- Email: [email protected]
Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy
Research Letters
Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy Keelin O’Donoghue, Jerry Chan, Josu de la Fuente, Nigel Kennea, Ann Sandison, Jonathan R Anderson, Irene AG Roberts, Nicholas M Fisk
Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0·0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation. Fetomaternal microchimerism, or fetal cell persistence after pregnancy, is implicated in the pathogenesis of autoimmune diseases that preferentially affect women after childbearing and resemble graft versus host disease (GVHD), a known chimeric condition.1 Since fetomaternal trafficking occurs in all pregnancies, microchimerism could take place in healthy women. Quantification has been hindered by a lack of consideration of alternative sources of chimerism and reliance on PCR to show gender-discordant Y-microchimerism, precluding identification of the responsible cell type. Fetal cells found in the CD34+ population 20 years after pregnancy,2 and differentiated male cells found in diseased female tissue suggests that fetal stem cells entering maternal blood engraft and differentiate in host tissues.3 Previously, we identified mesenchymal stem cells (MSC) in first trimester fetal blood, but were unable to isolate fetal MSC from pre-termination maternal blood.4 A range of evidence suggests that MSC engraft maternal tissues after transplacental passage: they express Patient
R1 R2 S1 S3 R5 S10 S2 S8 S9 R3 R4 S4 S5 S6 S7 R6
Age (years) Pregnancy history
59 51 65 80 72 68 61 83 63 79 68 46 67 82 76 53
Number of males (age in years*)
Number of females
Pregnancy losses
3 (28) 1 (13) 1 (32) 2 (45) 1 (43) 1 (36) 2 (34) 1 (51) 2 (38) 0 0 0 0 0 0 0
0 4 2 0 4 1 0 0 1 0 1 0 1 1 2 1
1 2 0 3 1 1 0 1 1 1 3 0 0 0 0 0
adhesion molecules and adhere avidly in vitro, they engraft widely in animal models, and suppress alloreactive lymphocyte proliferation. Here, we aimed to determine the incidence of fetal microchimerism in post-reproductive maternal tissues and investigated the cell type responsible. Women undergoing cardiopulmonary surgery gave written consent, approved by the institutional ethics committee, to a pre-operative interview and collection of marrow (n=16) and rib sections (n=6). Detailed histories addressed whether the women had male children, first trimester losses, other pregnancies, previous transfusion or organ transplantation. Nine women (median age 65 years, range 51–83) had one or more sons, two had a history of early pregnancy loss, and five (67, 46–82) had either full-term daughters or had never been pregnant; all had singleton pregnancies (table). Cell suspensions were prepared by flushing cells from rib sections (syringe and 22-gauge needle). Mononuclear cells collected from a Ficoll-1077 g/mL (Histopaque1077) interface were cultured until MSC colonies
Number of MSC slides analysed
Total male cells
Rate
Number of rib Total male cells slides analysed
28 30 22 18 26 21 22 23 26 20 23 23 22 20 19 23
11 10 9 7 11 12 10 2 10 0 2 0 0 0 0 0
1/101 818 1/120 000 1/97 777 1/102 857 1/ 94 545 1/ 70 000 1/ 88 000 1/460 000 1/104 000 ·· 1/460 000 ·· ·· ·· ·· ··
12 9 ·· ·· 12 ·· ·· ·· ·· 11 10 ·· ·· ·· ·· 10
Lancet 2004; 364: 179–82 Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology (K O’Donoghue MRCOG, J Chan MRCOG, N Kennea MRCPCH, Prof N M Fisk PhD) and Department of Haematology, Division of Investigative Sciences (J de la Fuente MRCPCH, Prof I A G Roberts MD), Imperial College London, Hammersmith Campus, London W12 0NN, UK; Department of Histopathology, Charing Cross Hospital, London W6 8RF (A Sandison MRCP); and Department of Cardiothoracic Surgery, Hammersmith Hospital, London W12 0NN (J R Anderson FRCS) Correspondence to: Dr Keelin O’Donoghue [email protected]
25 6 ·· ·· 7 ·· ·· ·· ·· 0 6 ·· ·· ·· ·· 0
*Age of youngest son. S1–S10 underwent sternotomy for coronary artery bypass grafting, valve replacement, or excision of a mediastinal mass. R1–R6 underwent thoracotomy for benign or malignant lung disease. S8 and S9 had received a blood transfusion on one occasion at least 10 years previously, and S2 had received multiple transfusions and renal allograft 10 years previously. The rate of microchimerism was calculated from an average of 40 000 cells per cytospin.
