Hum. Reprod. Advance Access originally published online on October 30, 2006
Human Reproduction 2007 22(3):654-661; doi:10.1093/humrep/del426
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Fetal cells participate over time in the response to specific types of murine maternal hepatic injury
1 Division of Genetics, Departments of Pediatrics and Obstetrics and Gynecology, Tufts-New England Medical Center, Boston, MA, USA 2 Laboratoire de Physiopathologie du Développement, Université Pierre et Marie Curie-Paris VI, Paris, France 3 Department of Surgery 4 Department of Pathology and 5 Institute for Clinical Research and Health Policy Studies, Tufts-New England Medical Center, Boston, MA, USA
6 To whom correspondence should be addressed at: Division of Genetics, Department of Pediatrics, Tufts-New England Medical Center, Box 394, 750 Washington Street, Boston, MA 02111, USA. E-mail: dbianchi{at}tufts-nemc.org
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Abstract |
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BACKGROUND: In humans, fetal microchimeric cells transferred to maternal tissues during pregnancy can adopt a hepatocyte phenotype. Our objective was to determine whether fetal cells participate in the response to specific murine post-partum hepatic injuries. METHODS: Wild-type female mice were bred to males transgenic for the enhanced green fluorescent protein (GFP) (n = 42). Following delivery, we created models of chemical or surgical injury with carbon tetrachloride (CCl4) injection or by performing partial hepatectomy. Liver injury was assessed histologically. Fetal cells in maternal liver were detected and measured by real-time PCR amplification of the gfp transgene and by immunofluorescence using anti-GFP antibodies. RESULTS: PCR results showed that in chemical but not surgical injury, fetal GFP+ cells were detectable in maternal liver and spleen and that fetal cell presence was significantly increased over time following injury (4 versus 8 weeks, P = 0.006 for liver and P = 0.0006 for spleen). In some animals, following chemical injury, GFP+ cells were detected by immunofluorescence. CONCLUSIONS: The results of this preliminary study suggest that specific types of injury may elicit different fetal cell responses in maternal organs. There is a significant effect of time on fetal cell presence in liver and spleen. Furthermore, real-time PCR amplification is more sensitive than immunofluorescence for the detection of microchimeric fetal cells.
Key words: carbon tetrachloride/fetal cell microchimerism/partial hepatectomy/pregnancy/stem cells
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Introduction |
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In humans, fetal stem cells enter the maternal circulation during pregnancy and persist for decades in blood and tissues, giving rise to fetal cell microchimerism (Bianchi et al., 1996
; Ariga et al., 2001; Osada et al., 2001). The long-term health consequences of this phenomenon are unclear. Some investigators have established an association between the number of fetal microchimeric cells and the occurrence of autoimmune diseases such as systemic sclerosis (Artlett et al., 1998; Nelson et al., 1998) or primary biliary cirrhosis (Rubbia-Brandt et al., 1999; Corpechot et al., 2000; Fanning et al., 2000). Fetal cells are also reliably detected in the circulation of 30–50% of healthy women post-partum (Artlett et al., 2002; Lambert et al., 2002). In addition, fetal cells have been detected in non-autoimmune diseases such as cervical cancer (Cha et al., 2003) and thyroid adenoma (Srivatsa et al., 2001). In the latter study, microscopic analysis of the fetal cells suggested that they had the morphology of fully mature and differentiated thyroid tissue. Using immunolabelling techniques, we have previously shown that decades after delivery, fetal microchimeric cells express markers of hepatocytic, epithelial or leukocyte differentiation in maternal liver, epithelial or haematopoietic organs, respectively (Khosrotehrani et al., 2004a). This observation led us to hypothesize that fetal microchimeric stem cells may home to maternal injured tissue as part of the maternal repair response to tissue injury. They may therefore be ‘helpful’ and not ‘harmful’ as previously proposed (Khosrotehrani and Bianchi, 2003).
