Fetomaternal cell traffic, pregnancy-associated progenitor cells, and autoimmune disease

Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 18, No. 6, pp. 959–975, 2004 doi:10.1016/j.bpobgyn.2004.06.007 available online at http://www.sciencedirect.com

9 Fetomaternal cell traffic, pregnancy-associated progenitor cells, and autoimmune disease Diana W. Bianchi* MD Division of Genetics, Departments of Pediatrics, Obstetrics and Gynecology, Tufts-New England Medical Center, Tufts University School of Medicine, Box 394, 750 Washington Street, Boston, MA 02111, USA Fetal cells in maternal blood are a potential source of fetal genetic material that can be obtained non-invasively. Efforts to isolate these cells from maternal peripheral blood are limited by their low circulating numbers (approximately 1 per ml of maternal blood in euploid pregnancies). Expansion of these cells by culture would provide more cells for diagnosis and give an opportunity to study fetal metaphase chromosomes. Despite extensive optimization of culture conditions, many groups have failed reproducibly to grow fetal cells from pre-procedural maternal samples. An unexpected benefit of this research has been the discovery of a novel population of fetal cells, the pregnancy-associated progenitor cell (PAPC), which remains in maternal blood and tissue for decades following delivery. These cells might play a role in some autoimmune diseases, such as scleroderma. PAPCs appear to have stem cell characteristics, such as the ability to proliferate and differentiate. Recently developed animal models will help to ascertain whether these cells cause disease, respond to disease, or have therapeutic applications. Key words: autoimmune disease; fetal cell microchimerism; fetal cells in maternal blood; fluorescence in situ hybridization; non-invasive prenatal diagnosis; polymerase chain reaction (PCR); pregnancy-associated progenitor cell (PAPC).

OVERVIEW OF FETAL CELLS IN THE MATERNAL CIRCULATION For almost 40 years it has been appreciated that fetal nucleated hematopoietic cells circulate within the maternal blood.1 Over the past 25 years many creative investigators have attempted to isolate and extract these cells with the goal of providing a noninvasive way to access the fetal genome (reviewed in Ref. 2). Despite significant advances in the development of molecular techniques sensitive enough to detect gene products at the single cell level, the use of fetal cells in maternal blood has not yet translated to clinical practice. Why is this? The major limitation appears to be the generally low number of fetal cells found in most maternal samples, which has been * Tel.: C1-617-636-1468; Fax: C1-617-636-1469. E-mail address: [email protected]. 1521-6934/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

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validated in four key studies using the techniques of fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (PCR). With a comprehensive and brute force approach, Hamada et al looked for the presence of male cells in unsorted maternal blood samples; fetal cells were identified by their nuclear hybridization to a Y-chromosome-specific probe.3 This team screened as many as 144 000 nuclei per pregnant woman to find evidence of a single YC cell. The data demonstrated that the frequency of YC cells increased as gestation progressed, from less than 1 in 105 during the first trimester to 1 in 104 at term. More recently, Krabchi et al4 examined nucleated cells in an entire 3-ml blood sample taken from each of 12 second-trimester pregnant women known to be carrying male fetuses. They prepared the blood smear with methanol and glacial acetic acid (Carnoy’s fixative), the same solution used in routine cytogenetic studies, and did not use any other methods of fetal cell enrichment. Fetal XYC nuclei were reproducibly detected at a concentration of 2–6 nuclei per ml of maternal whole blood. Whereas both of these studies conclusively demonstrated the presence of fetal cells in maternal blood, the approaches used are clearly impractical for large-scale clinical diagnosis; either an automated imaging system must be employed or some enrichment of the proportion of fetal cells present in a maternal sample must precede cytogenetic analysis. PCR is an alternative technique to measure the number of nucleated fetal cells present in a given maternal sample. Early versions of quantitative PCR measured the amount of P32-labeled deoxyribonucleoside triphosphate incorporated into maternal sample amplification products and compared them to standard curves generated from known concentrations of male DNA.5 In our laboratory, we performed an analysis of 230 mid-trimester maternal blood samples to determine the number of nucleated fetal cells present, and to explore the effect of fetal karyotype on fetomaternal cell transfusion.6 Of the 230 samples analyzed, 199 were from women who carried fetuses with a normal karyotype and 31 were from women who carried aneuploid fetuses. We calculated the number of male fetal cell equivalents in 16 ml of maternal whole blood. In samples obtained from the 90 pregnant women carrying 46,XY fetuses, the mean number of cells detected was 19 (range 0.1–91), or approximately 1 fetal cell per ml of maternal blood. Importantly, the results obtained when the fetus was 47,XY,C21 were sixfold elevated, or a mean of 110 fetal cells in 16 ml of maternal blood. The increased number of fetal cells detected when the fetus was aneuploid was highly significant (PZ 0.001). This study was the first to suggest that fetomaternal cell trafficking was affected by karyotype, and therefore that fetal cell isolation from maternal blood should be easier and more accurate for aneuploid fetuses. Using the more recently developed and more sensitive technique of real-time kinetic PCR amplification, Ariga et al7 analyzed serial blood samples taken every 2–4 weeks from 20 pregnant women carrying male fetuses. Two of the 20 women studied had evidence of male cellular DNA in their blood as early as 7 menstrual weeks. All of the women studied had detectable male cellular DNA by the third trimester of pregnancy. This group calculated that there were on average 2–40 fetal cells per ml of maternal whole blood. It is interesting that these four studies, performed at different times in different laboratories in different parts of the world, have derived remarkably similar estimates of the number of fetal cells present in a maternal sample. They all indicate that the expected number of fetal cells (except in the setting of aneuploidy or following termination of pregnancy) is low, and that enrichment methods are needed to detect them. The United States National Institute of Child Health and Human Development (NICHD) funded a prospective, multicenter clinical study, known as the ‘NIFTY’ trial, which has recently concluded (1994–2003). One of the purposes of this trial was to

