Fetal microchimerism in maternal mouse blood and brain 2

Chapter Fetal cell differentiation in maternal brain 4.1. Introduction A wide variety of fetal cells have been found in maternal peripheral blood circulation and brain. The cues that induce fetal cell migration and determine the destination of these fetal cells in maternal solid organs such as brain are still unclear. There is evidence that some populations of umbilical cord blood or bone marrow cells can transdifferentiate into cells expressing neural cell markers under in vitro culture conditions (SanchezRamos et al., 2000;Woodbury et al., 2000) and transplanted umbilical cord blood and bone marrow cells can appear to be able to transdifferentiate into neural cells in host brain (Azizi et al., 1998;Kopen et al., 1999). However, as discussed in Chapter 1, the evidence for transdifferentiation is still debated. Here we report fetal microchimerism in the maternal mouse brain and evidence for morphological and immunocytochemical differentiation of these fetal cells into perivascular macrophage-like and neural-like cell types in the maternal brain. 4.2. Materials and methods 4.2.1. Animals Eight- to 12-week-old male homozygous C57BL/6 Cr Slc TgN(act-EGFP) OsbC14-Y01FM131 mice (Green Mice) were crossed with wild-type young adult female C57BL/6 mice (8 to 10 weeks old). All the offspring were hemizygous Green Mice. Control wild- 98 type young adult female C57BL/6 mice remained virgins. Ex-breeder wild-type female C57BL/6 mice were purchased from the Laboratory Animals Centre, National University of Singapore. These mice had been held as breeding stock since the age of to weeks and were retired at the age of to months after weaning their last litter. They were used at the age of to months old, at least to months after delivering their last litter. All experiments were conducted under the institutional guidelines of the Animal Ethics Committee of the Singapore General Hospital and the Institutional Animal Care and Use Committee, Office of Life Sciences, National University of Singapore, in accordance with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health, and following the International Guiding Principles for Animal Research (Howard-Jones N 1985). 4.2.2. Fluorescence in situ hybridization (FISH) for the Y chromosome Four weeks after delivery, non-lesioned young adult mothers (n = 6) and ex-breeder stock females to months after delivering their last litter (n = 6) were perfused with 0.9 % saline followed by % paraformaldehyde in phosphate buffer (pH 7.4). Serial coronal sections (10 µm) were cut on a cryostat, recovered on glass slides, and washed briefly in phosphate buffered saline (PBS) before FISH with a Cy3-labelled mouse Y chromosome specific probe (Cambio) according to an adaptation of the manufacturer’s recommended protocol. Briefly, sections were incubated with methanol and acetic acid (3:1 vol/vol) for 45 s, air-dried, and incubated with sodium thiocyanate solution (1 M) for at 75O C. The sections then received two washes in deionised, distilled water and were postfixed in % paraformaldehyde in phosphate buffer (pH 7.4) for 10 min, and air-dried. 99 Next the sections were denatured by three minute washes with 60 % formamide in x SSC at 75O C, quenched in ethanol, dehydrated through graded alcohols, and air-dried. The Cy3-labelled mouse Y chromosome probe (Cambio) was pre-warmed to 37O C and 10-15 µl was applied to the centre of the sections, and the sections were individually covered with glass coverslips and sealed with rubber cement. The sections were further denatured in the presence of the probe by cycling to 90O C for on, off, on, off, and finally on. The slides were then hybridised overnight at 37O C in a humidified chamber. The coverslips were removed and the sections underwent three washes in 50 % formamide in x SSC, three washes with x SSC, and two 10 washes in 0.05 % Tween 20 in x SSC at 42O C. After FISH, the sections were immunostained as described below and mounted with an antifade mounting medium contain DAPI (Vectashield, Vector Laboratories). 4.2.3. Fluorescence immunocytochemistry Four weeks after delivery, mice (n = 10) were perfused with % paraformaldehyde in phosphate buffer (pH 7.4). Serial coronal sections (20 µm) were cut on a cryostat. Fluorescent EGFP-positive fetal cells were identified in parenchyma of the cerebral cortex, hippocampal formation, or subiculum and adjacent to blood vessels in these regions. Selected sections were immunostained. Primary antibodies used were rat antimouse CD11b Alexa Fluor 647-conjugated (1:100, BD Pharmingen), mouse monoclonal anti-CD45 (1: 100, Santa Cruz Biotechnology), rat anti-mouse F4/80 (1:100, Serotec), rabbit anti-GFAP (1:200, DakoCytomation), mouse monoclonal anti-NeuN (1:200, Chemicon), and polyclonal rabbit anti-von Willebrand Factor (anti-vWF, 1:100, 100 DakoCytomation). The secondary antibodies used were FluoroLink Cy2- and Cy3labelled goat anti-mouse IgG (Amersham Bioscience), Alexa Fluor 488 and Alex Fluor 568 goat anti-rat IgG (Molecular Probes), Alexa Fluor 488 and Alex Fluor 568 donkey anti-rabbit IgG (Molecular Probes), Alexa Fluor 633 goat anti-rabbit IgG (Molecular Probes) and Alexa Fluor 68 donkey anti-goat IgG (Molecular Probes). All the secondary antibodies were used at 1:200 dilutions. Briefly, sections were permeablized with 100% cold acetone, washed three times with phosphate buffer saline (PBS) and blocked with 5% BSA and 0.1% goat serum or % donkey serum, as appropriate for the secondary antibodies used, for hour. Slides were incubated with the primary antibody overnight at room temperature. The slides were then washed x in PBS and incubated with the appropriate secondary antibody for hr at room temperature and then washed x in PBS. For multiple labelling, the blocking and primary and secondary antibody incubations were repeated sequential. Some of the sections were further immunostained for EGFP with a mouse monoclonal antibody to GFP (1:100, ½ hrs, Chemicon) conjugated to the photostable fluorophore Alexa Fluor 488 (Zenon, Molecular Probes). Sections were examined by sequential scanning and optical sectioning with a confocal microscope (FV500, Olympus, or LSM510, Zeiss). For illustration, extended-focus XY confocal images were generated from the 3D image stacks and orthogonal slices through the Z-stacks along the X-, Y- and Z-axes are shown for selected cells. 101 4.3. Results 4.3.1. Location of fetal cells in the maternal brain Double-immunostaining with anti-GFP antibodies to identify Green Mouse fetal cells and anti-vWF antibodies to identify endothelial cells revealed perivascular fetal Green Mouse cells juxtaposed to blood vessels in the brains of non-lesioned young adult mothers weeks post-partum (Figure 9, A to F). Rarely such cells juxtaposed to blood vessels appeared to be binucleated (Figure 9, G to K). Other Green Mouse fetal cells were observed within the brain parenchyma with no obvious association to blood vessels (Figures 11 and 12) and occasionally aligned with the maternal brain cell layers (Figure 11, A to H). Likewise, fetal cells were identified in the brain parenchyma with no obvious association to blood vessels by FISH for the Y chromosome in the brains of exbreeder stock female mice at least to months after delivering their last litter 4.3.2. Differentiation of fetal Green Mouse cells in maternal brain Four weeks after delivery, Green Mouse fetal cells in the mothers’ brains were capable of morphologically and immunocytochemically differentiating into diverse cell types. Some of the Green Mouse fetal cells observed within the brain juxtaposed to blood vessels were found to immunostain with an antibody to the macrophage marker, F4/80, and would occasionally appear to wrap processes around adjacent endothelial cells (Figure 10, F and H). Occasionally, perivascular maternal cells immunolabelling for F4/80 were also observed with similar morphologies (Figure 10, I to L). However, there was no evidence of expression of CD11b by fetal cells in the maternal brain. No instances of 102 engulfment of host neural cells by fetal cells labelled with the anti-F4/80 antibody were observed. Double-labelling with antibodies to GFP and to the neuronal markers, MAP2ab and NeuN, provided immunocytochemical evidence for neuronal differentiation of Green Mouse fetal cells in the brains of young adult mothers weeks post-partum (Figure 11, A to H). These anti-GFP labelled cells also displayed morphological features consistent with neuronal cells and were occasionally found organotypically aligned with the cells of the host CA1 pyramidal layer (Figure 11, A to H). There was no evidence of F4/80 or CD45 immunoreactivity in Green Mouse fetal cells immunolabelled for these markers of neuronal cell type. Male fetal cells identified by FISH for the Y chromosome in the brains of ex-breeder stock female mice at least to months after delivery of their last litter also double-labelled with the anti-MAP2ab antibody (Figure 11, I to L). Although fetal cells were occasionally closely juxtaposed to NG2-positive host cells, no evidence was found for double-labelling of Green Mouse fetal cells by an antibody to NG2, a marker for immature oligodendrocytes (Figure 12, A to C). However, other Green Mouse fetal cells labelled with antibodies to MAG and GFAP, markers for oligodendrocytes and astrocytes, respectively (Figure 12, D to I). There was no evidence of F4/80 or CD45 immunoreactivity in Green Mouse fetal cells immunolabelled for these markers of neural cell type. Male fetal cells identified by FISH for the Y chromosome in the brains of ex-breeder stock female mice at least to months after delivery of their last litter were also double-labelled with the anti-GFAP antibody (Figure 12, J and K). 103 Figure 9: 104 Figure 9: Perivascular Green Mouse fetal cells in the maternal brain. Confocal images of sections from the brains of non-lesioned wild-type mothers of Green Mouse pups weeks after delivery. Sections were labelled with DAPI (blue) to identify nuclei (A and G) and by fluorescence immunocytochemistry with anti-GFP (green) and anti-vWF (red) antibodies shown in (B, H) and (C, I), respectively. D and J are merged images. Extended-focus confocal images from serial optical sectioning are shown in A to D and G to J. E and F are orthogonal slices through the cells in the regions identified by the white boxes in G. In E, the EGFP-positive fetal cell (green) is juxtaposed to the blood vessel, separated from the lumen (small “L”) of the vessel only by vWF-positive maternal endothelial cells with characteristically elongated somata and nuclei (F). A rare example of a putatively binucleated EGFP-positive fetal cell is shown in J. Orthogonal slices through the region identified by the white box in J shows that the cell is closely juxtaposed to the vWF-positive endothelial wall (K). Scale bars are (D) 25 µm, (E, F) µm, (J) 20 µm, and (K) 10 µm. 105 Figure 10: 106 Figure 10: F4/80 immunocytochemistry of perivascular Green Mouse fetal cells in maternal brain. Confocal images of sections from the brains of wild-type mothers of Green Mouse pups weeks after delivery. Sections were counterstained with DAPI (blue) to identify nuclei and labelled by fluorescence immunocytochemistry with anti-vWF (purple), anti-GFP (green), and anti-F4/80 (red) antibodies. Extended-focus confocal images from serial optical sectioning are shown in A, B, C, and D. E, F, G and H show orthogonal slices through the cells in the region identified by the white box in D. I, J, K, and L are orthogonal slices from another brain. The white arrowheads indicate large perivascular EGFP-positive fetal cells double-labelling for the