We have also reported a case in which a large number of male fetal cells repopulated the liver of a woman with hepatitis C (Johnson et al., 2002). This observation led us to ask whether microchimeric fetal cells could participate in the regeneration of maternal liver. Human studies are often limited by the number of subjects and the availability of healthy and diseased tissues. To better address this question, we developed a murine model of fetal cell microchimerism (Khosrotehrani et al., 2004b, 2005). We bred wild-type female mice to congenic males transgenic for the green fluorescent protein (GFP) reporter gene. Fetal cells from pups that inherit the transgene are easily detectable in maternal wild-type tissues. Carbon tetrachloride (CCl4) and partial hepatectomy are well-established injury models to study liver regeneration. CCl4 induces an acute injury in which the regeneration process involves hepatocyte cell division and oval cell activation (Theise and Krause, 2002; Wang et al., 2003). On the contrary, partial hepatectomy induces mitotic activity among remaining hepatocytes and does not recruit oval cells (Libbrecht and Roskams, 2002). In this preliminary study, our objective was to determine whether fetal cells participate in the maternal response to different post-partum hepatic injuries.
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Materials and methods |
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Mice
The Institutional Animal Care and Use Committee of the Tufts University School of Medicine Division of Laboratory Animal Medicine approved the present protocol. The enhanced GFP+ transgenic mouse (Jackson Laboratories stock no. 03291, Bar Harbor, ME, USA) has a C57BL/6J genetic background, with the gfp transgene under the control of a chicken
-actin promoter and a cytomegalovirus enhancer (Okabe et al., 1997). We purchased C57BL/6J (wild type) female retired breeders (Jackson Laboratories) that were reportedly bred to male GFP+ mice and gave birth to an average of three to four litters. The number of transgenic pups born to the retired breeders was unknown. We also bred 8-week-old C57BL/6J virgin female mice to GFP+ males. Female mice that did not deliver a litter were excluded to avoid confounding results due to microchimerism as a result of spontaneous abortion or resorption. After delivery, we recorded the total and transgenic number of pups for each mouse by using UV excitation to detect green fluorescence.
Liver injury models
Chemical injury
Mice were injected once with 0.1 ml of 20% CCl4 in vegetable oil i.p., as previously described (Recknagel, 1983; Koniaris et al., 2001). Retired breeders (n = 17) were studied in the first group of experiments (group 1). Animals were observed for 4 weeks (n = 5) or 8 weeks (n = 11) following injection and then sacrificed. An additional mouse was sacrificed 6 weeks following injection because of sick mouse syndrome and was not included in the statistical analyses. Liver was studied with histology, immunofluorescence and real-time quantitative PCR amplification. Owing to observed gross enlargement of the spleens in the chemically injured animals, which suggested an ongoing pathologic process, DNA from spleen was also isolated for PCR. Because PCR results in group 1 suggested a trend towards increasing fetomaternal microchimerism at the later observation point, a second group of experiments (group 2) were performed with 8-week-old C57BL/6J virgin females mated to GFP+ males (n = 18). In group 2, cases (10/18) were injected once with CCl4 and controls (8/18) were injected once with vegetable oil only. Investigators were not blinded as to the type of injection given. Mice were injected 5–6 weeks following delivery and observed for 4 weeks (n = 9) or 8 (n = 9) weeks after injection.
Surgical injury
Partial hepatectomy was performed 3–10 weeks after delivery on virgin female C57BL/6J mice (n = 7) that had been bred once to GFP+ male mice. Briefly, animals were anaesthetized using isoflurane inhalation and maintained under deep anaesthesia throughout the surgical procedure, with anaesthetic doses titrated as needed. A midline incision was made from xyphoid to mid-abdomen, and the liver was exposed. The anterior and medial segments of the liver were retracted into the wound and encircled with a 2–0 vicryl ligature and excised sharply. The excised liver was used as a baseline control for PCR only. The incision was closed with a running 3–0 vicryl suture, and animals were killed 7 days later.
Tissue collection
Mice were sacrificed using carbon dioxide inhalation. Liver and spleen were collected. Tissues were either fixed in 4% formaldehyde and 30% sucrose, in formalin, or immediately frozen in liquid nitrogen.