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compare the accuracy of non-invasive fetal aneuploidy detection by interphase FISH analysis using fetal cells in maternal blood to conventional prenatal metaphase cytogenetic diagnosis by chorionic villus sampling or amniocentesis; two different methods of fetal cell enrichment, the fluorescence-activated cell-sorter (FACS) and the magnetic-activated cell sorter (MACS) were compared. The interim results of this largest study of fetal cells to date, in which 2744 fully processed blood samples were obtained prior to an invasive procedure, have now been published.8 Of the study subjects, 1292 carried singleton male fetuses. The data showed that target fetal cell recovery and detection was better using MACS than FACS. However, one of the major limitations was that over the course of the study multiple sample processing protocols were used. In male fetuses, using blinded FISH analysis, at least one (presumptive fetal) cell with both X- and Y-chromosome signals was seen in 41% of cases (95% confidence interval [CI] 37, 46%). There was a higher than expected false-positive rate of gender detection of 11%, due primarily to indirectly labeled FISH probes that were used at one study site. Significantly, the sensitivity of detection of at least 1 fetal cell was higher (74%, CI 76, 99%), and the false-positive rate was lower (1–4%) when the fetus was aneuploid. As predicted by the quantitative PCR studies, fetal cells were easier to detect in the maternal samples when the fetus was aneuploid. At present it appears that technological advances are needed before fetal cell analysis can be used clinically for prenatal screening or diagnosis. Research efforts are currently focused on rare-event separation strategies, such as microfluidics and lasercapture microdissection, and development of novel markers to identify fetal cells. An important advance will be in the area of automated imaging, in which maternal blood samples can be processed minimally (thereby reducing the possibility for fetal cell loss) and placed on multiple slides that can be scanned for the presence of fetal chromosomal or cytoplasm identifiers. The ‘low purity’ approach taken by Krabchi and co-workers in their study4 required an enormous amount of human time at the microscope. Automated image analysis has the potential to significantly reduce that time, while simultaneously maximizing the potential for fetal cell retention and diagnosis.

EXPANSION OF FETAL CELLS FROM MATERNAL PERIPHERAL BLOOD As seen above, the efforts to isolate fetal cells reproducibly from maternal blood are limited by the absence of a universal fetal cell surface or cytoplasmic marker, and by their relative rarity in most maternal samples. Clonal expansion of fetal cells in maternal blood by cell culture potentially overcomes the limitation of the low target number of fetal cells present. Furthermore, successful culture of fetal cells would expand diagnostic capabilities by facilitating metaphase analysis of fetal chromosomes. Over the past 10 years, many publications have described both success and failure in the attempt to culture fetal cells from maternal peripheral blood (reviewed in Ref. 9). Each report used a different method or different culture medium and provided little discussion as to the rationale for selecting a particular approach. Erythroid progenitors In 1994 Lo and colleagues10 co-cultured male erythroid precursor cells derived from fetal liver with a 100-fold excess of peripheral blood mononuclear cells (PBMCs) from