Table: Analysis of mesenchymal stem cells in bone marrow and bone
www.thelancet.com Vol 364 July 10, 2004
179
Research Letters
B
A
D
C
G
F
E
H
x40 J
I
x10 K
L
M
Figure: Mesenchymal stem cells Mesenchymal stem cells from adult female marrow are XX on FISH (A), controls and male cells bearing a Y chromosome labelled with SpectrumGreen (arrowhead) are shown in MSC cultures from women with sons (B). The Y chromosome in male MSC (arrowhead) was identified with alternative Y FISH probes, DYZ1 (C) and SRY (D), labelled with SpectrumOrange. Vimentin-FITC+ cells are shown in (E), where SRY (arrowhead) is also labelled with SpectrumOrange. MSC morphology in culture is clearly visualised by staining with crystal violet (F), and MSC are CD45-FITC- (G). When grown in selective media, MSC differentiate into fat, as demonstrated by OilRed-O staining (H). Female cells (XX) are identified by FISH in sections of bone from a female rib (I). Male cells with an osteocyte phenotype are in cortical bone (J, L); the Y chromosome (arrowhead) is labelled with SpectrumGreen (J) and SpectrumOrange (L). Male cells are also in connective tissue near trabecular bone (K); the Y chromosome (arrowhead) is labelled with Spectrum Green. The X chromosomes in (I-K) are labelled with SpectrumOrange. A male cell bearing a Y chromosome (arrowhead) labelled with Spectrum Orange (M) was located in a section stained with laminin-FITC. Magnification x100 (except for F and H).
180
www.thelancet.com Vol 364 July 10, 2004
Research Letters
developed. Male peripheral blood lymphocytes or male fetal MSC were used as hybridisation controls for XY fluorescence in-situ hybridisation (FISH), as previously described.4 Rib sections preserved in 10% formaldehyde were decalcified in neutral EDTA, using X-ray on a Faxitron machine to establish the end-point. For each patient, 3–4 blocks were processed to paraffin wax, and from each 3–4 3 m sections analysed. FISH on paraffin-embedded bone was modified from published methods. Tissue sections were dewaxed and rehydrated, before sequential treatment with 0·2 mol/L hydrochloric acid for 10 min, 2SSC for 15 min at 80ºC, and 100 g/mL Proteinase-K (2 mg/mL) for 15 mins. Secondary fixation with 2:1 v/v methanol:acetone for 2 min preceded hybridisation. Paraffin-embedded trophoblast sections were hybridisation controls. For combined immunophenotyping and FISH, tissue pretreatment lasted 5 mins and 20 g/mL Proteinase-K was used pre-hybridisation. Male cells (range 0–3 per slide) were seen by XY FISH in MSC from all women with male children and no women who never had a male child (9/9 vs 0/5; p=0·0005). We also detected male MSC in one woman who had three miscarriages (sex unknown) and no sons. We located male cells over 50 years (median 36 years; range 13–51) after the birth of a son (table). Male cells were first identified using the chromosome-specific centromeric repeat probes DXZ1, labelled with SpectrumOrange, and DYZ1 (figure, B), labelled with SpectrumGreen (Vysis, Abbott Laboratories Ltd, Maidenhead) and then confirmed in all cases using at least two of three alternative probes for Y-sequences: centromeric repeat probes, DYZ1 (figure, C) and DYZ3, and a locus specific probe, SRY (figure, D), all labelled with SpectrumOrange. Repeat scoring of male cells by an observer who was unaware of reproductive histories yielded concordant results. Real-Time Taqman quantitative PCR with primers directed to a coding region of SRY, by an investigator who was unaware of the clinical details, showed male DNA in 3/3 samples from women with sons but in 0/2 with no sons. The microchimeric cells were identified as MSC by their morphology and immunophenotype (CD45-, CD14-, CD11a-, CD49b-, CD49dlow, SH2+, SH3+, Vimentin+, CD29+, CD49e+, CD106+, HLA-Class II-; determined by immunocytochemistry and flow cytometry), by their self-renewal in vitro and by osteogenic and adipogenic differentiation (figure, F–H). Combining immunocytochemistry and FISH,4 we identified cells with a Y chromosome with MSC markers in MSC cultures from all women with sons (figure, E). Male cells were identified in rib sections from the three women with male pregnancies (figure, J–L) and in the patient with three miscarriages, but not from two women with no sons (figure, I). Male cells (0–5 per section) were seen in similar locations in contiguous sections, mostly in the lamellae of cortical bone, and www.thelancet.com Vol 364 July 10, 2004