macrophage marker F4/80, but not labelling for the endothelial marker, vWF. In F and H, the yellow arrowheads indicate what appears to be evidence of an EGFP-positive process from a fetal perivascular macrophage wrapping around adjacent endothelial cells. The blue arrowheads indicate maternal cells labelling for vWF (A to D) and F4/80 (I to L). The F4/80-positive fetal cells exhibit a similar size and location to the maternal perivascular macrophage. The scale bars are (D) 20 µm and (H & L) 10 µm. 107 Fetal Microchimerism in the Maternal Brain 1446 0.1% goat serum or 5% donkey serum, as appropriate for the secondary antibodies used, for hour. Slides were incubated with the primary antibody overnight at room temperature. The slides were then washed three times for minutes in PBS-Triton and incubated with the appropriate secondary antibody for hour at room temperature and then washed three times for minutes in PBS-Triton. For multiple labeling, the blocking and primary and secondary antibody incubations were repeated sequentially. Some of the sections were further immunostained for EGFP with a mouse monoclonal antibody to GFP (1:100, 1.5 hours; Chemicon) conjugated to the photostable fluorophore Alexa Fluor 488 (Zenon; Molecular Probes). Sections were examined by sequential scanning and optical sectioning with a confocal microscope (FV500 [Olympus, Tokyo, http://www.olympus-global.com] or LSM510 [Carl Zeiss, Jena, Germany, http://www.zeiss.com]). For illustration, extendedfocus XY confocal images were generated from the 3D image stacks, and orthogonal slices through the Z-stacks along the X-, Y-, and Z-axes are shown for selected cells. Results within the block of tissue containing the site of the lesion, Block 3, the number of Green Mouse fetal cells was significantly greater in the lesioned maternal brain (29.9 ± 8.5 fetal cells per 1,000 maternal cells) than in the intact maternal brain (5.3 ± 2.6 fetal cells per 1,000 maternal cells; Fig. 2E; t-test, p < .05). In a separate experiment, real-time PCR of genomic DNA from the brains of young adult wild-type C57BL/6 mothers (n = 4) for the Y chromosome–specific sex-determining region of the mouse Y chromosome produced a similar estimate for the number of fetal cells in the intact maternal brain at weeks postpartum (5.5 ± 1.6 male fetal cells/1,000 maternal cells, which equates to approximately 11 fetal cells/1,000 maternal cells). Quantitative real-time PCR of genomic DNA from brains of C57BL/6 ex-breeder stock female mice at least 2–3 months after delivering their last litter (n = 9) for the sex-determining region of the mouse Y chromosome revealed male cells in the brains of four out of nine female mice. The male cells were found almost exclusively in Block 1, corresponding largely to the olfactory bulb. In those exbreeder stock females in which male cells were found, the mean number of male cells in Block was 95.8 ± 69.8 male cells per 1,000 maternal cells. Fetal Green Mouse Cells in Maternal Blood and Brain EGFP-positive Green Mouse fetal cells were detected by FACS in mononuclear cell fractions of maternal blood taken days after delivery from young adult mothers (n = 10) whose pups were fathered by Green Mice (Fig. 1A). When the FACS signals were corrected to the blood of young adult wild-type virgin females as negative controls (Fig. 1B) and the blood of 7-day-old Green Mouse pups as positive controls (Fig. 1C), 0.08 ± 0.02% of nucleated cells in the blood were found to be Green Mouse fetal cells. Samples of the nucleated cell fractions were also visualized directly by phase-contrast and epifluorescence microscopy. EGFP-positive Green Mouse cells were found in the blood of mothers of hemizygous Green Mouse pups (Fig. 1E). Four weeks after delivery, the mothers were euthanized and perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were serially sectioned. Small numbers of EGFP-positive green mouse fetal cells were found in the maternal brains under epiflourescence microscopy (Fig. 1H). Quantification of Fetal Mouse Cells in Maternal Brain Quantitative real-time PCR of genomic DNA revealed that, on the day of parturition, 7.1 ± 1.8 cells per 1,000 maternal cells were EGFP-positive Green Mouse fetal cells in the intact brain of young adult mothers (n = 4) whose pups were fathered by Green Mice. At weeks postpartum (n = 4), there were 17.5 ± 3.7 fetal cells per 1,000 maternal cells (Fig. 2D), which was significantly greater than on the day of parturition (t-test; p < .05). The number of Green Mouse fetal cells was not significantly greater in the lesioned brain overall (23.3 ± 4.3 fetal cells per 1,000 maternal cells; Fig. 2D). However, Figure 1. Green Mouse fetal cells enter maternal blood circulation and can be found in the maternal brain. Fluorescent-activated cell sorting revealed a small population (0.08% ± 0.02%) of enhanced green fluorescent protein–positive (EGFP+) cells in maternal blood from (A) the mothers of Green Mouse pups (n = 10) when normalized to (B) control wild-type blood from virgin females (n = 10) and (C) the blood of hemizygous Green Mouse pups (n = 10). M1 and M2 mark the regions selected to sort the cells into EGFP− and EGFP+ cells, respectively. Phase (D, F) and epifluorescence (E, G) photomicrographs showing EGFP+ fetal cells (arrows) in the blood of a wild-type mother of Green Mouse pups (D, E) and in a positive control blood sample from a hemizygous Green Mouse pup (F, G). Epifluorescence photomicrograph (H) showing EGFP+ fetal cells in the cortex of a wild-type mother of Green Mouse pups perfused weeks after giving birth and sectioned at 20 μm on a cryostat. Scale bar = 50 μm. Data are expressed as mean ± SEM. Downloaded from www.StemCells.com by on August 21, 2006 Tan, Liao, Sun et al. 1447 Location of Fetal Cells in the Maternal Brain Double-immunostaining with anti-GFP antibodies to identify Green Mouse fetal cells and anti-vWF antibodies to identify endothelial cells revealed perivascular fetal Green Mouse cells juxtaposed to blood vessels in the brains of non-lesioned young adult mothers weeks postpartum (Figs. 3, 4). Rarely, these cells juxtaposed to blood vessels appeared to be binucleated (Figs. 3G–3K). Other Green Mouse fetal cells were observed within the brain parenchyma with no obvious association to blood vessels (Figs. 5, 6) and occasionally aligned with the maternal brain cell layers (Figs. 5A–5H). Likewise, fetal cells were identified in the brain parenchyma with no obvious association to blood vessels by FISH for the Y chromosome in the brains of ex-breeder stock female mice at least 2–3 months after delivering their last litter. to wrap processes around adjacent endothelial cells (Figs. 4F, 4H). Occasionally, perivascular maternal cells immunolabeling for F4/80 were also observed with similar morphologies (Figs. 4I– 4L). However, there was no evidence of expression of CD11b by fetal cells in the maternal brain. No instances of engulfment of host neural cells by fetal cells labeling with the anti-F4/80 antibody were observed. Double-labeling with antibodies to GFP and to the neuronal markers MAP2ab and NeuN provided immunocytochemical evidence for expression of characteristics of neuronal cells by Green Mouse fetal cells in the brains of young adult mothers weeks postpartum (Figs. 5A–5H). These anti-GFP–labeled cells also displayed morphological features consistent with neuronal cells and were occasionally found organotypically aligned with Morphology and Immunocytochemistry of Fetal Cells in the Maternal Brain Four weeks after delivery, Green Mouse fetal cells in the mothers’ brains were capable of expressing morphological and immunocytochemical characteristics of diverse cell types. Some of the Green Mouse fetal cells observed within the brain juxtaposed to blood vessels were found to immunostain with an antibody to the macrophage marker F4/80 and would occasionally appear Figure 2. Quantitative real-time polymerase chain reaction (PCR) of fetal Green Mouse cells in the maternal brain. Four weeks after delivery, brains of wild-type mothers of Green Mouse pups were divided into (A) four blocks for extraction of genomic DNA for real-time PCR for the transgenic enhanced green fluorescent protein (EGFP) gene carried by the fetal Green Mouse cells. Block includes the location where the NMDA was injected in the lesioned mothers. A standard curve (B) for log EGFP cDNA copy number plotted against the cycle threshold (CT) was strongly linear (R = 0.97). The real-time PCR conditions adopted separated no template control (NTC), EGFP cDNA control, and genomic DNA samples from lesioned and intact maternal brain (C). Fetal cells were present in both intact and lesioned maternal brain (D), and in the lesioned brains there were more fetal cells in Block 3, the region corresponding to the site of the lesion (E). www.StemCells.com Figure 3. Perivascular Green Mouse fetal cells in the maternal brain. Confocal images of sections from the brains of non-lesioned wildtype mothers of Green Mouse pups weeks after delivery. Sections were labeled with DAPI (blue) to identify nuclei (A, G) and by fluorescence immunocytochemistry with anti-GFP (green) (B, H) and anti-vWF (red) (C, I) antibodies. (D, J): Merged images. (A–D, G–J): Extended-focus confocal images from serial optical sectioning. (E, F): Orthogonal slices through the cells in the regions identified by the white boxes in (D). (E): EGFP-positive fetal cell (green) is juxtaposed to the blood vessel, separated from the lumen (L) of the vessel only by vWF-positive maternal endothelial cells with characteristically elongated somata and nuclei (F). (J): A rare example of a putatively binucleated EGFP-positive fetal cell. Orthogonal slices through the region identified by the white box in (J) show that the cell is closely juxtaposed to the vWF-positive endothelial wall (K). Scale bars = (D) 25 μm, (E, F) μm, (J) 20 μm, and (K) 10 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; vWF, von Willebrand factor. Downloaded from www.StemCells.com by on August 21, 2006 Fetal Microchimerism in the Maternal Brain 1448 the cells of the host CA1 pyramidal layer (Figs. 5A–5H). There was no evidence of F4/80 or CD45 immunoreactivity in Green Mouse fetal cells immunolabeling for these markers of neuronal cell type. Male fetal cells identified by FISH for the Y chromosome in the brains of ex-breeder stock female mice at least 2–3 months after delivery of their last litter also double-labeled with the anti-MAP2ab antibody (Figs. 5I–5L). Although fetal cells were occasionally closely juxtaposed to NG2-positive host cells, no evidence was found for double-labeling of Green Mouse fetal cells by an antibody to NG2, a marker for immature oligodendrocytes (Figs. 6A–6C). However, other Green Mouse fetal cells labeled with antibodies to MAG and GFAP, markers for oligodendrocytes and astrocytes, respectively (Figs. 6D–6I). There was no evidence of F4/80 or CD45 immunoreactivity in Green Mouse fetal cells immunolabeling for these markers of neural cell type. Male fetal cells identified by FISH for the Y chromosome in the brains of ex-breeder stock female mice at least 2–3 months after delivery of their last litter also doublelabeled with the anti-GFAP antibody (Figs. 6J, 6K). Figure 4. F4/80 immunocytochemistry of perivascular Green Mouse fetal cells in maternal brain. Confocal images of sections from the brains of wild-type mothers of Green Mouse pups weeks after delivery. Sections were counterstained with DAPI (blue) to identify nuclei and labeled by fluorescence immunocytochemistry with anti-vWF (purple), anti-GFP (green), and anti-F4/80 (red) antibodies. (A–D): Extended-focus confocal images from serial optical