frequent in HC (63.6%) than in GD (33.3%) or HT (27.8%) women (P=0.0004 and P=0.001 respectively). A quantitative analysis confirmed that circulating male DNA was more abundant in HC than it was in GD or HT. Microchimeric cells were documented in vessels and in thyroid follicles. In neither GD/HT patients nor HC women was the HLA-G typing different between FCM-positive and FCM-negative cases. CONCLUSION: The higher prevalence of FCM in HC as compared to GD and HT patients suggests that it plays a possible protective role in autoimmune thyroid disorders. Placental factors have been excluded as determinants of the differences found. The vascular and tissue localization of microchimeric cells further highlights the ability of those cells to migrate to damaged tissues.

Maternal microchimerism: lessons learned from murine models.

The presence of maternal cells in the organs of the offspring is referred to as maternal microchimerism (MMc). MMc is physiologically acquired during pregnancy and lactation and can persist until adulthood. The detection of MMc in a variety of human diseases has raised interest in the short- and long-term functional consequences for the offspring. Owing to limited availability and access to human tissue, mouse models have become an essential tool in elucidating the functional role of MMc. This review compiles the detection techniques and experimental settings used in murine MMc research. It aims to summarize the potential mechanisms of migration of MMc, pre- and postnatal tissue distribution, phenotype and concatenated function, as well as factors modulating its occurrence. In this context, we propose MMc to be a materno-fetal messenger with the capacity to critically shape the development of the offspring's immunity.

Fetal Microchimerism in Cancer Protection and Promotion: Current Understanding in Dogs and the Implications for Human Health.

Fetal microchimerism is the co-existence of small numbers of cells from genetically distinct individuals living within a mother's body following pregnancy. During pregnancy, bi-directional exchange of cells occurs resulting in maternal microchimerism and even sibling microchimerism in offspring. The presence of fetal microchimerism has been identified with lower frequency in patients with cancers such as breast and lymphoma and with higher frequency in patients with colon cancer and autoimmune diseases. Microchimeric cells have been identified in healing and healed tissues as well as normal and tumor tissues. This has led to the hypothesis that fetal microchimerism may play a protective role in some cancers and may provoke other cancers or autoimmune disease. The long periods of risk for these diseases make it a challenge to prospectively study this phenomenon in human populations. Dogs get similar cancers as humans, share our homes and environmental exposures, and live compressed life-spans, allowing easier prospective study of disease development. This review describes the current state of understanding of fetal microchimerism in humans and dogs and highlights the similarities of the common cancers mammary carcinoma, lymphoma, and colon cancer between the two species. Study of fetal microchimerism in dogs might hold the key to characterization of the type and function of microchimeric cells and their role in health and disease. Such an understanding could then be applied to preventing and treating disease in humans.

Non-inherited maternal antigens, pregnancy, and allotolerance.

Non-inherited maternal antigens (NIMA) are those protein products derived from polymorphic genes that the mothers express but not the offspring. During normal human pregnancy, a bidirectional regulation occurs in such a way that the maternal immune system tolerates the inherited paternal antigens (IPA) expressed by the fetus and the developing fetal immune system tolerates NIMA. The process by which the described bidirectional regulation is developed is related to microchimerism, due to the bidirectional traffic of cells allowed by the decidua-trophoblast interface. An extensive body of knowledge from the transplantation and pregnancy physiology fields suggests a role for microchimerism and NIMA exposure in the development of NIMA-specific alloresponse regulation, which may include transforming growth factor ß (TGF-ß) as well as interleukin (IL)-10 and IL-35, producing peripheral T regulatory lymphocytes. The induction of this NIMA-specific allotolerance is called the "NIMA effect." Some experimental data suggest the existence of a "split tolerance" phenomenon associated with NIMA effect, in which regulation of NIMA-specific indirect pathway is induced without tolerogenic impact on the direct pathway. In this review, the most relevant literature about the immunological phenomena underlying the NIMA effect is discussed, including the most recent proposals about the role played by antigen-acquisition and the semi-direct pathway of allorecognition.

Maternal-fetal cellular trafficking: clinical implications and consequences.

