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- Materials and Methods
We investigated whether fetal cells can enter the maternal brain during pregnancy. Female wild-type C57BL/6 mice were crossed with transgenic Green Mice ubiquitously expressing enhanced green fluorescent protein (EGFP). Green Mouse fetal cells were found in the maternal brain. Quantitative real-time polymerase chain reaction (PCR) of genomic DNA for the EGFP gene showed that more fetal cells were present in the maternal brain 4 weeks postpartum than on the day of parturition. After an excitotoxic lesion to the brain, more fetal cells were detected in the injured region. The presence of fetal cells in the maternal brain was also confirmed by quantitative real-time PCR for the sex-determining region of the Y chromosome. Four weeks postpartum, EGFP-positive Green Mouse fetal cells in the maternal brain were found to adopt locations, morphologies, and expression of immunocytochemical markers indicative of perivascular macrophage-, neuron-, astrocyte-, and oligodendrocyte-like cell types. Expression of morphological and immunocytochemical characteristics of neuron- and astrocyte-like cell types was confirmed on identification of fetal cells in maternal brain by Y chromosome fluorescence in situ hybridization. Although further studies are required to determine whether such engraftment of the maternal brain has any physiological or pathophysiological functional significance, fetomaternal microchimerism provides a novel model for the experimental investigation of the properties of fetal progenitor or stem cells in the brain without prior in vitro manipulation. Characterization of the properties of these cells that allow them to cross both the placental and blood–brain barriers and to target injured brain may improve selection procedures for isolation of progenitor or stem cells for brain repair by intravenous infusion.
- Top of page
- Materials and Methods
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 anti-GFP antibody was also beneficial.
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 number 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 . 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 4 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 . 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 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 . However, at least in the rat brain, the rate of turnover of brain endothelial cells is very slow , 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 4 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 BM-derived perivascular cells have been reported to phagocytose host endothelial elements . 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 . 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 EGFP-positive 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 EGFP-positive donor cells to be readily distinguished from expression of neural markers by EGFP donor cells . 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 EGFP-positive 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 do 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 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 . It is known that hormonal changes during pregnancy can influence neurogenesis , 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.