Fetal microchimerism in maternal mouse blood and brain

FETAL MICROCHIMERISM IN MATERNAL MOUSE BLOOD AND BRAIN TAN XIAO WEI (MBBS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements I would like to extend my utmost gratitude to my supervisors, Dr Gavin Stewart Dawe and Associate Prof. Xiao Zhi Cheng for their advice, guidance, inspiration and patience during the period I have been working with them. I would like to thank Dr. Sun Li and Prof. Gerald Udolph for their proficient guidance and constant encouragement throughout the course of this study. I would also like to thanks Dr. Zeng Xiaoxia and Ms. Liao Hong for their invaluable help in the research work and the preparation of manuscripts for publication. I also wish to express my deepest appreciation and sincere thanks to all my lab mates in the Cellular and Molecular Neuropharmacology Laboratory, Department of Pharmacology, NUS and Neurobiology Laboratory, Department of Clinic Research, Singapore General Hospital, for their help during the course of this project. And last but not least to all my family members for their complete support throughout my study. Tan Xiao Wei 10th April 2007 Table of Contents Page Acknowledgements Table of Contents List of Abbreviations Summary 11 List of Tables 15 List of Figures 16 Chapter Introduction 1.1. Definition of microchimerism and fetal microchimerism 18 1.2. Fetal microchimerism in maternal peripheral blood 18 1.2.1. Free fetal DNA in maternal tissue 1.2.2. Fetal cells in maternal blood 1.2.3. Characterization of fetal cells in maternal blood 1.3. Fetal microchimerism in maternal solid organs 29 1.3.1. Free fetal DNA in maternal solid organs 1.3.2. Fetal cells in maternal solid organs 1.4. Neural differentiation of fetal cells 31 1.4.1. Neural differentiation of bone marrow cells 1.4.2. Neural differentiation of human umbilical cord blood cells 1.4.3. Cell fusion and microglial phagocytosis 1.5. Fetal microchimerism and maternal blood-brain barrier 36 1.5.1. Bone marrow cells and the blood-brain barrier 1.5.2. Fetal cells and the blood-brain barrier 1.6. Migration of chimerical fetal cells 39 1.6.1. The likely sites of spontaneous engraftment 1.6.2. Lesion-induced neurogenesis and migration 1.6.3. Pregnancy-related neurogenesis in the brain 1.7. Characterization of chimerical fetal cells in the brain 44 1.8. Factors influencing microchimerism 46 1.9. Possible roles of microchimerism in regulating human health and disease 49 1.10. Technical and study design considerations in evaluating microchimerism 51 1.11. Summary 52 Chapter Fetal microchimerism in the maternal mouse blood 2.1. Introduction 54 2.2. Materials and methods 54 2.2.1. Animals 2.2.2. Detection of Green Mouse cells in maternal blood by FACS 2.2.3. Visualization of fetal Green Mouse cells in maternal blood 2.2.4. Quantitative real-time PCR 2.3. Results 57 2.3.1. Fetal Green Mouse cells in maternal blood 2.3.2. Quantification of fetal mouse cells in maternal blood 2.4. Discussion 62 Chapter Fetal microchimerism in the maternal mouse brain: A novel population of fetal progenitor or stem cells able to cross the blood-brain barrier? 3.1. Introduction 71 3.2. Materials and methods 3.2.1. Animals 3.2.2. Excitotoxic lesions of the brain 3.2.3. 6-OHDA lesion of the brain 3.2.4. Behavioral testing 3.2.5. Fluorescent immunohistochemistry staining after 6-OHDA lesions 3.2.6. Quantitative real-time PCR 3.3. Results 79 3.3.1. Fetal Green Mouse cells in maternal brain 3.3.2. NMDA lesioning in young adult mice 3.3.3. Effect of 6-OHDA injections on dopaminergic neuronal viability in substantia nigra and thalamus region of young adult mice 3.3.4. Quantification of fetal mouse cells in maternal brain 3.4. Discussion 91 Chapter Fetal cell differentiation in maternal brain 4.1. Introduction 98 4.2. Materials and methods 98 4.2.1. Animals 4.2.2. Fluorescence in situ hybridization (FISH) for the Y chromosome 4.2.3. Fluorescence immunocytochemistry 4.3. Results 102 4.3.1. Location of fetal cells in the maternal brain 4.3.2. Differentiation of fetal Green Mouse cells in maternal brain 4.4. Discussion 112 Chapter Functional differentiation of fetal cells in PLP transgenic maternal mice 5.1. Introduction 119 5.2. Materials and methods: 121 5.2.1. Animals 5.2.2 Immunostaining 5.2.3 Behavioural test 5.3.Results 122 5.3.1. Differentiation of fetal cells in PLP transgenic maternal mice 5.3.2. Decreased number of T cells and microglia cells in PLP maternal mice 5.3.3. Motor function of PLP maternal mice was partially recovered 5.4.Discussion 125 Chapter 6: Conclusion and future work 132 References 136 Appendix 1: Selected protocols 163 Appendix 2: Published paper List of Abbreviations 6-OHDA: 