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a non-pregnant woman in medium enriched with erythropoietin (EPO). After 1 week, the absolute number of male fetal cells increased 26-fold. This was followed by experiments using blood samples obtained from five pregnant women in their second trimester known to be carrying male fetuses. Results, using early methods of quantitative PCR, showed that fetal cells were preferentially expanded over maternal cells. Subsequently, Valerio and co-workers described their experience using biotin labeled antibody to the EPO ligand and MACS to isolate fetal cells in eight secondtrimester maternal blood samples, which were then cultured for 6–10 days in a semisolid medium. Fetal BFU-E, CFU-E, and occasionally CFU-GEMM cells were identified by FISH and PCR.11 These investigators followed with two reports of the successful culture of fetal hematopoietic cells in the blood of women carrying aneuploid fetuses.12,13 As previously shown by quantitative PCR studies, fetal aneuploidy results in an increased amount of fetomaternal transfusion6, which suggests that the success of this group was due to a high baseline circulating level of fetal cells in the original maternal samples. Valerio’s impressive results were dampened by the negative findings of Chen et al14, who evaluated two different methods of fetal cell culture. In one, varying concentrations of EPO were investigated. The other method was based on the culture conditions described in the 1996 Valerio et al report.11 Fetal erythroid progenitors could not be demonstrated by either technique. Difficulty in reproducing results obtained by others is a consistent theme in this area of research. Han et al15,16 developed a novel method to separate cells dependent on EPO from cells independent of EPO in a two-phase liquid culture system. In the two reports, a total of 11 pregnant women were studied. In five of six women carrying male fetuses (at 8–14 weeks of gestation) evidence of male cells was detected by PCR. However, metaphase analysis of dividing cells demonstrated only cells with an XX karyotype, raising a question as to the validity of the PCR data. Larger-scale studies are needed to more fully evaluate the sensitivity and specificity of both of these methods. The field was further clouded by the report of Tutschek et al17, who described successful direct expansion of fetal clones from 12 non-enriched maternal blood samples obtained at 14–20 weeks of gestation. Following 14 days of culture, they micromanipulated single colonies and performed fluorescent PCR analysis to identify fetal cells. Results showed the presence of colonies of mixed (fetal/maternal) origin as well as apparently pure fetal colonies. Campagnoli et al18 challenged this report and questioned the existence of the mixed colonies (a result they had never encountered in their own model systems), as well as the accuracy of the fluorescent PCR data. Working independently in Switzerland, Zimmermann and colleagues19 tried to validate Tutschek’s work and could not. This group cultured 16 maternal blood samples under optimized conditions, isolated single colonies by micromanipulation, and performed real-time multiplex PCR. All colonies were maternal in origin. Zimmermann’s group ascribed Tutschek et al’s results to errors in their complex PCR assay, suggesting that alleles might have been incorrectly amplified and erroneously presumed to be fetal. Such amplification errors occur more frequently in the context of working with small cell numbers in the PCR. In any maternal blood sample, maternal erythroid progenitor cells will vastly outnumber fetal clonogenic cells. Therefore, in our laboratory, we spent a number of years trying to understand biologic differences between fetal and adult progenitor cells in culture. Bohmer and co-workers20 developed a quantitative two-color flow cytometric method of analysis and sorting using monoclonal antibodies to gamma and beta globin (hemoglobin F and A, respectively) (Figure 1). We observed that fetal and

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Day-7 colonies

Hemoglobin profile 104

Hb-F

103 102

101 100 100

101

102 Hb-A

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Figure 1. (Left) Photomicrograph of haemopoietic colonies grown from a mixed fetal and maternal blood sample after 7 days in culture on semisolid medium using the parameters optimized in Ref. 23. (Right) Flow cytometric analysis of hemoglobin F (Hb-F) and hemoglobin A (Hb-A) profiles resulting from this colony. The area in box at upper left of histogram indicates the fetal cell population that expresses high levels of fetal hemoglobin and no adult hemoglobin. Reprinted with permission.