sectioning. (E– H): Orthogonal slices through the cells in the region identified by the white box in (D). (I–L): Orthogonal slices from another mouse. The white arrowheads indicate large perivascular EGFP-positive fetal cells double-labeling for the macrophage marker F4/80 but not labeling for the endothelial marker vWF. (F, H): Yellow arrowheads indicate what appears to be evidence of an EGFP-positive process from a fetal perivascular macrophage wrapping around adjacent endothelial cells. The blue arrowheads indicate maternal cells labeling for (A–D) vWF and (I–L) F4/80. The F4/80-positive fetal cells exhibit a similar size and location to the maternal perivascular macrophage. Scale bars = (D) 20 μm and (H, L) 10 μm. Abbreviations: DAPI, 4,6-diamidino2-phenylindole; EGFP, enhanced green fluorescent protein; vWF, von Willebrand factor. Discussion Fetal Green Mouse cells were found to enter wild-type maternal blood and brain, and some of these cells expressed immunocytochemical markers for neural cell types. The presence of fetal Green Mouse cells in maternal blood confirms that, as has been previously demonstrated for crossings of other mouse strains [3, 4], fetal Green Mouse cells can enter the maternal circulation in C57BL/6 mice. Although we also confirmed our findings by using Y chromosome–specific markers to identify male fetal cells, the use of Green Mice ubiquitously expressing EGFP facilitated detection of the fetal cells in maternal blood and tissues. However, EGFP should never be visualized in frozen sections without prior fixation because leaching of soluble EGFP can lead to false negatives. Neither ethanol nor acetone fixatives are sufficient to stabilize EGFP in tissue and, as previously described by others [16, 17], we found it important to fix the EGFP with a formaldehyde-based fixative before sectioning to avoid leaching of soluble EGFP. Subsequent immunofluorescence with an antiGFP antibody was also beneficial. Figure 5. Fetal cells can express neuronal immunocytochemical markers in the maternal brain. Confocal images of sections from the brains of lesioned young adult wild-type mothers of Green Mouse pups weeks after delivery (A–H) and a wild-type ex-breeder female (I–L). (A–C, E– G): Extended-focus confocal images generated by serial optical sectioning. Orthogonal slices through the cells in the regions identified by the white boxes in (C, G, K) are shown in (D, H, L), respectively. Fetal cells were identified by fluorescence immunocytochemistry with an anti–green fluorescent protein (anti-GFP) antibody (A, E) or by Y chromosome–specific fluorescence in situ hybridization (FISH) (I). Sections were immunostained (red in B–D, F–H; green in J–L) for neural cell type markers for neuronal cells (MAP2ab in B–D, J–L and NeuN in F–H). White arrowheads indicate fetal cells double-labeled either by anti-GFP immunocytochemistry (A, E) or Y chromosome FISH (I) and neural cell type markers MAP2ab (B, J) and NeuN (F). Orthogonal slices through the cells in the regions identified by the white boxes in (C, G, K) are shown in (D, H, L), respectively. Scale bars = (C, G) 100 μm and (K) 20 μm. Downloaded from www.StemCells.com by on August 21, 2006 Tan, Liao, Sun et al. 1449 We found that at least some of the fetal cells that spontaneously enter maternal circulation during pregnancy are capable of entering the maternal brain. Although fetal Green Mouse cells were rare in the brain, the number recovered is not insubstantial. However, although the blood was removed by perfusion, the num- Figure 6. Fetal cells can express oligodendrocytic and astrocytic immunocytochemical markers in the maternal brain. Confocal images of sections from the brains of lesioned young adult wild-type mothers of Green Mouse pups weeks after delivery (A–I) and a wild-type ex-breeder female (J, K). (A–I): Extended-focus confocal images generated from image stacks of serial optical sectioning. (J, K): Orthogonal slices. Fetal cells were identified by fluorescence immunocytochemistry with an anti–green fluorescent protein (antiGFP) antibody (A, D, G) or by Y chromosome–specific fluorescence in situ hybridization (FISH) (J, K). Sections were immunostained (red in B–D, F–H; green in J, K) for neural cell–type markers for oligodendrocytic cells (NG2 in B, C and MAG in E, F) and astrocytic cells (GFAP in H–K). Fetal cells did not double-label for NG2 (C). White arrowheads indicate fetal cells double-labeled either by antiGFP immunocytochemistry (A, D, G) or Y chromosome FISH (J, K) and the oligodendrocytic cell–type marker MAG (E) or the astrocytic cell–type marker GFAP (H, J, K). Blue arrowheads indicate cells labeling for neural cell–type markers but not double-labeling for enhanced green fluorescent protein (EGFP). Yellow arrowheads indicate EGFP-positive fetal cells not double-labeling for neural cell–type markers. Scale bars = (C, F, I) 100 μm and (J, K) 20 μm. www.StemCells.com ber of cells identified in brain tissue by the real-time PCR analysis does not reflect only neural cells but also includes cells engrafted into other niches, such as the perivascular environment. Studies of ex-breeder stock females showed that in the intact brain, fetal cells are present at least 2–3 months after pregnancy in some but not all individuals. In humans, individual differences in engraftment of the blood and skin of mothers by fetal cells have also been reported [2]. These individual differences may be due to differences in traffic across the placental barrier between pregnancies or to the mothers’ immune systems and the degree of histocompatibility between the fetal cells and the mothers. The larger numbers of cells observed in some of the individual mice that retained fetal cells suggests that fetal cells can accumulate over multiple pregnancies or can proliferate in the mothers. That greater numbers of fetal cells are found in the maternal brain at weeks postpartum than on the day of parturition could suggest that these cells are progenitor or stem cells capable of proliferation. However, this does not necessarily mean that proliferation occurs within the brain because it has been reported that, at least in humans, fetal cells engraft the BM [18]. In the intact brains of non-lesioned young adult mice and the ex-breeder stock females, the fetal cells were preferentially found in the region of the olfactory bulb. The subventricular zone has been reported to support survival and limited proliferation, migration, and immunocytochemical differentiation of umbilical cord blood cells and BM cells [19, 20]. Perhaps the subventricular zone and the rostral migratory stream to the olfactory bulb offer a niche facilitating fetal cell incorporation into the maternal brain, fetal cell proliferation, or fetal cell survival. When the maternal brain is injured, these cells preferentially enter the region of the injury. Previous studies had shown that umbilical cord blood cells can cross the blood–brain barrier to enter the injured brain, where they can express some immunocytochemical markers of neural cell types [11, 12]. Injury to the brain could increase the entry of cells from maternal blood circulation by compromising the blood–brain barrier or by releasing signaling molecules that cause fetal cells to be recruited to the brain. In both intact and lesioned brains, fetal cells were found both closely juxtaposed to blood vessels and within the brain parenchyma with no obvious association to blood vessels. Fetal cells expressing morphological and immunocytochemical features characteristic of neuronal cells were also found organotypically aligned with host neurons in the pyramidal cell layer of CA1 of the hippocampus (Figs. 5A–5H). Together, these data suggest that the fetal cells may have migrated within the host brain and developed in response to cues from the host. Within the brain, fetal cells were capable of expressing morphological and immunocytochemical characteristics of various cell types. Some fetal cells remained in close association with blood vessels, but we found no evidence for endothelial cells of fetal origin in the maternal brain. It might have been predicted that fetal engraftment of the endothelium of maternal vessels Downloaded from www.StemCells.com by on August 21, 2006 Fetal Microchimerism in the Maternal Brain 1450 would be observed because it has been reported that after focal ischemia the adult mouse brain can recruit endothelial progenitors cells from BM for neovascularization [21]. However, at least in the rat brain, the rate of turnover of brain endothelial cells is very slow [22], and it may be that unless injury triggers neovascularization, circulating fetal endothelial cells will not be recruited. Besides, at least in humans, it appears that endothelial cells in the blood of pregnant women are of maternal rather than fetal origin [23, 24]. Fetal cells were also observed within the brain in close juxtaposition to the endothelial cells of the host blood vessel wall. The location and morphology of these fetal cells resemble those of BM-derived perivascular macrophages [25–28]. Consistent with differentiation into macrophages, some of these fetal cells were immunolabeled for the macrophage marker F4/80. However, there was no evidence at weeks postpartum in the intact maternal brain that such fetal cells expressed CD11b. This may suggest incomplete differentiation or maturation of perivascular macrophages of fetal origin. In the intact mouse brain, such BMderived perivascular cells have been reported to phagocytose host endothelial elements [29]. It may be that the apparent wrapping of processes around neighboring endothelial cells observed here (Figs. 4F, 4H) is evidence of similar phagocytosis by fetal-derived perivascular macrophages. After brain injury, BM-derived macrophages and microglia are also reported to infiltrate the brain parenchyma [26–28]. It has been reported that such BM-derived parenchymal microglia can engulf host neural cells [26]. Observations of such engulfment events would show that the donor cells are functional, but we saw little evidence of infiltration of the intact maternal brain parenchyma by fetal-derived macrophages or microglia and did not observe any instances of engulfment of host neural cells by fetal cells. We observed a rare example of a seemingly binucleated EGFPpositive cell adopting a putatively perivascular macrophage-like location juxtaposed to a blood vessel. This may represent evidence of cell fusion either between two fetal cells or between a host cell and a fetal cell. Alternatively, it may represent a fetal cell caught in the act of division or the development of a polynucleated cell type. Binucleated and multinucleated microglia and macrophages frequently occur in association with central nervous system injury, especially chronic inflammatory injury [30, 31]. It may be that the presence of fetal cells in the maternal brain has led to an inflammatory response. If this is a fusion event, we cannot determine whether this cell fused because it became perivascular macrophage–like or whether it became perivascular macrophage–like because it fused with a host perivascular macrophage. Fetal cells in the maternal brain were also capable of developing gross morphological similarities to neural cell types and expressing immunocytochemically labeled protein markers normally associated with neural cell types (Figs. 5, 6). As some fetal cells were observed with macrophage-like characteristics, it is important that it has been demonstrated that serial confocal sectioning allows engulfment of differentiated host cells by EGFPpositive donor cells to be readily distinguished from expression of neural markers by EGFP donor cells [26]. The presence of EGFP within the soma and arborizations of cells colabeling for neural markers is inconsistent with macrophage engulfment (e.g., Figs. 5D, 5H). Further studies might determine whether these cells become electrophysiologically functional neurons. We also found no evidence of EGFP-positive fetal cells with neural-like morphologies or immunocytochemically labeling for neuronal markers colabeling for F4/80 or CD45. The lack of expression of these markers suggests that these cells were not macrophages. The lack of expression of CD45 by fetal cells in the maternal brain may also suggest that the fetal cells that infiltrate the maternal brain and adopt characteristics typical of neural cell types are not of hematopoietic origin. However, we cannot exclude the possibility that these cells expressed CD45 before entry into the maternal brain and then stopped expressing CD45 when in the brain. No Green Mouse fetal cells were found expressing the immature oligodendrocyte marker NG2, although occasionally EGFPpositive cells were found in close juxtaposition to NG2-positive cells (Fig. 5C). This could suggest that, when they differentiate along an oligodendrocytic path, the Green Mouse fetal cells not form an active oligodendrocyte progenitor population in the maternal brain but rather differentiate into nonproliferating mature oligodendrocytes. The Green Mouse fetal cells observed in the maternal brain usually occurred in clusters. Frequently, these clusters contained a number of EGFP-positive cells with different morphological and immunocytochemical profiles (e.g., Fig. 6). This clustering may indicate that a single fetal progenitor or stem cell entered the brain and then proliferated to produce daughter cells following various differentiation pathways in subsequent generations. Alternatively, clustering may indicate that multiple fetal cells enter the brain at particular locations, perhaps attracted by release of signaling molecules or because of local variations in the permeability of the blood–brain barrier to infiltration by fetal cells, and that these multiple fetal cells then followed different differentiation pathways or fused with different types of maternal cells. Further studies are required to investigate the mechanisms of this clustering and why these cells express markers for different cell types. These data suggest that pregnancy is a minimally invasive model allowing entry of fetal cells into the maternal brain. This model will facilitate comparisons of the fate of fetal cells and endogenous adult progenitor or stem cells in the adult brain. It has been speculated that in vitro isolation and culture can alter the properties of progenitor or stem cells. The pregnancy model will also allow investigation of the fate of fetal cells in the adult brain without in vitro manipulation. Further characterization of fetal cells capable of crossing the blood–brain barrier may improve Downloaded from www.StemCells.com by on August 21, 2006 Tan, Liao, Sun et al. 1451 selection procedures for isolation of progenitor or stem cells from other sources, for example, directly from umbilical cord blood, for brain repair by intravenous infusion. Recently, there has been much speculation that fetal microchimerism may have implications in maternal health [32–35]. It is possible that fetal microchimeric cells may participate in the maternal response to injury [35]. It is known that hormonal changes during pregnancy can influence neurogenesis [36], and it may be that pregnancy makes certain niches in the brain a more receptive environment for fetal cells. Further studies are required to determine whether there are any functional or pathological implications of the engraftment of the maternal brain with fetal cells during pregnancy. Acknowledgments References 16 Jockusch H, Voigt S, Eberhard D. Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor. J Histochem Cytochem 2003;51: 401–404. Herzenberg LA, Bianchi DW, Schroder J et al. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Natl Acad Sci U S A 1979;76:1453–1455. Bianchi DW, Zickwolf GK, Weil GJ et al. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci U S A 1996;93:705–708. Liegeois A, Gaillard MC, Ouvre E et al. Microchimerism in pregnant mice. Transplant Proc 1981;13:1250–1252. Philip PJ, Ayraud N, Masseyeff R. Transfer, tissue localization and proliferation of fetal cells in pregnant mice. Immunol Lett 1982;4:175–178. Johnson KL, Nelson JL, Furst DE et al. Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis. Arthritis Rheum 2001;44:1848–1854. Invernizzi P, De Andreis C, Sirchia SM et al. Blood fetal microchimerism in primary biliary cirrhosis. Clin Exp Immunol 2000;122:418–422. Tanaka A, Lindor K, Gish R et al. Fetal microchimerism alone does not contribute to the induction of primary biliary cirrhosis. Hepatology 1999;30:833–838. Ha Y, Choi JU, Yoon DH et al. Neural phenotype expression of cultured human cord blood cells in vitro. Neuroreport 2001;12:3523–3527. Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002;69:880–893. 10 Zigova T, Song S, Willing AE et al. Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 2002;11:265–274. 11 Chen J, Sanberg PR, Li Y et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001;32:2682–2688. 12 Lu D, Sanberg PR, Mahmood A et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 2002;11:275–281. 13 Howard-Jones N. A CIOMS ethical code for animal experimentation. WHO Chron 1985;39:51–56. 14 Nakanishi T, Kuroiwa A, Yamada S et al. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002;80: 564–574. 15 Nagamine CM. The testis-determining gene, SRY, exists in multiple copies in Old World rodents. Genet Res 1994;64:151–159. www.StemCells.com This research was supported by the National University of Singapore Young Investigator Award to G.S.D. and by grants from the National Medical Research Council of Singapore, Singapore Health Services Pte Ltd., and the Department of Clinical Research, Singapore General Hospital, to Z.C.X. We thank Prof. Catherine J. Pallen for helpful comments on the draft manuscript. We thank the Department of Experimental Surgery, Singapore General Hospital, for provision of animal housing facilities. Disclosures The authors indicate no potential conflicts of interest. 17 Kusser KL, Randall TD. Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues. J Histochem Cytochem 2003;51:5–14. 18 O’Donoghue K, Chan J, Dela FJ et al. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy. Lancet 2004;364:179–182. 19 Walczak P, Chen N, Hudson JE et al. Do hematopoietic cells exposed to a neurogenic environment mimic properties of endogenous neural precursors? J Neurosci Res 2004;76:244–254. 20 Hudson JE, Chen N, Song S et al. Green fluorescent protein bone marrow cells express hematopoietic and neural antigens in culture and migrate within the neonatal rat brain. J Neurosci Res 2004;76:255–264. 21 Zhang ZG, Zhang L, Jiang Q et al. Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 2002;90:284–288. 22 Robertson PL, Du BM, Bowman PD et al. Angiogenesis in developing rat brain: an in vivo and in vitro study. Brain Res 1985;355:219–223. 23 Gussin HA, Bischoff FZ, Hoffman R et al. Endothelial precursor cells in the peripheral blood of pregnant women. J Soc Gynecol Investig 2002;9:357–361. 24 Gussin HA, Sharma AK, Elias S. Culture of cells from maternal circulation, in conditions favoring fetal endothelial cell expansion, does not facilitate the preferential expansion of circulating fetal cells. Fetal Diagn Ther 2005;20:64–69. 25 Bechmann I, Priller J, Kovac A et al. Immune surveillance of mouse brain perivascular spaces by blood-borne macrophages. Eur J Neurosci 2001;14:1651–1658. 26 Hess DC, Abe T, Hill WD et al. Hematopoietic origin of microglial and perivascular cells in brain. Exp Neurol 2004;186:134–144. 27 Tanaka R, Komine-Kobayashi M, Mochizuki H et al. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience 2003;117:531–539. 28 Vallieres L, Sawchenko PE. Bone marrow-derived cells that populate the adult mouse brain preserve their hematopoietic identity. J Neurosci 2003;23:5197–5207. Downloaded from www.StemCells.com by on August 21, 2006 Fetal Microchimerism in the Maternal Brain 1452 29 Galimi F, Summers RG, Van PH et al. A role for bone marrow-derived cells in the vasculature of non-injured CNS. Blood 10.1182/blood-200402-0612. 30 Lee TT, Martin FC, Merrill JE. Lymphokine induction of rat microglia multinucleated giant cell formation. Glia 1993;8:51–61. 31 Leskovar A, Turek J, Borgens RB. Giant multinucleated macrophages occur in acute spinal cord injury. Cell Tissue Res 2001;304:311–315. 32 Bianchi DW. Fetomaternal cell trafficking: a new cause of disease? Am J Med Genet 2000;91:22–28. 33 Bianchi DW. Fetomaternal cell traffic, pregnancy-associated progeni- tor cells, and autoimmune disease. Best Pract Res Clin Obstet Gynaecol 2004;18:959–975. 34 Johnson KL, Bianchi DW. Fetal cells in maternal tissue following pregnancy: what are the consequences? Hum Reprod Update 2004;10: 497–502. 35 Khosrotehrani K, Bianchi DW. Fetal cell microchimerism: helpful or harmful to the parous woman? Curr Opin Obstet Gynecol 2003;15: 195–199. 36 Shingo T, Gregg C, Enwere E et al. Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 2003;299: 117–120. Downloaded from www.StemCells.com by on August 21, 2006 [Cell Adhesion 1:1, e1-e9, EPUB Ahead of Print: http://www.landesbioscience.com/journals/celladhesion/abstract.php?id=4082; January/February/March 2007]; ©2007 Landes Bioscience Review Cell Migration from Baby to Mother Gavin S. Dawe1,* Xiao Wei Tan2 Zhi-Cheng Xiao2,3,4,* Abstract *Correspondence to: Gavin S. Dawe; Department of Pharmacology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore 117597; Tel.: 65.6516.8864; Fax: 65.6873.7690; Email: gavindawe@nus.edu.sg / Zhi‑Cheng Xiao; Department of Clinical Research; Singapore General Hospital; Block A, No.7; Hospital Drive; Singapore 169608; Tel.: 65.6326.6195; Fax: 65.6321.3606; Email: xiao.zhi.cheng@sgh.com.sg Original manuscript submitted: 02/27/07 Manuscript accepted: 02/28/07 Microchimerism is the presence of a small population of genetically distinct and s­ eparately derived cells within an individual. This commonly occurs following ­transfusion or transplantation.1‑3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)4‑7 and from the mother to the fetus.8‑10 Similar exchange may also occur between monochorionic twins in utero.11‑13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many childbearing women.7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of ­ pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy‑acquired ­ microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells. Trophoblasts were the first zygote‑derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.15 Later, trophoblasts were also observed in the maternal circulation.16‑20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.21,22 These fetal cell types included lymphocytes,23 ­erythroblasts or nucleated red blood cells,24,25 haematopoietic progenitors7,26,27 and putative ­mesenchymal progenitors.14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,29‑31 trophoblast release does not appear to occur in all pregnancies.32 Likewise, in mice, fetal cells have also been reported in maternal blood.33,34 In the mouse, fetomaternal transfer also appears to occur during all ­pregnancies.35 IEN This manuscript has been published online, prior to printing for Cell Adhesion & Migration, Volume 1, Issue 1. Definitive page numbers have not been assigned. The current citation is: Cell Adhesion & Migration 2007; 1(1): http://www.landesbioscience.com/journals/celladhesion/abstract.php?id=4082 Once the issue is complete and page numbers have been assigned, the citation will change accordingly. RIB of Anatomy; Yong Loo Lin School of Medicine; National University of Singapore; Singapore IST 4Department OT D 3Department of Clinical Research; Singapore General Hospital; Singapore ON 2Institute of Molecular and Cell Biology; Singapore .D of Pharmacology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore CE 1Department UT E . Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood‑brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood‑brain barriers and the persistence and distribution of fetal cells in the mother. SC Key words © 20 07 LA ND ES BIO fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migration e1 Anatomy of the Placenta Human and rodent placentation is hemochorial, the fetomaternal interaction between the two blood circulations involving direct physical interaction between maternal blood and the chorionic trophoblasts.36 The fetal and maternal blood circulates in channels lined by these zygote‑derived cells within the placental region known as the labyrinth in mice or the fetal placenta in humans (Fig. 1). In the human, the channels through which the fetal blood flows, the chorionic villi, form trees with numerous branches and sub‑branches terminating in villous blunt‑endings. The maternal blood flows in the ­ relatively open ­intervillous space. In contrast in the mouse, the maternal blood flows through a Cell Adhesion & Migration 2007; Vol. Issue Fetomaternal Microchimerism Figure 1. A simplified diagrammatic representation of the structure of the human placenta (adapted from Georgiades et al.36) and hypothesized mechanisms of fetomaternal cell traffic. From the end of the first trimester, maternal blood flows into the fetal placenta via the maternal spiral arteries, through the intervil‑ lous space bathing the branches of the villous trees and out through the maternal veins (red arrows on left‑hand side). The fetal blood enters via the umbilical cord and circulates to the fetal capillaries in the villous trees. A layer of zygote‑derived trophoblasts, in humans a syncytium of syncytiotrophoblasts, on the surface of the villous trees (dark green) forms the barrier between the fetal tissues and the maternal blood. Zygote‑derived trophoblasts also progressively invade the placental bed and line the maternal vasculature. By the third trimester the maternal spiral arteries are lined through to the (im), while the maternal veins are lined to the border between the decidua basalis (db) and basal plate (bp). In the mouse, the analogue of the fetal placenta is labyrinthine and the trophoblastic invasion of the maternal blood vessels does not extend beyond the junctional zone analogous to the basal plate. Hypothesized mechanisms of fetomaternal cell traffic include (i) deportation of trophoblasts lining the maternal vessels and intervillous space; (ii) microtraumatic hemorrhage; and (iii) cell adhesion and transmigration across the placental barrier. l­abyrinthine network of interconnected cavities or lacunae.36 A layer of ­ trophoblast cells forms the interface between the maternal blood and the fetal tissues. It is these trophoblast cells that form the placental barrier between maternal and fetal circulation. In the human, this interface consists of a syncytium of ­syncytiotrophoblasts directly contacting the maternal blood (Fig. 2B). In the first trimester, there is also a layer of replicating mononuclear cytotrophoblasts beneath the syncytiotrophoblasts. In contrast, in mice there are three layers of trophoblasts. The outer layer consists of mononuclear cytotrophoblasts while the middle and inner layers are syncytiotrophoblastic.36 Between the trophoblasts and the fetal blood there are a trophoblastic basement membrane, in some but not all interfaces a core of ­extracellular matrix and/or pericytes, an ­endothelial basement membrane, and fetal capillary endothelial cells36 (Fig. 2B). Fetal blood enters and leaves the fetal placenta/labyrinth via the umbilical cord, whereas maternal blood enters and leaves the fetal placenta/ labyrinth via the utero‑placental circulation. The zone bordering the maternal surface of the fetal placenta/ labyrinth is known as the basal plate in humans and the ­junctional zone or spongiotrophoblast zone in mice. This region is not perfused by fetal blood but is crossed by maternal blood channels lined by zygote‑derived trophoblast cells through which the maternal blood flows in and out of the fetal placenta/labyrinth.36 This zone in turn is bordered by the maternal uterine tissue on the maternal www.landesbioscience.com side. The maternal uterine tissue becomes progressively invaded by ­ zygote‑derived trophoblast cells. In particular, these cells line the maternal blood vessels in the maternal uterine tissue. The maternal uterine tissues of this region, known as the placental bed in humans, can be divided into the decidua basalis adjacent to the basal plate/junctional zone and the myometrium on the maternal side. In humans, ­trophoblast invasion extends to the inner third of the myometrium but in mice, trophoblast invasion is shallow and is limited to the decidua basalis.36,37 Even within the decidua basalis, maternal arteries and veins remain lined by maternal endothelium rather than trophoblasts in the mouse.38,39 While in the human the trophoblasts stimulate arterial remodeling in the mouse uterine natural killer cells are more important.39‑41 The cells of the placenta itself comprise both ­zygote‑derived and maternal cells. In mice, the zygote‑derived cells include ­trophoblasts derived from the polar trophectoderm of the outer cell mass; fetal blood vessels and mesenchyme derived from the allantoic ­mesenchyme, which in turn is derived from the primitive ectoderm of the inner cell mass; and fetal blood cells of mesodermal lineage. Meanwhile, the maternal cells of the mouse placenta include uterine cells and cells coming from the maternal blood.36 It is generally assumed that the origin of human placental cells is similar to those in the mouse, although not lineage studies have been performed on human placentae.36 However, there is debate over whether the Cell Adhesion & Migration e2 Fetomaternal Microchimerism role of invasion by zygote‑derived trophoblasts in the maternal circulation in the human placenta. Cell Traffic Across the Placenta Figure 2. Simplified diagrammatic representations of blood‑brain and ­placental barriers and hypothesized molecular mechanisms of cell adhesion and transmigration. (A) A simplified diagrammatic representation of multistep lymphocyte recognition and capture from blood at the blood brain barrier (adapted from Engelhardt48). Cells expressing a4b1 are captured by VCAM‑1 expressed by endothelial cells. There is a rapid activation phase (seconds) that may involve lymphoid chemokines CCL19/ELC and CCL21/SLC. There is a prolonged adhesion phase (hours) followed by slow transmigration (hours) dependent upon binding of LFA‑1 to ICAM‑1 and/or ICAM‑2 on the endothelial cells. It is hypothesized that a similar molecular mechanism may explain fetal cell migration across the blood‑brain barrier and the ­placental barrier. (B) A simplified diagrammatic representation of the human placental barrier showing a hypothetical mechanism of fetal cell ­capture, adhesion and transmigration. The placental barrier comprises of fetal ­capillary endothelial cells (fcec), an endothelial basement membrane (ebm), the villous core (vc) which at some interfaces contains pericytes (p) and extracellular matrix, a trophoblastic basement membrane (tbm), in the first trimester a layer of proliferative cytotrophoblasts (ct), and a multinucleated syncytium of syncytio‑ trophoblasts (ss). In the mouse, the trophoblastic layers differ in that there are two syncytiotrophoblastic layers and the cytotrophoblastic layer is outermost facing the intervillous interface. It is hypothesized that fetal cells may adhere and transmigrate across the placental barrier in a similar manner to that by which lymphocytes cross the blood‑brain barrier. human allantoic vasculature, through which the fetal blood passes, is of trophectodermal or epiblast/hypoblast origin.36,42 The similarities in the anatomy of placentation and placental blood flow in mice and humans36,39 and the role of analogous genes in mouse and human placentation43 make mouse placentation a good model for many aspects of human placentation. However, there are important anatomical differences,36,39 in particular the ­difference between the villous nature of the human fetal placenta and the ­labyrinthine nature of the analogous mouse labyrinth and the greater e3 The mechanism by which cells are exchanged across the placental barrier is unclear. Possible explanations include deportation of trophoblasts, microtraumatic rupture of the placental blood channels or that specific cell types are capable of adhesion to the trophoblasts of the walls of the fetal blood channels and migration through the placental barrier created by the trophoblasts (Fig. 1i–1iii). Intervillous thrombi containing mixed maternal and fetal cells occur in the fetal placenta/ labyrinth.44,45 Histological defects in the continuity of the trophoblasts lining the vasculature of the placenta are also reported.46,47 Together these observations suggest the possibility that fetomaternal hemorrhage within the fetal placenta/labyrinth may allow exchange of cells between the fetal and maternal circulation. Microtraumatic dislodgment of trophoblasts from the trophoblast‑lined blood ­channels through which the maternal blood passes may also explain why trophoblasts appear in maternal circulation. The microtraumatic hypothesis of cell exchange does not appear consistent with the hypothesis that fetomaternal microchimerism may be of adaptive value to the fetus but fits well with the hypothesis that fetomaternal microchimerism is an epiphenomenon of pregnancy with potential pathological consequences. An alternative hypothesis is that cells cross the placental barrier by mechanisms akin to the active adhesion and transmigration that occurs across high endothelial venule (HEV) endothelium in peripheral lymph nodes and at the blood‑brain barrier (BBB).48 Intriguingly, in the mouse at least some of the fetal cells that enter the mother are also capable of crossing the blood brain barrier into the brain.35,49 At the BBB and HEV, lymphocyte migration across the ­endothelial membrane involves a multistep process of recognition and recruitment from the blood involving tethering/rolling or capture, activation, adhesion and finally transmigration (Fig. 2A). In both HEV endothelium and BBB, the final stage of transmigration involves binding of LFA‑1 expressed by the lymphocytes to ICAM‑1 in HEV endothelium and to ICAM‑1 and/or ICAM‑2 at the BBB.48,50,51 In the HEV endothelium, the ICAM‑1 also appears to be involved in the adhesion preceding transmigration, whereas at the blood brain barrier VCAM‑1 is involved in lymphocyte capture and adhesion. Fetal cells crossing the placental barrier must transmigrate both the fetal capillary endothelial cell layer and the trophoblast cell layers (Fig. 2B). The fetal capillary endothelial cell layer expresses a number of cell adhesion molecules including PECAM‑1 and ICAM‑1.52,53 While, there is VCAM‑1 expression in umbilical cord endothelium there appears to be no evidence for VCAM‑1 expression on fetal capillary endothelium in normal placenta at term.52,53 As PECAM‑1 plays a role in integrin‑mediated neutrophil adhesion and endothelial transmigration,54‑56 including migration of CD34+ positive cells57 such as the fetal cells in maternal blood,7 