Fetal Microchimerism in the Maternal Mouse Brain: A Novel Population of Fetal Progenitor or Stem Cells Able to Cross the Blood–Brain Barrier
http://www.100md.com
《干细胞学杂志》
a Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
b Department of Clinical Research, Singapore General Hospital, Singapore;
c National Drug Screening Laboratory, Chinese Pharmaceutical University, Nanjing, China;
d Genome Information Research Center, Osaka University, Osaka, Japan;
e Institute of Molecular and Cell Biology, Singapore
Key Words. Fetomaternal microchimerism ? Maternal brain ? Fetal ? Neural differentiation ? Neural stem cell ? Neural progenitor cell ? Pregnancy
Correspondence: Gavin S. Dawe, Ph.D., Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, MD2, 18 Medical Drive, Singapore 117597. Telephone: 65-6516-8874; Fax: 65-6873-7690; e-mail: gavindawe@nus.edu.sg; and Zhi-Cheng Xiao, M.D., Ph.D., Department of Clinical Research, Singapore General Hospital, Singapore 169608. Telephone: 65-6326-6195; Fax: 65-6321-3606; e-mail: gcrxzc@sgh.com.sg
ABSTRACT
Fetal cells can enter maternal blood circulation during pregnancy , persist in maternal circulation after pregnancy , and engraft many maternal tissues, including bone marrow (BM), spleen, and liver in both mice and humans . In humans, such cells have been found to persist in maternal blood as long as 27 years postpartum . However, it was not known whether fetal cells capable of crossing the placental barrier to enter maternal blood during pregnancy could also cross the blood–brain barrier to enter the maternal brain. Under certain conditions, umbilical cord blood cells have been reported to be capable of expressing some proteins characteristic of neural cell types and, when injected intravenously into rats with traumatic brain injury or stroke, of entering the brain and expressing immunocytochemical markers for neural cell types . This evidence suggests the hypothesis that fetal cells may enter the maternal brain during pregnancy and differentiate into neural cells. In this study we report fetal microchimerism in the maternal mouse brain together with morphological and immunocytochemical evidence that these fetal cells can express characteristics of perivascular macrophage-like and neural-like cell types in the maternal brain.
MATERIALS AND METHODS
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 7 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).
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 4 weeks after giving birth and sectioned at 20 μm on a cryostat. Scale bar = 50 μm. Data are expressed as mean ± SEM.
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 4 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, 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).
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 3 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 (R2 = 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).
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 4 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 ex-breeder stock females in which male cells were found, the mean number of male cells in Block 1 was 95.8 ± 69.8 male cells per 1,000 maternal cells.
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 4 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.
Figure 3. 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 4 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) 5 μm, (J) 20 μm, and (K) 10 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; vWF, von Willebrand factor.
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 4 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. (EH): 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-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; vWF, von Willebrand factor.
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 4 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.
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 4 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 (anti-GFP) 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 anti-GFP 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.
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 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 4 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 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 double-labeled with the anti-GFAP antibody (Figs. 6J, 6K).
DISCUSSION
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.
FOOTNOTES
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b Department of Clinical Research, Singapore General Hospital, Singapore;
c National Drug Screening Laboratory, Chinese Pharmaceutical University, Nanjing, China;
d Genome Information Research Center, Osaka University, Osaka, Japan;
e Institute of Molecular and Cell Biology, Singapore
Key Words. Fetomaternal microchimerism ? Maternal brain ? Fetal ? Neural differentiation ? Neural stem cell ? Neural progenitor cell ? Pregnancy
Correspondence: Gavin S. Dawe, Ph.D., Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, MD2, 18 Medical Drive, Singapore 117597. Telephone: 65-6516-8874; Fax: 65-6873-7690; e-mail: gavindawe@nus.edu.sg; and Zhi-Cheng Xiao, M.D., Ph.D., Department of Clinical Research, Singapore General Hospital, Singapore 169608. Telephone: 65-6326-6195; Fax: 65-6321-3606; e-mail: gcrxzc@sgh.com.sg
ABSTRACT
Fetal cells can enter maternal blood circulation during pregnancy , persist in maternal circulation after pregnancy , and engraft many maternal tissues, including bone marrow (BM), spleen, and liver in both mice and humans . In humans, such cells have been found to persist in maternal blood as long as 27 years postpartum . However, it was not known whether fetal cells capable of crossing the placental barrier to enter maternal blood during pregnancy could also cross the blood–brain barrier to enter the maternal brain. Under certain conditions, umbilical cord blood cells have been reported to be capable of expressing some proteins characteristic of neural cell types and, when injected intravenously into rats with traumatic brain injury or stroke, of entering the brain and expressing immunocytochemical markers for neural cell types . This evidence suggests the hypothesis that fetal cells may enter the maternal brain during pregnancy and differentiate into neural cells. In this study we report fetal microchimerism in the maternal mouse brain together with morphological and immunocytochemical evidence that these fetal cells can express characteristics of perivascular macrophage-like and neural-like cell types in the maternal brain.
MATERIALS AND METHODS
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 7 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).
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 4 weeks after giving birth and sectioned at 20 μm on a cryostat. Scale bar = 50 μm. Data are expressed as mean ± SEM.
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 4 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, 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).
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 3 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 (R2 = 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).
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 4 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 ex-breeder stock females in which male cells were found, the mean number of male cells in Block 1 was 95.8 ± 69.8 male cells per 1,000 maternal cells.
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 4 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.
Figure 3. 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 4 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) 5 μm, (J) 20 μm, and (K) 10 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; vWF, von Willebrand factor.
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 4 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. (EH): 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-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; vWF, von Willebrand factor.
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 4 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.
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 4 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 (anti-GFP) 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 anti-GFP 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.
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 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 4 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 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 double-labeled with the anti-GFAP antibody (Figs. 6J, 6K).
DISCUSSION
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.
FOOTNOTES
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