adult progenitor cells developed distinct hemoglobin expression profiles in co-culture. For up to 7 days in culture, fetal cells made only HbF, and expressed HbA only after the levels of intracellular HbF had reached a maximum amount. Using the HbFCHbA-flowsorting criteria, we were able to isolate fetal cells with 50% purity in post-termination blood samples and O90% purity in artificial mixtures consisting of 1% fetal blood and 99% maternal blood. The flow-sorting criteria allowed further exploration of different variables in culture conditions. For example, we analyzed the effects of different serum supplementation on fetal and adult erythropoiesis in semi-solid culture medium.21 Charcoal-treated human umbilical cord serum resulted in an expansion of fetal red cells and minimized the proportion of adult cells that expressed HbF. In addition, human cord serum was shown to be a more powerful stimulator of cellular proliferation than fetal calf serum, a commonly used medium supplement. We then provided ‘proof of principle’ that fetal cells could be cultured from the blood of a pregnant woman at 18 weeks carrying a fetus with a normal karyotype but multiple sonographic abnormalities. After 10 days in culture supplemented by 1% human cord serum, 63% of flow-sorted HbFCHbA-cells were fetal. However, we could not completely suppress the growth of all adult HbFCHbA-cells. We further showed that interleukin-3 (IL-3), when added to the erythrocyte culture, strongly stimulates the growth of adult HbFC cells with relatively little effect on fetal HbFC cells.22 We were able to demonstrate an increased purity of fetal cells in the expanded population by omitting IL-3 from the culture medium. We later

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demonstrated that this effect was based on indirect action via the maternal monocyte population. Thus, removal of monocytes from the cell culture achieved the same effect. After extensive efforts to develop the ‘optimal’ conditions to promote the growth of fetal erythroid progenitors, we embarked upon a preliminary clinical trial.23 These conditions included adding charcoal-adsorbed human umbilical cord serum and omitting IL-3 from the culture medium over a time course of 7–9 days, followed by flow-sorting of HbFCHbA-cells. Eighteen mid-trimester blood samples were studied (11 women carrying a known 46,XY fetus, 5 women carrying fetuses with trisomy 21, and 2 women carrying fetuses with trisomy 18). As a positive control, blood samples were taken from 6 women immediately following elective termination of pregnancy, which is known to cause fetomaternal hemorrhage.24 None of the cultures from the ongoing pregnancies yielded fetal cells, whereas most of the samples obtained posttermination did. This demonstrated that the culture methods were appropriate to expand fetal erythroid progenitors if present. We concluded that there was likely to be an extremely low number of fetal cells with clonogenic potential in maternal blood. Data from cordocentesis and cardiocentesis studies show that in pure fetal blood, somewhere between 0.05 and 10% of nucleated fetal cells express the CD34 antigen.25,26 Using our own quantitative PCR data6, and estimating that there are, on average, 20 nucleated fetal cells in maternal blood, only 0.01–2 fetal CD34C cells would be expected to be present in that sample. Thus, it is not surprising that it has been so difficult to culture fetal erythroid cells from maternal blood. Cells that express the CD34 antigen Despite the above statistics, fetal cells that express the CD34 antigen remain an attractive target because of data that suggest that they proliferate more rapidly in culture than similar cells of adult origin.27 In model systems using small numbers of CD34C cells derived from umbilical cord blood mixed into 400 000 adult female CD34C cells, Jansen et al were able to demonstrate a 1500-fold preferential expansion of fetal over adult cells.28 However, using the same protocol on maternal blood samples obtained at 7–16 weeks, they were not able selectively to expand fetal stem cells. Manotoya and coworkers29 were unable to find evidence of male fetal cells by either FISH or PCR when they cultured CD34C cells from the peripheral blood of 11 pregnant women, 2 women following termination of pregnancy, and 4 non-pregnant controls. By contrast, Little et al30 detected between 0 and 93 male fetal CD34C cells in fresh enriched maternal blood samples after culturing them for 5 days. More recently, Guetta and co-workers found evidence of male fetal CD34C cells in all samples obtained from women carrying male fetuses.31 They processed 15–20 ml of maternal blood within 18 hours of venipuncture, performed a density gradient separation using Ficoll 1.077, isolated CD34C cells by MACS, and targeted the resulting enriched samples for either direct analysis or culture. A mean of 13 fetal cells was found in the non-cultured samples and 31 cells in the cultured specimen, a significant difference (PZ0.04). These investigators concluded that the CD34C cells were more resilient in the maternal circulation than cells that had already committed to the erythroid differentiation pathway. Endothelial cells Alternative fetal cell types in maternal blood have also been studied. For example, Gussin et al32 hypothesized that endothelial precursor cells might enter the maternal