we hypothesize that it is also a candidate for contribution to fetal cell transmigration across the fetal capillary endothelium (Fig. 2B). The functional ligand for PECAM‑1 in transmigration is unknown, but it is possible that it is an avb3 integrin.58 It is possible that multiple fetal cell types cross the placental barrier by different mechanisms. Once the fetal cells have crossed the fetal capillary ­endothelium, they must next cross the trophoblast layer. Trophoblasts express Cell Adhesion & Migration 2007; Vol. Issue Fetomaternal Microchimerism Figure 3. Time course of fetal cell engraftment and persistence in the mouse brain. Adult female mice received intraventricular injection of the excitotoxic NMDA to produce a diffuse brain lesion or were untreated. The mice were crossed with adult male enhanced green fluorescent protein (EGFP) trans‑ genic Green Mice. Fetomaternal microchimerism in the brain was assayed at various time points: gestational days (GD) and 14, the day of parturi‑ tion (P0), and at seven days (P7), four weeks (P4W) and eight weeks (P8W) post partum (n = 3–8 per group at each time point). The number of fetal cells relative to total cells present in a brain block centered about the site of the injection was quantified by real‑time PCR for the EGFP gene in genomic DNA. Procedures were as previously described.49 There are great individual differences, however, in those mothers in which fetal cells were detected in the brain, the number of fetal cells detected in the brain increases by four weeks post partum and declines again by eight weeks post partum. Overall, in those mothers in which fetal cells persist at four weeks and eight weeks post partum, there are greater numbers of fetal cells in the lesioned brains. ICAM‑1 in vitro and in vivo59‑61 and monocytes bind to ICAM‑1 expressed by trophoblasts in an LFA‑1‑dependent manner.60 Similarly, the migration of Toxoplasma gondii across epithelial barriers, including the placental barrier comprised of trophoblast cells, involves ­interaction of the parasite adhesion molecule, MIC2, with the intercellular adhesion molecule (ICAM‑1).62 Together these studies suggest that the molecular apparatus for maternofetal transmigration may be present at the placental barrier. Although there is evidence for greater in vivo expression of ICAM‑1 on the apical surface of the villous syncytiotrophoblasts exposed to the maternal blood,60 ICAM‑1 is also present throughout the stroma of the chorionic villi,60,61 although it has not been clearly established that it is expressed on the basal surface of the trophoblasts facing the villous core. Trophoblasts also express VCAM‑1.63‑65 Thus the molecular apparatus for ­ fetomaternal transmigration of fetal cells expressing LFA‑1 may also be present at the trophoblast cell layer. Once the fetal cells have crossed the fetal capillary endothelial cell layer, we hypothesize that they cross the trophoblast cell layer again in a manner similar to that in which lymphocytes cross the BBB (Fig. 2B). We hope that this speculative hypothesis regarding the mechanisms of fetomaternal cell traffic may stimulate further research and that future studies will determine whether active fetomaternal adhesion and transmigration occurs and elucidate the molecular mechanisms involved. Timing of Onset of Fetomaternal Traffic In mice, fetal cells generally first appear in the mother in the second week of pregnancy35 (see also Fig. 3). Numbers of fetal cells www.landesbioscience.com are present in maternal blood by GD10 to GD12 days (gestational days, the day of vaginal plug detection being designated GD0) in pregnancies from syngenic and allogenic crosses; however the cells not appear in blood in until GD13 to GD16 in pregnancies from outbred crosses.66 The appearance of fetal cells in maternal blood at GD10 to GD12 in syngenic and allogenic crosses is consistent with the establishment of uteroplacental circulation. Maternal blood first appears in the labyrinth between GD9 and GD10 and extensive fetal capillary formation occurs by GD12.39,67 This coincides with the onset of fetal circulation on the completion of organogenesis at GD9 to GD10.36 In humans, fetal DNA has been detected in maternal blood as early as four weeks and five days after ­conception and both fetal cells and DNA are consistently detected from seven weeks.68,69 Thus in humans, the first appearance of fetal cells in maternal blood occurs slightly before the completion of fetal organogenesis, the onset of fetal circulation to the placenta, and the appearance of maternal blood within the fetal placenta. Plugs of invading trophoblast cells, which block the tips of the uteroplacental spiral arteries, are progressively dislocated after 10–12 weeks70 and blood only becomes evident in the intervillous space of the fetal placenta after ten weeks gestation.71 Effective arterial circulation of the placenta is not established until around the twelfth week of gestation39,72,73 when the human embryo has largely completed the organogenesis stage.36 In the mouse, the timing of the appearance of fetal cells in maternal blood is consistent with the hypothesis that fetomaternal exchange occurs between fetal and maternal blood at the placental barrier in the fetal placenta/labyrinth. In the fetal placenta/labyrinth, the maternal blood comes into direct contact with the zygote‑derived trophoblast and it has been proposed these may also be deported into the maternal circulation.66 The fetal placenta/labyrinth is also very rich in fetal hematopoietic stem cells74‑76 and it has been suggested that these cells might able to migrate into the maternal blood.66 The earlier appearance of fetal cells in maternal blood in humans may suggest more active migration of certain fetal cells. Potentially there may be multiple cell types and phases of migration involved. More detailed investigation of the time course of the appearance of maternal blood in the placenta and the appearance of fetal cells in maternal blood in humans may be informative. The reason for the delay in the appearance of fetal cells in maternal blood in outbred mouse crosses is at present unknown. Outbred crosses were also observed to result in delayed and reduced trophoblast invasion of the decidua basalis.66 It may be that the appearance of fetal cells in maternal blood on outbred crosses is due to a more aggressive immune response; alternatively the delay may be due to a delay in the maturation of the placenta and maternal ­circulation to the labyrinth. It is hoped that further studies may elucidate the issue. Intriguingly in syngenic pregnancies, fetal cells were detected in mouse lungs and to a lesser extent spleen and kidney in the first week of gestation before they robustly appear in detectable numbers in maternal circulation.35,66 One explanation might be that, consistent with the appearance of trophoblasts in maternal lungs in humans,15 these cells are trophoblasts. Thus one might hypothesis that the earliest phase of fetomaternal microchimerism involves deportation of zygote‑derived trophoblasts as they invade the decidua basalis to line the maternal blood vasculature. In particular, the fate of the trophoblasts that plug the ends of the maternal arteries of the uteroplacental circulation may be to become dislodged into maternal circulation as maternal blood flow begins to break through into the fetal placenta/labyrinth. Trophoblasts being large are rapidly cleared Cell Adhesion & Migration e4 Fetomaternal Microchimerism from maternal blood as they become lodged in the microvasculature of the lung and to a lesser extent other organs. While the studies discussed here have made important contributions to establishing the time course of fetomaternal traffic, the question of whether different zygote‑derived cell types show different time courses of traffic has not been investigated in depth. It is hoped that future studies will address this important issue. Frequency and Persistence of Fetomaternal Microchimerism Fetomaternal microchimerism appears to occur with great frequency following human pregnancy. It has been suggested that fetomaternal traffic occurs in all pregancies.14 Moreover fetal cells are reported to persist in the mother for decades. Male cells have been found in maternal blood even decades after pregnancy,7,77 including in one case in which the women was last pregnant with a male child 27 years earlier.7 Fetal cells also may persist for even longer after engrafting maternal bone marrow14 and perhaps other organs. By engrafting into niches such as the bone marrow, fetal cells may also be able to proliferate and reinfiltrate blood or other tissues later. There is strong evidence that fetal cells with the characteristics of ­mesenchymal cells engraft the bone marrow. Male DNA was detected in 48% of CD34‑enriched apheresis products from nonpregnant female marrow donors.1 Male cells were also detected in all bone marrow samples from women who had previously been ­pregnant with males, including one woman who was last pregnant with a son 51 years earlier.14 The absence of Y chromosome markers in samples from women who had never born sons in some studies14 strongly supports the ­argument that the male cells observed originate from the fetus. However, it is important to note that there are crucial caveats in the use of the Y chromosome alone as a marker for fetomaternal ­microchimerism that may have led to over estimation of the ­incidence and persistence of fetomaternal microchimerism in humans. Male cells have been found in the blood of women without sons.78,79 Male cells may occur in the blood of as many as 8–10% of healthy women without sons and no known history of abortion.79 It has been speculated that the male cells arise from unrecognized ­spontaneous abortions, vanished male twins, an older brother transferred by the maternal circulation, or sexual intercourse. However, a history of unrecognized spontaneous abortions or sexual intercourse cannot explain all cases of the presence of male cells in females as another study detected the presence of the Y chromosome in normal liver from seven of eleven female fetuses and five of six female children.80 Such microchimerism may be best explained, by fetofetal transfer from an undetected vanishing male twin or maternofetal transfer of male cells harbored by the mother. Estimates of the frequency of vanishing twins range from 3.7–100% of pregnancies81 however not all twins share connected placenta vasculature, especially at the early stages of development at which many twins disappear. Maternofetal transfer to the mother may also have occurred if the mother’s mother had a history of blood transfusion, transplantation or previous pregnancy with a male fetus. It is difficult to estimate how frequently male cells in females could arise as a result of fetofetal or maternofetal transfer. Although one might expect such events to be rare, the incidence may be high enough to have biased estimates of the incidence of fetomaternal microchimerism in humans. While the possibility that the Y chromosome could also enter the mother via microchimerism as a consequence of previous blood transfusion or transplantation e5 has been considered in most studies, the possibility that male cells detected in the mother may have arrived via fetofetal or maternofetal transfer to the mother in utero has not be ­ systematically excluded. Conclusive proof of fetomaternal microchimerism in humans would require the use of other paternal markers that differentiate between the father of the fetus and the father of the mother. One scenario might be to investigate cases where the mother and the mother’s father share a genetic