were osteocyte-like: small angular cells with eccentric nuclei, arranged within pericellular lacunae (figure, J–L). We also showed male cells in periosteum and in connective tissue adjacent to trabecular bone (figure, K). Although limited by technical difficulties in combining immunostaining and FISH in bone, we identified several vimentin-positive and laminin-positive cells with a Y chromosome (figure, M). We showed that male cells of putative fetal origin can be identified years after pregnancy in all women who had sons. This finding implies that fetal cells transferred into maternal blood during pregnancy engraft marrow and bone, where they persist for decades. The cell type responsible for microchimerism has not been established, although transplacental passage of fetal stem cells has been suggested.5 Our data confirm that fetal cells entering maternal blood after early fetal loss might be primitive progenitors, and show that the progenitor type persisting in women is mesenchymal in origin. The observation of single male cells encased in bone, like true osteocytes, from patients with known male pregnancies, but not in controls, suggests that fetal MSC differentiate in vivo and integrate into bone. Clinical variables are important in interpretation of microchimerism, since male cells could originate from other sources: trafficking at delivery, twin pregnancy, blood transfusion, or stem cell or organ transplantation.1 We obtained histories by interview, which is a more accurate method than retrospective chart survey or questionnaire. In patients with male children (n=9), three had had a blood transfusion, one of whom had a renal transplant. Compared with those with no alternative source of chimerism (n=6), these women had similar numbers of male MSC. Miscarriage is a source of microchimeric fetal cells and might allow more fetal cells to enter maternal blood than in a term pregnancy.5 We used stringent measures to minimise contamination as a source of false positive results, and controls were consistently negative. We relied on FISH for male cell detection, which is less subject to contamination than PCR, and also allows quantification and morphologic assessment. Since a repeating unit of the DYZ1 family (pHY10), often used in microchimerism studies, has varying male specificity, and because some Y chromosome sequences cross-react with autosomes, we used different Y chromosome sequences (DYZ1, DYZ3, SRY). We suggest that fetal microchimerism might be more common than estimated, and propose that fetal MSC are responsible. Microchimerism could be an incidental result of normal pregnancy without biological significance or could have long-term consequences. Persisting fetal stem cells in marrow and other tissues might explain why women make poor donors for organ and marrow transplants. Whether fetal MSC can, as suggested experimentally, be activated by chemical stimuli or by further pregnancy, or induce disease by 181
Research Letters
altering the maternal immune system remains unknown. However, fetal stem cells in maternal marrow could also act as a long-term reservoir of stem cells and might even explain why women live longer than men and why pregnancy protects against susceptibility to some diseases. Contributors K O’Donoghue designed the study, interviewed patients and collected the samples, developed the FISH and immunocytochemistry methods, undertook the laboratory analyses, and drafted the report. J Chan assisted with cell culture and MSC differentiation, and did the masked repeat scoring of slides. J de la Fuente performed flow cytometry of MSC. N Kennea assisted in developing the methods and with cell culture and epifluorescence microscopy. A Sandison prepared and provided the rib sections. J R Anderson managed the patients and provided the samples intraoperatively. I A G Roberts and N M Fisk secured grant funding, supervised the project and helped design the study, analyse data, and prepare the report. Role of the funding source The sponsors of the study had no role in study design, collection or analysis of data, interpretation of results, or in writing of the report.
182
Conflict of interest statement None declared. Acknowledgments KOD’s salary was funded by the Institute of Obstetrics & Gynaecology Trust (Registered Charity No. 292518) and consumables by a grant from the Hammersmith Hospital Trust Research Committee. 1 2
3
4
5
Nelson JL. Pregnancy and Microchimerism in Autoimmune Disease: Protector or Insurgent? Arthritis Rheum 2002; 46: 291–97. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996; 93: 705–08. Srivatsa B, Srivatsa S, Johnson KL, Samura O, Lee SL, Bianchi DW. Microchimerism of presumed fetal origin in thyroid specimens from women: a case-control study. Lancet 2001; 358: 2034–38. O’Donoghue K, Choolani M, Chan J, de la Fuente J, Kumar S, Campagnoli C, Bennett PR, Roberts IA, Fisk NM. Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis. Mol Hum Reprod 2003; 9: 497–502. Khosrotehrani K, Bianchi DW. Fetal cell microchimerism: helpful or harmful to the parous woman? Curr Opin Obstet Gynecol 2003; 15: 195–99.
