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.

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.

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.

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.