mutation or polymorphism not carried by the father of the fetus. In such cases, evidence of genetic markers derived from the father of the fetus in the mother could provide more conclusive evidence of fetomaternal microchimerism in humans. If the genetic mutation or polymorphism caused disease the presence of fetal cells in the diseased tissue could also offer evidence of the potential of fetomaternal tissue repair. In contrast, to the suggestion that fetal cells are retained for decades after nearly every human pregnancy,7,14 the retention of fetal cells in mice appears more sporadic and rarely persists for more than a few weeks post partum. The use of mice bearing unique genetic markers such as, the cytogenetic marker chromosome, T626,33 and more recently transgenic mice bearing genetic markers such as enhanced green fluorescent protein (EGPF)35,49,66 has conclusively demonstrated fetomaternal microchimerism. The number of mice in which fetal cells can be detected in maternal blood and the number of fetal cells in maternal blood declines towards the end of gestation, at least in syngenic and allogenic crosses.66 Beyond the first week postpartum, fetal cells are rarely detected in maternal blood;35,66 although they have been found in some mice at 21 days post partum following allogenic crosses and at 42 days post partum, but not 21 days post partum, following outbred crosses.66 Likewise, in maternal bone marrow, spleen, liver, heart, lung and kidney fetal cells not appear to be retained by maternal mice beyond the first week post partum.35 Even within the first week post partum, the retention of fetal cells is sporadic and highly variable between i­ndividuals.35 Our own observations suggest that there might be greater retention of fetal cells within the brain as although fetal cell numbers are low, cells persist to weeks post partum49 (see also Fig. 3). However, by 6–8 weeks post partum, the number of fetal cells has fallen below the limits of detection in blood and all organs studied, including uninjured brain66 (see also Fig. 3). Although the numbers of fetal cells present were very low, fetal cells did persist at eight weeks post partum in some of the lesioned maternal brains (Fig. 3). Together, these data suggest the possibility that, although fetal cells are cleared from the blood and some organs within a few weeks postpartum in mothers of syngenic and allogenic crosses, some fetal cells may remain harbored longer‑term in certain niches. In contrast, fetal cells have been detected in the blood of some mice at 42 days post partum following outbred crosses.66 Additionally, there is limited evidence that in some, but not all mice, repeated pregnancies may lead to greater retention of fetal cells,35,49 which may suggest that in some mothers there is longer‑term retention of fetal cells. However, the duration of fetal cell retention in those few mice in which fetal cells persist has not been systematically investigated. The reasons for the large individual differences in the numbers of fetal cells retained and the duration of retention are not known. During pregnancy the mother develops immune tolerance to the fetus but after pregnancy this suppression of the maternal immune response to the fetus is lifted.82 It is conceivable that, although ­fetomaternal cell traffic probably occurs in every pregnancy, ­persistence of microchimeric fetal cells after pregnancy depends upon the immunocompatibility between the mother and fetus. Cell Adhesion & Migration 2007; Vol. Issue Fetomaternal Microchimerism This might explain why fetomaternal microchimerism does not persist in all mothers. The greater preservation of fetal cells in the brain than the blood would be consistent with an immune rejection hypothesis, the brain being an immune privileged site.83 However, it is difficult to reconcile the hypothesis that immune rejection explains the great inter‑individual variability and low rate of fetal cell persistence in syngenically crossed mice66 as there is less immune rejection on transplantation between syngenic mice. Although some ­differences between the mother and fetus may be an advantage as it has been noted that, despite reducing placental expression of major ­histocompatibility complex (MHC) genes, major histocompatibility complex expression is often reestablished in the most invasive ­trophoblast cells and may contribute to an immunoprotective effect on the fetus.84 In conclusion, although it has not been studied systematically and there are obvious methodological differences between the mouse and human studies, there appears to greater likelihood of long‑term retention of microchimeric fetal cells in humans than in mice. This difference in the retention of fetal cells may be consistent with the hypothesis that fetomaternal microchimerism has developed as a mechanism by which the fetus ensures maternal fitness. As mice wean their offspring by 3–4 weeks postpartum, there would be no need for the fetal cells to continue to survive. In contrast, human mothers nurse their offspring for many months and thereafter continue to nurture their offspring for many decades so there may be an ­adaptive advantage to fetal cell persistence. Alternatively, if fetal cells have adverse effects on the mother, it may be that rodents have developed greater maternal resistance to fetal cell infiltration as they have far more offspring over a far shorter life span. Intriguingly, there may in fact be greater retention of fetal cells in outbred mice than in syngenic or allogenic crosses.66 That humans, who are generally outbred, retain fetal cells may be further evidence against the immunocompatibility hypothesis for ­fetomaternal ­ microchimeric persistence. It is hoped that future studies may investigate the determinants of fetal cell retention. The immunological hypothesis would predict that immunosuppression from late pregnancy and through the post‑partum period would increase fetomaternal microchimerism. Another hypothesis might be that hormonal changes coinciding with the later stages of ­pregnancy and the post partum period lead to rejection of fetal cells. This hypothesis would predict greater fetomaternal microchimerism in mother who did not complete the normal hormonal sequela of delivery and peri‑ and post‑partum hormonal changes. In humans, there is indeed evidence that spontaneous and induced abortions increase the frequency and level of male microchimerism,79,85 but this may equally be explained by trauma associated with abortion leading to greater fetomaternal exchange. Distribution of Microchimeric Fetal Cells The microchimeric fetal cells in the mother appear to be of ­ ultilineage potential. Y chromosome bearing cells have been m identified in numerous tissues, including skin, liver, kidney and bone marrow, in healthy women and in women with autoimmune diseases86‑92 and other none immune diseases such as hepatitis C93 and cervical cancer.94 There is now a large literature on fetomaternal microchimerism, especially in autoimmune disease, and overall there appears to be evidence of increased fetal cell presence in diseased tissues than healthy tissues.27,95 It is debatable whether ­ microchimerism plays a role in triggering autoimmune disease,86‑89,91 perhaps by www.landesbioscience.com stimulating graft‑host disease or host‑graft disease,96 or whether fetal cells home in on diseased tissue and contribute to tissue repair.27,96 In systematic lupus erythematosus, for example, it appears that microchimeric fetal cells are more likely to be found in severe cases than in mild cases97 suggesting that the fetal cells are not causing the disease but rather are targeting the diseased maternal tissue once the damage reaches a threshold level.27 Similarly, in an animal model of excitotoxic brain injury we found greater numbers of fetal cells in the injured brain region.49 Fetal cells may also persist longer at sites of injury than in uninjured tissue (Fig. 3). This suggests the possibility that fetal cells may target to specific tissues and contribute to tissue repair or function. There are various manners in which fetal cells might come to target damaged tissue. Sometimes the mechanism by which the zygote‑derived cells are sequestered in particular tissues may be mechanical as has been hypothesized for the entrapment of large trophoblast cells in the capillaries of the microvasculature of the lung.15 Likewise, targeting of injured tissues may simply be a mechanical process whereby tissue damage is associated with microdamage to the blood vessels and cells of all types are more likely to leak out into the damaged tissue. Another hypothesis is that fetal cells invade all maternal tissues but only find a niche conducive to survival in damaged tissues. Alternatively, if this is a process that has evolved to allow the fetus to treat the mother to enhance fetal survival, the fetal cells may actively invade the damaged tissue by a physiological mechanism of adhesion and transmigration across the blood vessel walls followed by active migration through the tissue to sites of damage. Recently, Khosrotehrani and colleagues98 have used in vivo ­bioluminescence imaging of fetal cells in which the paternal marker was VEGF receptor promoter controlled luciferase gene expression to demonstrate that fetal cells contribute to neoangiogenesis. This in vivo bioimaging approach will be extremely valuable in determining the extent to which fetal cells invade damaged tissues. Tracking genetically modified fetal cells or the behaviour of fetal cells in genetically modified mothers it may be possible to address important questions about the mechanisms by which fetal cells engraft maternal tissues and home in on injured tissue. Types of Fetal Cells Involved in Fetomaternal Microchimerism The fetal cell type or types responsible for fetomaternal ­ icrochimerism are unknown. Candidates include all cell types m in fetal blood and trophoblasts. However, considerable evidence points towards the conclusion that fetal stem or progenitor cells may also be involved. Subsequent pregnancies appear to trigger further ­proliferation and mobilization to maternal blood of fetal cells acquired during previous pregnancies.34 The very fact that fetal cells can be detected decades after pregnancy7,14,99 is strong evidence that these cells are replicating in the mother. Moreover, women with older sons have a greater number of male cells suggesting proliferation over time.93 Although fetal cells were not detected in all ex‑breeder mice those mice that had fetal cells in the brain tended to have higher numbers than in mice that had only delivered one litter suggesting ­accumulation or proliferation of fetal cells.49 The numbers of fetal cells detected in the maternal brain also showed marked postnatal increase between the last day of gestation and four weeks post partum (Fig. 3). This evidence that fetal cells can proliferate in the mother is fairly persuasive, but the alternative possibility that the fetal cells Cell Adhesion & Migration e6 Fetomaternal Microchimerism engraft in one niche and then subsequently remobilize to another niche without increasing in number has not been excluded. Fetal cells appear indistinguishable from maternal tissues years after pregnancy and can bear epithelial, leukocyte, ­ hematopoietic, ­hepatocytic, renal or cardiomyocytic markers.27,95,100 That ­microchimeric fetal cells also appear to be able to differentiate to adopt cellular characteristics of various host organs suggests that they may be stem or progenitor cells. In injured mouse brain, we have found fetal cells expressing various morphologies, localization and immunocytochemically stained protein