www.thelancet.com Vol 364 July 10, 2004
Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy Keelin O’Donoghue, Jerry Chan, Josu de la Fuente, Nigel Kennea, Ann Sandison, Jonathan R Anderson, Irene AG Roberts, Nicholas M Fisk
Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0·0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation. Fetomaternal microchimerism, or fetal cell persistence after pregnancy, is implicated in the pathogenesis of autoimmune diseases that preferentially affect women after childbearing and resemble graft versus host disease (GVHD), a known chimeric condition.1 Since fetomaternal trafficking occurs in all pregnancies, microchimerism could take place in healthy women. Quantification has been hindered by a lack of consideration of alternative sources of chimerism and reliance on PCR to show gender-discordant Y-microchimerism, precluding identification of the responsible cell type. Fetal cells found in the CD34+ population 20 years after pregnancy,2 and differentiated male cells found in diseased female tissue suggests that fetal stem cells entering maternal blood engraft and differentiate in host tissues.3 Previously, we identified mesenchymal stem cells (MSC) in first trimester fetal blood, but were unable to isolate fetal MSC from pre-termination maternal blood.4 A range of evidence suggests that MSC engraft maternal tissues after transplacental passage: they express Patient
R1 R2 S1 S3 R5 S10 S2 S8 S9 R3 R4 S4 S5 S6 S7 R6
Age (years) Pregnancy history
59 51 65 80 72 68 61 83 63 79 68 46 67 82 76 53
Number of males (age in years*)
Number of females
Pregnancy losses
3 (28) 1 (13) 1 (32) 2 (45) 1 (43) 1 (36) 2 (34) 1 (51) 2 (38) 0 0 0 0 0 0 0
0 4 2 0 4 1 0 0 1 0 1 0 1 1 2 1
1 2 0 3 1 1 0 1 1 1 3 0 0 0 0 0
adhesion molecules and adhere avidly in vitro, they engraft widely in animal models, and suppress alloreactive lymphocyte proliferation. Here, we aimed to determine the incidence of fetal microchimerism in post-reproductive maternal tissues and investigated the cell type responsible. Women undergoing cardiopulmonary surgery gave written consent, approved by the institutional ethics committee, to a pre-operative interview and collection of marrow (n=16) and rib sections (n=6). Detailed histories addressed whether the women had male children, first trimester losses, other pregnancies, previous transfusion or organ transplantation. Nine women (median age 65 years, range 51–83) had one or more sons, two had a history of early pregnancy loss, and five (67, 46–82) had either full-term daughters or had never been pregnant; all had singleton pregnancies (table). Cell suspensions were prepared by flushing cells from rib sections (syringe and 22-gauge needle). Mononuclear cells collected from a Ficoll-1077 g/mL (Histopaque1077) interface were cultured until MSC colonies
Number of MSC slides analysed
Total male cells
Rate
Number of rib Total male cells slides analysed
28 30 22 18 26 21 22 23 26 20 23 23 22 20 19 23
11 10 9 7 11 12 10 2 10 0 2 0 0 0 0 0
1/101 818 1/120 000 1/97 777 1/102 857 1/ 94 545 1/ 70 000 1/ 88 000 1/460 000 1/104 000 ·· 1/460 000 ·· ·· ·· ·· ··
12 9 ·· ·· 12 ·· ·· ·· ·· 11 10 ·· ·· ·· ·· 10
Lancet 2004; 364: 179–82 Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology (K O’Donoghue MRCOG, J Chan MRCOG, N Kennea MRCPCH, Prof N M Fisk PhD) and Department of Haematology, Division of Investigative Sciences (J de la Fuente MRCPCH, Prof I A G Roberts MD), Imperial College London, Hammersmith Campus, London W12 0NN, UK; Department of Histopathology, Charing Cross Hospital, London W6 8RF (A Sandison MRCP); and Department of Cardiothoracic Surgery, Hammersmith Hospital, London W12 0NN (J R Anderson FRCS) Correspondence to: Dr Keelin O’Donoghue [email protected]
25 6 ·· ·· 7 ·· ·· ·· ·· 0 6 ·· ·· ·· ·· 0
*Age of youngest son. S1–S10 underwent sternotomy for coronary artery bypass grafting, valve replacement, or excision of a mediastinal mass. R1–R6 underwent thoracotomy for benign or malignant lung disease. S8 and S9 had received a blood transfusion on one occasion at least 10 years previously, and S2 had received multiple transfusions and renal allograft 10 years previously. The rate of microchimerism was calculated from an average of 40 000 cells per cytospin.