6-hydroxydopamine bFGF: basic fibroblast growth factor β-Gal: β-galactosidase BHA: butylated hydroxyanisole BM: bone marrow BNDF: brain-derived neurotrophic factor Bp: base pair BNDF: brain-derived neurotrophic factors CNS: central nervous system DAPI: 4’, 6-diamidino-2-phenylindole DdH2O double distilled water DG: dentate gyrus DMEM: Dulbecco’s modified eagle’s medium DMSO: dimethyl sulfoxide DNA: deoxyribonucleic acid EGF: epidermal growth factor EGFP: enhanced green fluorescence protein ELISA: enzyme-linked immunosorbent assay ESC: embryonic stem cell FACS: Fluorescent activated cell sorting FBS: fetal bovine serum FCS: fetal calf serum FGF: fibroblast growth factor FISH: fluorescence in situ hybridization GAP43: growth associated protein 43 GD gestational day GFAP: glial fibrillary acidic protein (poly) GFP: green fluorescent protein GS goat serum GVHD: graft-versus-host disease HLA: human leukocytes antigen HSC: haematopoietic stem cell HUCB: human umbilical cord blood IIM: idiopathic inflammatory myopathies IMEM: improved minimum essential medium KHCO3: potassium bicarbonate MACO: middle cerebral artery occlusion NaEDTA: sodium ethylenediaminetetraacetate NCAM: neural cell adhesion molecule NeuN: neuronal-nuclear protein NCAM: neural cell adhesion molecule NSC: neural stem cell NeuN: neuronal-nuclear protein NGF: nerve growth factor NMDA: N-methyl-D-aspartate NOD: non-obese diabetic NPC: neural progenitor cell NSC: neural stem cell NT3: 3’-nucleotidase PBC: primary biliary cirrhosis PBS: phosphate buffer saline PCR: polymerase chain reaction PD: Parkinson disease PEP: polymorphic eruption of pregnancy PLP: proteolipid protein PBC: primary biliary cirrhosis QPCR: Quantitative Polymerase Chain Reaction RA: retinoic acid RT-PCR: reverse transcriptase polymerase chain reaction SGZ: subgranular zone SLE: system lupus erythematosus SN: substantia nigra SRY: sex-determining region Y gene SSc: systemic sclerosis SSC: saline-sodium citrate SVZ: subventricular zone Figure 6: A B C D Cell number/1mm² E Saline 50000 NMDA 3.7x104 100um 40000 2.1x104 30000 2.0x104 20000 *0 10000 CA DG 82 Figure 6: Toxic effect of NMDA on neuronal viability in the hippocampal region of young adult mice. Upper two panes are representative micrographs of the hippocampus of a normal control mouse treated with saline (Figure A and 6C) and a mouse treated with 1mg/ml NMDA (6B and 6D). Arrows indicate that the neurons in hippocampal CA fields are significantly degenerated comparing with the control mice. Viable cells were evaluated by estimating the mean profile number per mm2. Data are expressed as the percent of the corresponding control values and represent the mean ± sem of at least 12 sections per each brain. Scale bars are 100 µm. 83 A B C D Figure 7: Effect of 6-OHDA on dopaminergic neuronal viability in the substantia nigra and dopaminergic fibers in striatal region of young adult mice. Representative photographs of tyrosine hydroxylase positive (TH+) fibres in contralateral intact striatum (A), striatum ipsilateral to the lesion (B) and TH+ cells in the contralateral substantia nigra (C) and 6-OHDA lesioned substantia nigra (D). Scale bars are 100 µm. 84 Table 10: Apomorphine induced rotations in 6-OHDA-lesion animals. The percentage improvement in rotational behavior (three 2-min periods) before and after pregnancy was not significantly different (Student’s t-test) between the 6-OHDA injection mice and the sham lesion group, which received saline injection. 85 3.3.4. Quantification of fetal mouse cells in maternal brain Quantitative real-time PCR of genomic DNA revealed that in Block1 of the brains of normal female mice crossed with Green male mice there were around ± 1.7 fetal cells per 10,000 cells at gestational day (GD7) and 12 ± 6.9 fetal cells per 10,000 cells at GD14. The fetal cells number reached a peak of around 18.4 ± 5.6 fetal cells per 10,000 cells on the day of delivery and decreased to ± 3.4 fetal cells per 10,000 cells at postpartum day (PD7) followed by another peak of ± 5.2 fetal cells per 10,000 cells at weeks postpartum. Fetal Green Mouse cells could not be detected at weeks after delivery. The number of fetal cells in the NMDA lesioned group showed similar trends, but there were significantly greater numbers of fetal cells (36 ± 11 fetal cells per 10,000 cells; P < 0.05, Student’s t-test against intact control) at weeks after delivery (Figure 8A). Fetal cells were very rare in all blocks of the maternal brain at weeks after delivery (Figure 8). There were significant difference in the number of fetal cells in Block (the block contains the site of the NMDA lesion) between the intact control group and the NMDAlesioned