markers characteristic of various brain cell types including perivascular macrophages, neurons, astrocytes and oligodendrocytes.49 While the evidence for differentiation may appear persuasive, important alternative hypotheses have yet to be excluded. Notably there have yet to be clear‑cut examples of functional differentiation of microchimeric fetal cells. For example, it would be important to show that apparent neuronal differentiation does not just involve location, morphology and expression of a few protein markers but instead that this differentiation leads to functional neuronal characteristics such as the capacity to fire action potentials and synaptic connectivity to repair damaged circuitry. Likewise in the case of apparent oligodendrocytic differentiation, morphology and protein expression should be accompanied by functional wrapping of axons, and recovery of motor function in demyelination models. At present there is little evidence for or against fusion as a ­mechanism of the apparent differentiation in microchimeric fetal cells. While a binucleated fetal cell was observed juxtaposed to a blood vessel in the brain in a niche in which other fetal cells adopted a perivascular macrophage‑like character,49 it is unclear whether this represents a fusion event, a cell division event, or a multinucleated cell type. Systematic and careful study of fusion events in fetomaternal microchimerism will be important in interpreting whether apparent differentiation of fetal cells is in fact the result of cell fusion. Typically cell fusion in iatrogenic microchimerism following transplantation has been studied by fluorescent in situ hybridization (FISH) for X and Y chromosome markers. The presence of multiple X chromosomes in the cells bearing Y chromosomes has been taken as evidence of fusion. However, the study of cell fusion by this method in fetomaternal microchimerism is complicated. Not only may the Y chromonsome not be a specific marker for fetal cells as discussed above, but the trophoblasts, one of the cell types which contribute to fetomaternal microchimerism, can be ­ multinucleated and due to the mosaic nature of the placenta could naturally carry multiple X chromosomes together with the Y chromosome in cases of vanishing female twins or in the rodent model where most litters contain both male and female offspring. Other strategies will be required to investigate fusion in fetomaternal microchimerism. For example, combining labeling for paternal‑specific and maternal specific markers (e.g., crossing male EGFP transgenic mice with DsRed transgenic mice). Alternatively, Cre/lox recombination might be used to detect cell fusion events101 but this approach would require in utero implantation of homozygous embryos, which may alter fetomaternal cell traffic. If the multilineage differentiation capacity of microchimeric fetal cells does prove to be genuine and functional this suggests that the fetal cells responsible are stem cells. The type of stem cell or stem cells involved is controversial. There is some evidence implicating ­haematopoietic stem cells. For example, male cells that persist in maternal blood after pregnancy are CD34+/CD38+,7 behave like proliferative haematopoeitc progenitor cells in vitro e7 culture,102 and in haematopoietic tissues, such as the lymph nodes and spleen, the majority of microchimeric male cells express CD45.95 In contrast, there is also evidence suggesting that fetal mesenchymal stem cells (fMSC) are involved. Fetal MSCs have been identified in maternal blood during pregnancy.28,103 Fisk and colleagues appear to favor the interpretation that these cells are fetal ­ mesenchymal stem cells because, at least when found in the bone marrow, male cells in mothers were immunophenotypically mesenchymal.14 However, it has been pointed out that the extent of the ­ multilineage ­ differentiation of microchimeric male cells argues against a strictly mesenchymal lineage.104 Indeed, unless one accepts the still ­controversial concept of stem cell plasticity and transdifferentiation, neither haematopoietic nor mesenchymal stem cells could explain the full range of differentiation, for example into neural cell types,49 that has been reported. The diversity of cell types into which ­microchimeric fetal cells can apparently differentiate suggests that, if a single stem or progenitor cell type is involved, it is a very early stem cell type.27,95,100 Bianchi and colleagues have referred to these cells as pregnancy‑associated progenitor cells (PAPC) and appear to favor the interpretation that they may be a relatively early stem cell type retaining multilineage potential.27,91,95,100 The alternative possibility that numerous cell types of different lineage enter the mother has not been excluded. Perhaps the involvement of a number of cell types including various types of early stem cells could better explain the diversity of ­differentiation reported. Conclusions and Future Prospects Fetal cells exhibit a remarkable ability to migrate across the placenta into the mother and to integrate with diverse maternal tissues and organs, apparently homing in particularly to sites of damage and disease.49,97 Much remains to be learned about the basic biology of fetomaternal microchimerism. The cell type or types involved have yet to be conclusively characterized. If various cell types are involved, it will be important to understand the time course of the migration of the various cell types and their persistence in the mother. Studies of the process of cellular adhesion and migration that allow the cells to cross the placental barriers, infiltrate tissues and organs, cross the BBB and migrate to sites of damage will be especially informative. Although long‑term persistence of fetal cells may be less frequent in the mouse, the mouse appears to offer a useful model for ­investigating aspects of fetomaternal traffic during pregnancy. In the longer‑term, elucidation of the biology of fetomaternal microchimerism may have important implications for understanding autoimmunity and graft‑host interactions. Moreover, knowledge of the cell types and molecular mechanisms that allow for the remarkable migratory and multilineage differentiation capacity of microchimeric fetal cells in the mother may improve strategies for cytotherapeutic repair. Harnessing the capabilities of microchimeric fetal cells may enhance the prospects for minimally invasive ­ intravenous delivery of stem cells. References 1. Adams KM, Nelson JL. Microchimerism: An investigative frontier in autoimmunity and transplantation. JAMA 2004; 291:1127‑31. 2. Reed W, Lee TH, Norris PJ, Utter GH, Busch MP. Transfusion‑associated microchimerism: A new complication of blood transfusions in severely injured patients. Semin Hematol 2007; 44:24‑31. 3. Starzl TE. Chimerism and tolerance in transplantation. Proc Natl Acad Sci USA 2004; 101(Suppl 2):14607‑14. 4. 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Cell Adhesion & Migration 2007; Vol. Issue [...]... studied in the rodent’s brain In the next step, an excitotoxic drug, NMDA, was infused into the hippocampus of maternal mice to create lesions in the maternal brains and facilitate the migration and differentiation of fetal cells Numbers of fetal cells in leisoned maternal mice were significantly increased compared with those in normal maternal mice Fetal cells near blood vessels of maternal mouse brains... then checked Immunostaining results show the migration of fetal cells across blood vessels in the brain which strongly supported the original hypothesis that fetal cells are capable of crossing the blood- brain barrier F4/80 staining of these fetal cells indicated that these fetal cells may come from the macrophage cells in maternal blood Also, immunostaining and Y chromosome in situ hybridization double... of fetal cells in maternal blood and brain, an immunosuppressant drug was injected into maternal mice and the maternal mice were housed with babies, keeping the maternal mice weaning Preliminary data indicate that the hormonal changes associated with weaning may have some effect on facilitating the survival of fetal cells This is the first time that environmental or internal factors effecting fetal microchimerism. .. reporter has already been widely accepted 1 32 To determine whether fetal cells are able to engraft into maternal peripheral blood, the maternal blood samples were scanned and screened by FACS and epifluorescence microscopy Existence of fetal cells in maternal peripheral blood was verified Fetal cells were found in the maternal brain by observing maternal brain tissue slices under a microscope To study... expression of CD45 by fetal cells in the maternal brain may also suggest the fetal cells that infiltrate the maternal brain and adopt characteristics typical of neural cell types are not of haematopoietic origin However, it cannot be excluded the possibility that these cells expressed CD45 before entry into the maternal brain and then stopped expressing CD45 when in the brain No Green Mouse fetal cells were... suggest that the fetal cells may have migrated within the host brain and developed in response to cues from the host Within the brain, fetal cells were capable of expressing morphological and immunocytochemical characteristics of various cell types Some fetal cells remained in close association with blood vessels but no evidence for endothelial cells of fetal origin in the maternal brain was found It... unless the injury triggers neovascularization, circulating fetal endothelial cells will not be recruited Besides, at least in humans, it appears that 1 12 endothelial cells in the blood of pregnant women are of maternal rather then fetal origin (Gussin et al., 20 02; Gussin et al., 20 05) Fetal cells were also observed within the brain in close juxtaposition to the endothelial cells of the host blood vessel... after delivering, maternal PLP transgenic mice, the same age virgin PLP transgenic mice and virgin wide type mice were sacrificed for immunofluorescence staining and DAB staining At 1 week, 2 week and 3 week after delivery, maternal PLP transgenic mice, virgin PLP transgenic mice and same age virgin wide type mice were grouped to conduct rotarod studies 5 .2. 2 Immunostaining Cryosections of spinal cord... difficulty in achieving good and reproducible surgical lesions in mice indicated that genetic lesioning should be used in future to compare cells targeting different types of cell loss in the central nervous system We therefore investigated the fetomaternal microchimerism in a transgenic mouse model of MS-like demyelinating injury In this mouse model, overexpression of PLP leads to demyelination, apparently... differentiation of fetal cells has been observed in maternal blood and with that in mind, the present studies were aimed at determining whether fetal cells are able to cross the maternal blood- brain barrier and differentiate into neural cells In this study, wild type female mice were crossed with EGFP transgenic male mice Conventional methods to identify fetal cells consist of PCR and Y chromosome in situ hybridization . 4 Fetal cell differentiation in maternal brain 4.1. Introduction A wide variety of fetal cells have been found in maternal peripheral blood circulation and brain. The cues that induce fetal. three 5 min washes in 50 % formamide in 2 x SSC, three 5 min washes with 2 x SSC, and two 10 min washes in 0.05 % Tween 20 in 4 x SSC at 42 O C. After FISH, the sections were immunostained as. after delivering their last litter 4.3 .2. Differentiation of fetal Green Mouse cells in maternal brain Four weeks after delivery, Green Mouse fetal cells in the mothers’ brains were capable