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circulation. This group took PBMC from 13 mid-trimester pregnant women, 10 nonpregnant women, and 2 men, and cultured them for 8–10 weeks under conditions that promoted endothelial cell differentiation. So-called ‘early outgrowth’ cells appeared after 1 week in culture in all samples. ‘Late outgrowth’ cells bearing endothelial markers were observed in 8/13 samples taken from pregnant women but in 0/10 non-pregnant controls. FISH analysis of the late outgrowth samples only revealed nuclei with XX signals whether the women were carrying a male or a female fetus. This study showed that primitive endothelial precursor cells are present in the blood of most pregnant women but they are maternal in origin. Mesenchymal cells Relatively recently, Campagnoli et al33 identified a novel population of mesenchymal stem/progenitor cells (MSCs) that circulate in first-trimester fetal blood, are present in fetal liver and bone marrow, and can be cultured and differentiate into fat, bone, and cartilage (see Chapter 3). The frequency of these cells declines with gestational age. This group subsequently developed optimal protocols for fetal MSC enrichment from the peripheral blood of pregnant women.34 In artificial mixtures, O’Donoghue and her collaborators could successfully culture 1 male fetal MSC in as many 25 million adult female nucleated cells. Using post-termination samples as a biological model in which an increased fetomaternal hemorrhage would have been expected, they were able to detect male fetal MSC in only 1 of 20 samples studied. Thus, the frequency of fetal MSCs in the blood of pregnant women appears to be quite low and it is probably dependent on the gestational age at time of sampling. This probably precludes their clinical use for non-invasive prenatal diagnosis but does not eliminate the chance that they play a role in post-pregnancy health issues. It is possible that they rapidly engraft in maternal tissues after transplacental passage.

POST-PARTUM DEVELOPMENT OF FETAL CELL MICROCHIMERISM In the context of evaluating different monoclonal antibodies for the purpose of isolating fetal cells from maternal blood, our group made the surprising and novel observation that fetal CD34C and fetal CD34C CD38C hematopoietic stem cells could persist in the circulation of their healthy, non-transfused mothers for as long as 27 years postpartum.35 Previously, other investigators had documented that fetal leukocytes that contained a Y body in their interphase nuclei were present in the peripheral blood of healthy primigravidas for up to 1 year after delivery.36 Subsequently, Ciaranfi et al37 extended these observations, studying 62 women following the birth of a male infant. They showed that more than half of the women had circulating male lymphocytes 2 years post-partum. In some cases metaphase spreads with a quinacrine-stained Y chromosome were found up to 5 years following the birth of a male infant. In our study35, we sorted PBMCs from 32 pregnant women and 8 non-pregnant women using monoclonal antibodies that recognized cell surface antigens present on stem cells, T cells, and B cells. DNA within the sorted cells was amplified via nested PCR for Y chromosome-specific sequences. Male DNA was found in CD34C cells from 4 pregnant women carrying female fetuses, all of whom had been previously pregnant, and either underwent a termination of pregnancy or delivered a male infant. Unexpectedly, male DNA was also detected in the sorted CD34C CD38C cells of

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6 of the 8 non-pregnant women; all had sons. Male DNA was not detected in the 2 women who had delivered their sons most recently (6 and 10 months prior to blood sampling). In this study, we made no attempts to quantify the number of male cells present, but as they were detectable only by Southern hybridization and/or nested PCR, they were rare. Our results suggested that the persistence of fetal stem and/or progenitor cells for decades post-partum established a long-term, low-grade microchimeric state in the human female. The term ‘microchimerism’ was originally proposed by Lie´gois to describe the apparently stable long-term survival and proliferation of allogenic fetal cells in a maternal mouse without the induction of graft-versus-host disease (GVHD).38 Seven years later, in their study of circulating fetal CD34C cells in maternal blood, Guetta and colleagues31 validated the persistence of fetal (male) CD34C cells in 10 women who delivered sons 4–21 years earlier and in 23 women who had previously delivered one or more sons but were currently pregnant with a female fetus. They also quantified the number of the circulating fetal cells using FISH and nested PCR for the Y chromosome. In 2 of the 10 non-pregnant women, a single male CD34C cell was detected in 15–20 ml of peripheral blood. In the pregnant women carrying female fetuses, 3 of 11 women had 1–3 male CD34C cells from prior pregnancies in their uncultured blood and 3 of 12 women had 18–60 male CD34C cells in their cultured blood sample. Adams and colleagues sought to determine if male (presumed fetal) DNA was present in the apheresis products of parous female hematopoietic stem cell donors.39 In growth-factor-mobilized PBMC products from 29 women, 34% were positive for male DNA, with a range of 0–35 genome equivalents (GE)/ml. In the CD34C-enriched fractions obtained from 21 women, 48% were positive for male DNA, with a range of 0–357 GE/ml. These authors speculated that the presence of fetal cells in the donor population might explain the increased risk of GVHD when the stem cell donor is a parous woman. Their data provide further evidence as to the long-term persistence of fetal stem cells in the mother after pregnancy.