group at weeks postpartum. Around 10.4 ± fetal cells / 10,000 maternal cells in the intact control group were detected, while the number in the NMDA-lesioned group was 29.9 ± 14.1 fetal cells / 10,000 cells (Figure 8C). RT-PCR of fetal cells in Block and Block of maternal brain did not reveal significant differences between the intact control group and the NMDA-lesioned group at all seven 86 Figure 8: Quantitative real-time PCR of fetal Green Mouse cells in the maternal brain at different time points. Fetal cells were present in both intact and lesioned maternal brain and in the lesioned brains there were more fetal cells in Block (A) and Block (C). Block (B); Block (D). * p fetal cell / 10,000 cells) varied between to positive case per mice across the brain blocks in both groups. In a different batch of experiments, where the maternal mice were injected with cyclosporine, it was found that, compared with saline-treated control mice, cyclosporine injection did not change the frequency of detection of fetal Green Mouse cells in maternal brain. In all groups there were to positive cases out of mice at weeks after delivery (Table 12). Another two batches of mice were induced to continue nursing pups till or weeks postpartum. Real time-PCR of genomic DNA from the maternal brain samples collected at weeks after delivery revealed that there was more microchimerism at weeks in the control mice than in the mice continuing to nurse (Table 13). However, when the maternal mice were kept nursing until weeks after delivery, the incidence of fetomaternal microchimerism was higher than in the control group weaning at to weeks as normal. The control group showed a frequency of to positive case out of 88 Table 11: Results of real-time PCR of brain samples from maternal mice housed with pups. Test group mice were housed with their female pups till weeks after delivery. Control group mice were housed with pups till 2-3 weeks after delivery. Positive (+) represents cell number > fetal cell / 10,000 cells. Negative (-) represents cell number < 0.5 fetal cell / 10,000 cells. Positive/negative (±) represents cell number 0.5 fetal cell / 10,000 cells. 89 Table 12: Results of real-time PCR of the brain samples from cyclosporine injection maternal mice. Control mice were treated with saline till or weeks after delivery. Test group mice were treated with cyclosporine till or weeks after delivery. Positive (+) represents cell number > fetal cell / 10,000 cells. Negative (-) represents cell number < 0.5 fetal cell / 10,000 cells. Positive/negative (±) represents cell number 0.5 fetal cell / 10,000 cells. 90 mice, while the experimental group showed a frequency of to positive cases out of mice. In a separate experiment, real-time PCR of genomic DNA from the brain samples of young adult wild-type C57BL/6 mothers crossed with adult male Green Mice (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 post-partum (5.5 ± 1.6 male fetal cells/1,000 cells, which equates to approximately 11 fetal cells/1,000 cells). Quantitative real-time PCR of genomic DNA from brains of C57BL/6 ex-breeder stock female mice at least to 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 out of female mice. The male cells were found almost exclusively in Block 1, corresponding largely to the olfactory bulb (Table 14). In those ex-breeder stock females in which male cells were found, the mean number of male cells in Block was 95.8 ± 69.8 male cells/1,000 cells. 3.4. Discussion Fetal cells were found to enter wild-type maternal blood and brain and the use of Green Mice ubiquitously expressing EGFP facilitated detection of the fetal cells in maternal blood and tissues. Previous studies have reported that free GFP is soluble and very easily to leak out from liquid-covered cryostat sections (Jockusch et al., 2003;Kusser and Randall, 2003). In the present experiments, the tissue block was fixed by perfusion with 91 Table 13: Results of real-time PCR of brain samples from maternal mice kept nursing. Control group mice nursed their pups for 2-3 weeks and were sacrificed at or weeks after delivery. Test group mice were kept nursing with substitution of surrogate pups and were sacrificed at or weeks after delivery. Positive (+) represents cell number > fetal cell / 10,000 cells. Negative (-) represents cell number < 0.5 fetal cell / 10,000 cells. Positive/negative (±) represents cell number 0.5 fetal cell / 10,000 cells. 92 Table 14: Results of real-time PCR of Sry+ male cells in brain samples from ex-breeder maternal mice. Positive (+) represents cell number > 1/10000 total cells. Positive/negative (±) represents cell number > 0.5/10000 but < 1/10000 total cells. Negative (-) represents cell number [...]