Histology
Paraffin-embedded sections of liver specimens were obtained and stained with haematoxylin–eosin or Trichrome Blue to detect fibrosis. Sections were then assessed histologically, focusing on general structure, number of mitoses, amount of inflammation and presence of steatosis or necrosis. Histologic assessment was used to track differences in the recovery process at 4 and 8 weeks.
DNA extraction and real-time PCR amplification
Genomic DNA extraction was performed on all samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed as previously described using an ABI 7700 Sequence Detection System with the SDS v1.9 software (Khosrotehrani et al., 2004b). To estimate the total amount of DNA in liver and spleen specimens, we measured the apolipoprotein B gene (ApoB) as previously described (Khosrotehrani et al., 2005). Based on these measurements and using a conversion factor of 6.25 pg of DNA per C57BL/6J genome (Capparelli et al., 1997), between 100 000 and 200 000 maternal genome equivalents (GE) were added to each PCR reaction for the measurement of the gfp transgene as previously described (Khosrotehrani et al., 2005). Using this mouse genome conversion factor and the total number of cells as determined by PCR amplification of ApoB, we converted the results to the number of GFP+ cells per 1 x 106 GE of maternal (total) cells (see Appendices 1 and 2). All GFP PCR amplification was performed in triplicate, and a tissue sample was considered to be positive for fetal cell microchimerism if the mean amount of gfp transgene detected was equivalent to at least one genome in a background of 1 million maternal GE. Each reaction included a positive control, which was used to generate the standard curve, and a ‘no template added’ negative control.
Detection of GFP fluorescence in maternal tissues
Anti-GFP rabbit polyclonal antibodies (Chemicon International, Temecula, CA, USA) were used on frozen sections to detect fetal GFP+ cells. Goat anti-rabbit immunoglobulin G labelled with fluorescein isothiocyanate (Jackson Immunoresearch, West Grove, PA, USA) was used as a secondary antibody. Briefly, after rehydration, sections were blocked using 20% normal goat serum. Primary antibody was used at a dilution of 1:100 and incubated overnight at 4°C. After washes, secondary antibody was used at a dilution of 1:50 and incubated for 40 min. Slides were then washed, counterstained with 0.3 µg/ml of 4,6-diamidino-2-phenylindole and observed with a fluorescence microscope (Zeiss Axioskop).
Statistical analyses
Each mouse was analysed for the presence of microchimerism, defined by the presence of transgenic cells by microscopy or the number of microchimeric GE by quantitative PCR in the liver or spleen. Fisher’s exact test was used to assess the proportion of samples with transgenic cells among cases and controls. The number of microchimeric GE in cases and controls as well as between the two time points (4 and 8 weeks) in the tissues was analysed using the non-parametric Wilcoxon rank sum test. All statistical analyses were performed using SAS/STAT software (SAS Institute, Inc., Cary, NC, USA). A P value of <0.05 was considered significant.
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Histology
Chemical injury
In group 1 mice, CCl4 injection induced massive (60–90%) liver necrosis at 4 weeks after the injection (Figure 1A, Table I). At 8 weeks after the injection, all mice showed histologic evidence of recovery from their liver injury. The livers were slightly fibrotic with excess microvesicular steatosis (Figure 1B and C). The injured livers also had more inflammatory cells (Figure 1D), sometimes organized in aggregates. In addition, 10 of 16 exposed mice had gross evidence of splenic enlargement; spleens were not available for histology.
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Surgical injury
Post-mortem analysis of livers from the four surviving animals in group 1 that were electively sacrificed showed healthy regenerating liver tissue, with mitotic activity present (Figure 2).