DO FETAL CELLS PLAY A ROLE IN THE ETIOLOGY OF AUTOIMMUNE DISEASE? Following our discovery of the persistent CD34C and CD34C CD38C cells in the peripheral blood of parous women, we became interested in the long-term health consequences of these fetal circulating cells for women who had been pregnant.40,41 In our original paper35 we questioned whether there was any relationship between pregnancy and the subsequent development of autoimmune disease. At around the same time, J. Lee Nelson, a rheumatologist, was also exploring the role of pregnancy in autoimmunity.42 In an initial collaboration between our laboratory groups we tested the hypothesis that circulating fetal cells would be detectable in the peripheral blood of post-partum women with scleroderma, a disease that occurs almost exclusively in women.43 In the peripheral blood of affected women who had previously delivered at least one son, significantly higher numbers of male cells were found, as compared with healthy controls, although the percentage of each group with detectable microchimerism was not significantly different. At least two other studies have shown that the number of

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Figure 2. Lymph node section from a woman who died from complications of scleroderma. FISH was performed using fluorescently labeled probes to the X and Y chromosome. Arrow indicates a single male cell within a cluster of female cells. Photograph courtesy of Kirby L. Johnson PhD.

male, presumed fetal cells in the peripheral blood of non-pregnant women with scleroderma is significantly elevated.44,45 In subsequent studies we wished to determine if circulating fetal cells were capable of migration to maternal organs and could participate in the disease process. Artlett et al had originally shown that male cells were detectable by FISH in the skin sections of women affected by scleroderma.46 In autopsy specimens taken from women with scleroderma and lupus, we demonstrated that male (fetal) cells tend to cluster in the liver, spleen, and/or in organs affected by the disease process, such as the small intestine (Figure 2).47,48 Evidence from our laboratory and others showed that in biopsy or autopsy tissue taken from parous women with specific autoimmune diseases, fetal cell microchimerism is consistently present in levels above that found in tissue from controls (Table 1).49–61 To date, some investigators are exploring the hypothesis that the fetal cells are involved in the pathogenesis of autoimmune disease, possibly through a graft-versus-host phenomenon.62–64

PREGNANCY-ASSOCIATED PROGENITOR CELLS: A NOVEL SOURCE OF STEM CELLS? Preliminary studies from our laboratory Our laboratory is now focused on an alternative hypothesis. Perhaps the fetal cells are not causing disease but are found in the clinically affected organs because they are attempting to repair the disease. We developed this hypothesis because of intriguing

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Table 1. Fetal cell (FC) microchimerism in the post-partum woman: summary of diseases studied Condition Polymorphic eruption of pregnancy49 Scleroderma 43–45,47,50–52 Autoimmune thyroid disease53–55 Cervical cancer56 Primary biliary cirrhosis57,58 Lupus erythematosus48 Sjogren’s syndrome59–61

FC in maternal blood Unknown Present Absent Unknown Unknown Absent Present/? Same as controls

FC in maternal tissue Present Present Present Present Present/? Same as controls Present Present if condition is secondary to other autoimmune conditions such as scleroderma