PURPOSE OF REVIEW: Maternal-fetal cellular trafficking (MFCT) is the bidirectional passage of cells between mother and fetus during pregnancy. This results in the presence of fetal cells in the maternal circulation, known as fetal microchimerism, and maternal cells in the fetal circulation, known as maternal microchimerism. The biologic role of this transplacental cellular trafficking during pregnancy is not known, although it has been implicated in development of the fetal immune system, tolerance mechanisms during pregnancy, tissue repair in autoimmune disease and cancer, and immune surveillance. RECENT FINDINGS: Clinical utility of MFCT has been identified in prenatal testing for aneuploidies and prediction of pregnancy complications. Additionally, this transplacental passage of cells has been implicated in the delicate balance between immunologic priming and tolerance, which can influence the occurrence of autoimmune disease and transplantation outcomes. Ongoing studies are evaluating the utility of microchimerism in predicting the risk of graft rejection in transplantation. SUMMARY: In this review, we will discuss the clinical implications of MFCT in pregnancy, fetal surgery, autoimmune disease, transplantation, and cancer.

Maternal microchimerism in biliary atresia: are maternal cells effector cells, targets, or just bystanders?

The etiology of biliary atresia (BA) is unknown; however, the liver histology is similar to that observed in immune-mediated hepatic disorders. Liver fibrosis in BA progresses even after bile drainage has been achieved by the Kasai operation. Maternal microchimerism has been purported to play a part in the pathogenesis of BA as well as certain autoimmune diseases. However, the role of maternal cells has not yet been determined in BA. Specifically, it is unknown whether these maternal cells function as maternal effector T lymphocytes, or targets or bystanders. We currently hypothesize that the first hit is due to GvHD interaction by engrafted maternal effector T lymphocytes. Furthermore, we suggest that the secondary effects that are manifested by progressive cirrhosis are caused either by maternal chimeric effector T lymphocytes (e.g., GvHD interaction) or targets (e.g., HvGD interaction). Based on our hypothesis, mixed lymphocyte reactions between patients and their mothers might shed light on the etiopathogenesis and prognostic indicators.

Biphasic recruitment of microchimeric fetal mesenchymal cells in fibrosis following acute kidney injury.

Fetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-ß/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis.

Fetal cell microchimerism in the maternal mouse spinal cord.

Fetal cell microchimerism refers to the persistence of fetal cells in the maternal tissues following pregnancy. It has been detected in peripheral organs and the brain, but its existence in the spinal cord has not been reported. Our aim was to detect fetal cell microchimerism in the spinal cord of maternal mice. C57BL/6 female mice were crossed with GFP transgenic male mice and sacrificed after their first or third delivery. GFP-positive cells, which were presumably from fetuses whose fathers were GFP transgenic, were detected in the spinal cord by fluorescence microscopy and immunohistochemistry. PCR was also performed to detect GFP DNA, which must come from GFP hemizygous fetuses. We found GFP-positive cells and detectable GFP DNA in most of the maternal spinal cords. Twenty percent (1/5) of the mice that were only pregnant once had detectable fetal cells, while 80% (4/5) of those that were pregnant three times had detectable fetal cells. Some fetal cells, which not only emitted green fluorescence but also expressed NeuN, were detected in the spinal cords from maternal mice. These results indicate that fetal cells migrate into the spinal cord of a maternal mouse during and/or after the gestational period, and the fetal cells may differentiate into neurons in the spinal cord.

Chimerism in transfusion medicine: the grandmother effect revisited.

Transfusion therapy is complicated by the production of alloantibodies to antigens present in the donor and lacking in the recipient through the poorly-understood but likely multi-factorial process of alloimmunization. The low prevalence of alloimmunization in transfused patients (6.1%) (1) suggests that processes central to immunologic tolerance may be operating in the vast majority of transfused patients who do not produce alloantibodies. Using RhD as a prototype, evidence is reviewed that the ability to make antibodies to red blood cell (RBC) antigens may result in part from immunologic tolerance acquired in utero. These ideas are extended to other examples of maternal microchimerism (MMc) of other non-inherited maternal antigens (NIMA). An evolutionary argument is offered that multi-generational immunity supports the hypothesis that MMc may partly explain the "non-responder" phenotype in RBC alloimmunization.