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Real-time PCR amplification of fetal transgenes
Chemical injury
Results of real-time PCR amplification for groups 1 and 2 are summarized in Tables I and II. Raw data are summarized in Appendices 1 and 2. No amplification of either ApoB or GFP was ever observed in the no-template control wells. For group 2, a comparison of the median number of fetal GE in maternal liver at 4 weeks for CCl4 exposed (n = 5) versus vegetable oil control (n = 4) was not significant in either liver or spleen (P = 0.44 and P = 1.0, respectively). At 8 weeks, the comparison of exposed (n = 5) to control (n = 4) was significant for spleen (P = 0.016) but not for liver (P = 0.29), probably because of the small sample size. We also examined the effect of time in all CCl4-exposed mice (groups 1 and 2) for both the median number of fetal GE and the relative frequency of organs with detectable fetal cell microchimerism. Both were highly significant. In the comparison of 4 weeks (n = 10) versus 8 weeks (n = 16), both liver and spleen had more fetal cells present at 8 weeks (P = 0.006 and P = 0.0006, respectively, by Wilcoxon rank sum test). The frequencies in liver were 2/10 (4 weeks) versus 11/16 (8 weeks) (P = 0.04, by Fisher’s exact test). Similar results were observed in spleen: 1/10 (4 weeks) versus 12/16 (8 weeks) (P = 0.004, by Fisher’s exact test).
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Surgical injury
For PCR experiments, 3-lobe hepatectomies were performed on seven mice, four of which were electively sacrificed 7 days following surgery and three of which died 1–4 days following surgery. All mice that were electively sacrificed had evidence of mitotic activity present, indicating active liver regeneration (Table III). No GFP+ cells were detected in any of the post-hepatectomy livers nor in their corresponding baseline liver lobes removed, which served as the internal control for each mouse. Six of seven spleens had no GFP+ cells detected. In one spleen, one of the triplicate experiments had 4.32 GFP GE per 1 x 106 maternal GE. The other two reactions had 0.
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Effect of the number of transgenic pups
PCR results from the livers and spleens of group 2 CCl4-exposed animals at 8 weeks (n = 5) were used to determine whether the number of transgenic pups delivered affected the results. Although liver (r2 = 0.22, P = 0.72) and spleen (r2 = 0.45, P = 0.45) fetal cell numbers moderately correlated with the number of transgenic pups, the results did not reach statistical significance.
Immunofluorescence
Immunofluorescence analysis of liver sections allowed us to detect GFP+ cells in 2 of 11 CCl4-exposed livers when studied at 8 weeks (Table I). Figure 3 shows an example of a GFP+ cell observed in liver tissue of a mouse injected with CCl4. No fetal GFP+ cells were detected in regenerated livers following partial hepatectomy.
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Discussion |
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The persistence of fetal cells in maternal tissues after human pregnancy is well known. We have recently proposed that fetal cells, once in the maternal circulation, may participate in the maternal response to tissue injury for years post-partum (Khosrotehrani and Bianchi, 2003
). We have also shown that fetal cell microchimerism occurs during all pregnancies in mice; however, by 3 weeks after delivery, no fetal cells can be detected in maternal tissues (Khosrotehrani et al., 2005). In the present study, we demonstrate that fetal GFP+ cells are found in the chemically injured liver and spleen in the post-partum female mouse. Exposure to CCl4 results in an increase in the frequency and number of fetal cells. Although the experiments were designed with liver toxicity in mind, CCl4 is also known to affect the spleen (Alexeyeva et al., 1994). Our mice had gross evidence of splenic enlargement, so it is not surprising that fetal cells were found in this injured tissue. Our study was not designed to determine whether fetal cells migrate to the injured organs or proliferate in situ. Future planned experiments monitoring blood trafficking of GFP+ cells following injury will help to distinguish between these two possibilities.
Our results also demonstrate that there is a significant effect of time on fetal cell presence in liver and spleen. An increase in the median number of fetal cells in the maternal livers occurred some time between 4 and 8 weeks after the chemical injury. According to our histological findings, this corresponds to the period when most of the liver regeneration took place. We used another injury model, partial hepatectomy, in which the regeneration takes place in the first few days but did not observe any increase in the number of microchimeric cells in the regenerating liver by PCR or by immunostaining. It is possible that we did not allow our mice to survive long enough following surgical injury to detect fetal cell microchimerism, although the histologic results suggest that active repair had already taken place. Liver regeneration after CCl4 injection involves haematopoietic stem cells and hepatic oval cells (Wang et al., 2003), whereas the regeneration after partial hepatectomy is based on hepatocyte cell division (Libbrecht and Roskams, 2002). Furthermore, we did not perform partial hepatectomies on retired breeders, which might have had an increased likelihood of demonstrating the presence of fetal cells. However, prior data from our laboratory have shown that even in healthy retired breeders, only 40% of them have detectable microchimerism (Khosrotehrani et al., 2005).