data from a case that we studied. We analyzed a liver biopsy specimen from a woman with hepatitis C and a history of having had male children. This woman had a high viral load but was taking her medication inconsistently, and ultimately stopped treatment against medical advice. Despite this, she did well clinically, and her disease abated. Her liver specimen demonstrated hundreds of male cells, by dual color FISH studies using probes for the X and Y chromosome. She had never received a blood transfusion and was not a twin. The result of follow-up studies using DNA polymorphism analyses of the woman, her son, and several sexual partners, suggests that the likely source of the male cells in her liver was a pregnancy that she terminated 17–19 years earlier.65 It is remarkable that fetal cells could persist in this woman’s body for such a long time. Our findings have not been limited to the liver. In a study of biopsy material from 29 women with thyroid disorders we found the expected increased incidence of fetal cell microchimerism in the women with Hashimoto disease.53 An unexpected finding, however, was the detection of large numbers of male fetal cells in an otherwise healthy woman with a benign thyroid adenoma. Using DNA probes that map to the X and Y chromosomes we showed that mature follicles from the woman’s thyroid sample were partly male and partly female. Our interpretation was that fetal stem cells from her previous male-bearing pregnancy had persisted, and eventually developed into part of her thyroid. She had no other sources of microchimeric cells. She had never been transfused, had never had an organ transplant, and had no history of being a twin.66 The presence of large numbers of male cells in the liver and thyroid of these women suggests two conclusions: (i) that fetal cell microchimerism is not limited to autoimmune disorders; and, more importantly (ii) that endogenous fetal cells acquired from a woman’s own pregnancies might play a therapeutic role later in life. Fetal cells apparently express non-hematopoietic antigens in maternal tissue Although we can clearly document the presence of male PAPCs within maternal host tissue (see Figure 2), until recently we did not know if the microchimeric cells were hematopoietic or non-hematopoietic in origin. Because we were able to isolate circulating fetal CD34C cells from maternal blood we suspected that they were hematopoietic cells. Surprisingly, preliminary evidence suggests that fetal (XYC) PAPCs in paraffin-embedded tissue obtained from women with a history of having had

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at least one son suggests that fetal cells in maternal thyroid express cytokeratin AE1/3 (a marker of epithelial cells) and some fetal cells in maternal liver express heppar-1.67 However, in lymph node and spleen, 90% of fetal cells express the common leukocyte antigen CD45. Thus it appears that fetal cells express the appropriate antigens for the tissue in which they reside and this is a non-random process. We do not yet know whether this is occurring by differentiation or fusion. Existing stem cell literature fails to incorporate information on pregnancy history Within the stem cell literature there are major discrepancies in the reported frequencies of donor stem cell engraftment into human recipients. For example, in three studies in which male Y-chromosome-positive cells were detected in female donor hearts transplanted into male recipients, the reported frequencies of male cells ranged from 0.04 to 0.2 to 20%.68–70 In all of these studies the interpretation of the results was that the recipient’s own pluripotent stem cells migrated into the donor heart and differentiated there, even within a time span of as short as 4 days between transplant and recipient death.69 An alternative explanation might be that the female heart donors had themselves been pregnant at one time with a male fetus, and that the donor’s fetus was actually the source of the male Y-chromosome-positive cells. These male cells could then differentiate at any time between the donor’s pregnancy and her death. We believe that is important to consider the donor and/or the recipient’s pregnancy history in the interpretation of clinical data suggesting stem cell plasticity.71 Unfortunately, this is not generally done. Role of pregnancy loss in the development of microchimerism To acquire PAPCs and to develop microchimerism it is not necessary to continue a pregnancy to full-term and deliver a live born infant. We performed a study in which we obtained peripheral blood samples from 40 women immediately after elective termination of pregnancy. Using quantitative PCR we showed that in 42 of 64 samples, a median number of 1246 males were present in 16 ml of blood.24 When extrapolated to the full circulating blood volume, this data implied that as many as 500 000 nucleated fetal cells could be transfused into a woman following termination. Subsequently, we performed a meta-analysis of all published studies of fetal cell microchimerism that documented reproductive history. We were unable to distinguish a history of miscarriage from elective termination and included both under the category of ‘fetal loss’.72 Women with a history of fetal loss had a statistically increased chance of developing fetal cell microchimerism. We speculated that this was due to a large fetomaternal hemorrhage resulting from the fetal loss, or that the fetal cells transferred at an earlier point in gestation may have been better tolerated by the mother due to their earlier gestational age.

NEED FOR ANIMAL MODELS To be able to answer the question of whether the PAPCs are acting more like stem cells or mediators of autoimmune disease, we need to develop animal models. Because of