... infused into maternal mice to investigate the hypothesis that injury would trigger migration and differentiation of fetal cells in maternal brain Real-time PCR was also used to study the spatial and temporal distribution of fetal cells in maternal blood and brain during different stages of pregnancy 11 and at various postpartum time-points A relatively high number of fetal cells were found in the brain. .. nursing for a prolonged period by fostering new pups More fetal green mouse cells were found in the brains of mice which were kept nursing for 4 and 8 weeks than in mice which were kept nursing for only 2 to 3 weeks Immunofluorescent staining and FISH experiments further confirmed the migration and differentiation of fetal cells in maternal brain Fetal cells had differentiated into cells expressing... found in the adult mouse brain: DG dentate gyrus (DG) and subventricular zone (SVZ) Figure 2: Green Mouse fetal cells enter maternal blood circulation Figure 3: Sagittal representation of blocks for extraction of total genomic DNA from maternal mouse brain Figure 4: Quantitative real-time PCR of fetal Green Mouse cells in the maternal mice brain Figure 5: Green Mouse fetal cells can be found in the maternal. .. Perivascular Green Mouse fetal cells in the maternal brain Figure 10: F4/80 immunocytochemistry of perivascular Green Mouse fetal cells in maternal brain Figure 11: Fetal cells can express neuronal immunocytochemical markers in the maternal brain Figure 12: Fetal cells can express oligodendrocytic and astrocytic immunocytochemical markers in the maternal brain Figure 13: Fetal cells can express immature... certain culture conditions Based on this previous work, we hypothesized that fetal cells in maternal peripheral blood may cross the blood- brain barrier and differentiate into cells capable of expressing neural markers in the maternal brain EGFP transgenic male mice were crossed with wild-type female mice, and the resultant fetal cells inheriting the EGFP gene and could easily be tracked Through FACS and. .. the existence of fetal mouse cells in maternal peripheral blood was confirmed Through real-time PCR amplification of the EGFP gene and the male SRY gene, fetal cell DNA fragments were also found in maternal blood and brain The discovery of fetal cell clusters in maternal cerebral cortex near the lateral ventricle and in the cerebellum indicated the possibility of chimeric fetal cells being able to proliferate... and separation methods are required to eliminate the overwhelming excess of contaminating maternal cells Also, additional studies with more standardized techniques, procedures and detailed experimental designs are needed 28 1.3 Fetal microchimerism in maternal solid organs 1.3.1 Free fetal DNA in maternal solid organs Fetal cell DNA has been found not only in maternal peripheral blood but also in maternal. .. unclear SSc 1 in 25 cases 0-1/1x105 total cells >21.6 year unclear (Artlett et al., 1998) (Sanberg et al., 2005) 22 salivary gland salivary gland PCR in situ hybridization skin QPCR skin intestine PCR immunostaini ng and FISH spleen immunostaini ng and FISH skin thyroid FISH immunostaini ng and FISH immunostaini ng and FISH immunostaini ng and FISH thyroid ELISA thyroid PCR thyroid whole blood PCR liver... immunocytochemical markers in the maternal spinal cord Figure 14: Fetal cells encircle the axons in the maternal spinal cord Figure 15: Decreased number of T cells and microglia cells in PLP maternal mice 16 Figure 16: Falling latency of maternal PLP transgenic mice were delayed compared with virgin PLP transgenic mice 17 Chapter 1 Introduction 1.1 Definition of microchimerism and fetal microchimerism Microchimerism... peripheral blood peripheral blood QPCR tissue resource Liver peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood lymphocyte s peripheral mononucle ar blood peripheral mononucle ar blood PMBC PCR PCR nested PCR PCR and ELISA PCR and ELISA unclear 1445.3±204 2.9 copies 2 in 6 cases 3 in 23 cases 4 in 29 cases 3 in 12 cases FISH PCR and ELISA PCR and ELISA 12 in 47 cases 3 in . fusion and microglial phagocytosis 1.5. Fetal microchimerism and maternal blood- brain barrier 36 1.5.1. Bone marrow cells and the blood- brain barrier 1.5.2. Fetal cells and the blood- brain barrier. cells in maternal blood 2.3.2. Quantification of fetal mouse cells in maternal blood 2.4. Discussion 62 Chapter 3 Fetal microchimerism in the maternal mouse brain: A novel population of fetal. from maternal mouse brain. Figure 4: Quantitative real-time PCR of fetal Green Mouse cells in the maternal mice brain. Figure 5: Green Mouse fetal cells can be found in the maternal brain.