Our data agree with previous studies using rodent models that demonstrate the presence of fetal cells in maternal tissue following injury. Christner et al. (2000) described a higher number of fetal cells in the circulation of mice injected with vinyl chloride to induce skin fibrosis. Imaizumi et al. (2002) induced thyroiditis in the mouse and noted an increase in the number of microchimeric cells in the thyroid. Wang et al. (2004) exposed post-partum female rats to ethanol and gentamicin; fetal GFP+ cells were detected in liver and kidney. The previous studies, however, did not have controls matched for reproductive history. Furthermore, they did not study animals at different time points in the recovery period.
In humans, we have shown that fetal cells bearing hepatocyte markers can be found in the livers of women with hepatitis C and autoimmune hepatitis (Johnson et al., 2002; Khosrotehrani et al., 2004a). In the present study, we did not attempt to identify the type of fetal cell in the liver, given their relative rarity in tissue sections. The fetal GFP+ cells in the maternal livers after CCl4 injury were, however, mononuclear. Some of them were isolated, and some were grouped in aggregates. They were not perivascular. Ongoing studies in the laboratory are directed towards determining the type of fetal cell involved in the injury response.
Of note, 7 of 16 mice and 4 of 10 mice in groups 1 and 2, respectively, that were exposed to CCl4 had no detectable fetal cell microchimerism. One explanation could be that the number of fetal cells present was below the sensitivity of 1 per 1 x 106 maternal cells. A more likely explanation, however, is that the fetal cells are not homogeneously distributed throughout the maternal liver. The PCR experiments shown here were performed on a relatively small subsection of the maternal liver (100 000–200 000 GE). They therefore do not reflect the contents of the entire liver. Thus, it is possible that we analysed an area without transgenic cells, although they were present elsewhere in the liver. Furthermore, because group 1 animals were bred at the Jackson Laboratories, we can never prove that they gave birth to transgenic pups.
In this study, more fetal cells were detected by real-time PCR than by immunohistochemistry. We do not think that the PCR results are due to contamination, because reactions were run in triplicate, and positive and negative controls were run each time. Still, a discrepancy exists between the immunofluorescence and PCR data. It has been suggested previously that the detection of gene sequences by in situ hybridization and PCR is more sensitive than the detection of protein markers by immunohistochemistry (Mezey et al., 2003). We think that the major issue here was the lack of a high-quality anti-GFP monoclonal antibody and the innate autofluorescence of liver.
In conclusion, this is a preliminary study in a mouse model that demonstrates that fetal microchimeric cells are transferred during pregnancy and can be detected in the maternal liver and spleen as a result of chemical but not surgical injury. The statistically significant differences in fetal cell microchimerism observed at different time points suggest that these cells play a dynamic role in the repair process. Further studies are needed to address whether the fetal cells involved in this process have a well-defined cellular phenotype, whether they migrate to the injury or proliferate on site and whether their presence ultimately improves maternal liver and splenic function.
Appendix 1. PCR data from group 1 mice
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CCl4, carbon tetrachloride; GE, genome equivalent; GFP, green fluorescent protein.
Appendix 2. PCR data from group 2 mice
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CCl4, carbon tetrachloride; GE, genome equivalent; GFP, green fluorescent protein.
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Acknowledgements |
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This study was supported by an internal grant from the Tufts-New England Medical Center, by a pilot project award from the GRASP Center at Tufts-New England Medical Center (NIH NIDDK P30 DK34928), by NIH R01 HD049469-01, and by the Tufts Post-Baccalaureate Research Education Program (NIH 5R25GM066567). The authors also thank the staff of the Department of Laboratory Animal Medicine (Courtney A. Bowker, Matthew S. Gale and Karrie E. Southwell) for their helpful advice.
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* These authors contributed equally to this work.
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References |
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Submitted on May 17, 2006; resubmitted on August 2, 2006; resubmitted on September 26, 2006; accepted on October 2, 2006.