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a relatively short gestation time, ease of manipulation, genetic homology with humans, and excellent genetic characterization, the mouse is a logical first choice. Mouse Gaillard et al73 studied the transmission of fetal cells bearing a uniquely paternal cytogenetic marker, the T6 chromosome, in the mouse. They showed that during murine pregnancy there was a considerable concentration of T6C fetal cells in the maternal spleen (3–6% of total cells). Additionally, these cells were shown to be capable of division, survived post-partum, and increased in number during a second pregnancy. Lie´gois et al74 further showed that T6-positive cells were demonstrable in maternal spleen and bone marrow even when the subsequent pregnancies resulted from mating with other murine species that did not carry this cytogenetic marker. Thus, they demonstrated that in the mouse, fetal cells could persist from prior pregnancies. They also suggested that the maternal lymphoid organs were an important location where fetal cell surface antigens were recognized. Trying to better understand the connection between microchimerism and autoimmune disease, Christner and co-workers75 demonstrated an increased number of fetal cells in the peripheral blood of adult female mice following exposure to vinyl chloride, which is associated with dermal fibrosis. Imaizumi et al76 showed that fetal cells accumulate within the thyroid glands of mice that develop experimental autoimmune thyroiditis during pregnancy and the early post-partum period. These studies suggest that microchimerism might be variable in a healthy mouse and might increase in diseased or injured organs. We have developed a mouse model for microchimerism, using the wild-type C57Bl6 female mated to a syngeneic male who is carrying an enhanced green fluorescence protein (GFP) transgene. The GFP sequence is transmitted as a dominant gene and all pups that inherit this sequence will fluoresce green in all of their cells except for erythrocytes and fur. This permits us to track fetal cells in the mother mouse’s body during pregnancy and post-partum. Using GFP detection by both real-time PCR and immunohistochemistry we are in the process of generating data on the natural history of fetal cell microchimerism in the mouse. We have developed chemical, surgical, and genetic models of injury in which to test the fetal cell response in maternal tissue. In doing so, we aim to prove that pregnancy results in the acquisition of a novel type of stem cell by the maternal host.

SUMMARY Intact fetal cells in maternal blood during pregnancy are rare but have attracted interest as a source of fetal genetic material for non-invasive prenatal diagnosis. Efforts to isolate and expand these cells by culture are limited by the low circulating numbers of clonogenic cells. This work, however, led to the unexpected discovery that fetal progenitor cells persist in maternal blood and tissue for decades following delivery. Data obtained from the human adult, non-transfused female shows that fetal cells (identified on the basis of the Y chromosome as well as fetal-specific DNA polymorphisms) are detectable in peripheral blood and clinically diseased organs. Initial studies focused on the hypothesis that the fetal cells were immunocompetent, and could initiate a graft-versus-host reaction against the mother that would explain

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the higher incidence of autoimmune disease in women. More recently, we hypothesized that the persisting fetal cells might have stem-cell-like qualities, with potential for therapeutic applications. We have demonstrated that fetal cells in maternal tissues also express non-hematopoietic markers. Experimental animals, such as the mouse, are beginning to be used in model systems to understand the mechanisms involved in fetal cell microchimerism. Preliminary data suggest that fetal cells are capable of migration to sites of injury and differentiation, implying that they might play a role in tissue repair. Pregnancy results in the physiologic acquisition of cells with stem-cell capabilities, the PAPC, which might influence long-term health.

Practice points † at present, the inability to reproducibly culture fetal erythroid progenitor cells is primarily due to their low circulating number in maternal blood samples † clear-cut developmental and biological differences exist between fetal and adult erythropoiesis. The variable experimental results observed in the papers cited might be due to the different selection methods, culture media, and cytokines used by investigators † pregnancy induces synthesis of maternal endothelial cell progenitors. The clinical significance of this is presently unknown † fetal mesenchymal cells cannot be routinely cultured from preprocedural maternal blood samples taken in the first trimester † our data suggests that human and murine pregnancy results in the transfer and persistence of a new type of stem cell–the PAPC † PAPCs are not truly ‘adult’ but are present in the adult female. Therefore, they have the potential advantage of fetal cell plasticity but could be acquired via simple venipuncture on a consenting adult † future work needs to consider the role of pregnancy history in stem cell donors and recipients

Research agenda † determine if circulating fetal CD34C CD38C cells differentiate or fuse with maternal cells in tissue † determine if PAPCs encompass one or multiple cell types † perform additional studies to determine the origin of fetal cells in maternal tissue

ACKNOWLEDGEMENTS This work is supported by an internal grant from the Tufts-New England Medical Center, a grant from the Digestive Diseases Center (NIDDK, P30 DK34928), and NIH support for the NIFTY trial (NICHD, N01 HD43204). The author would also like to acknowledge the substantial primary research work performed by members of her

972 D. W. Bianchi

laboratory, most notably Kirby L. Johnson PhD, Kiarash Khosrotehrani MD, Helene Stroh, Sarah Gue´gan MD, and Dong Hyun Cha MD.

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