Table: Analysis of mesenchymal stem cells in bone marrow and bone
www.thelancet.com Vol 364 July 10, 2004
179
Research Letters
B
A
D
C
G
F
E
H
x40 J
I
x10 K
L
M
Figure: Mesenchymal stem cells Mesenchymal stem cells from adult female marrow are XX on FISH (A), controls and male cells bearing a Y chromosome labelled with SpectrumGreen (arrowhead) are shown in MSC cultures from women with sons (B). The Y chromosome in male MSC (arrowhead) was identified with alternative Y FISH probes, DYZ1 (C) and SRY (D), labelled with SpectrumOrange. Vimentin-FITC+ cells are shown in (E), where SRY (arrowhead) is also labelled with SpectrumOrange. MSC morphology in culture is clearly visualised by staining with crystal violet (F), and MSC are CD45-FITC- (G). When grown in selective media, MSC differentiate into fat, as demonstrated by OilRed-O staining (H). Female cells (XX) are identified by FISH in sections of bone from a female rib (I). Male cells with an osteocyte phenotype are in cortical bone (J, L); the Y chromosome (arrowhead) is labelled with SpectrumGreen (J) and SpectrumOrange (L). Male cells are also in connective tissue near trabecular bone (K); the Y chromosome (arrowhead) is labelled with Spectrum Green. The X chromosomes in (I-K) are labelled with SpectrumOrange. A male cell bearing a Y chromosome (arrowhead) labelled with Spectrum Orange (M) was located in a section stained with laminin-FITC. Magnification x100 (except for F and H).
180
www.thelancet.com Vol 364 July 10, 2004
Research Letters
developed. Male peripheral blood lymphocytes or male fetal MSC were used as hybridisation controls for XY fluorescence in-situ hybridisation (FISH), as previously described.4 Rib sections preserved in 10% formaldehyde were decalcified in neutral EDTA, using X-ray on a Faxitron machine to establish the end-point. For each patient, 3–4 blocks were processed to paraffin wax, and from each 3–4 3 m sections analysed. FISH on paraffin-embedded bone was modified from published methods. Tissue sections were dewaxed and rehydrated, before sequential treatment with 0·2 mol/L hydrochloric acid for 10 min, 2SSC for 15 min at 80ºC, and 100 g/mL Proteinase-K (2 mg/mL) for 15 mins. Secondary fixation with 2:1 v/v methanol:acetone for 2 min preceded hybridisation. Paraffin-embedded trophoblast sections were hybridisation controls. For combined immunophenotyping and FISH, tissue pretreatment lasted 5 mins and 20 g/mL Proteinase-K was used pre-hybridisation. Male cells (range 0–3 per slide) were seen by XY FISH in MSC from all women with male children and no women who never had a male child (9/9 vs 0/5; p=0·0005). We also detected male MSC in one woman who had three miscarriages (sex unknown) and no sons. We located male cells over 50 years (median 36 years; range 13–51) after the birth of a son (table). Male cells were first identified using the chromosome-specific centromeric repeat probes DXZ1, labelled with SpectrumOrange, and DYZ1 (figure, B), labelled with SpectrumGreen (Vysis, Abbott Laboratories Ltd, Maidenhead) and then confirmed in all cases using at least two of three alternative probes for Y-sequences: centromeric repeat probes, DYZ1 (figure, C) and DYZ3, and a locus specific probe, SRY (figure, D), all labelled with SpectrumOrange. Repeat scoring of male cells by an observer who was unaware of reproductive histories yielded concordant results. Real-Time Taqman quantitative PCR with primers directed to a coding region of SRY, by an investigator who was unaware of the clinical details, showed male DNA in 3/3 samples from women with sons but in 0/2 with no sons. The microchimeric cells were identified as MSC by their morphology and immunophenotype (CD45-, CD14-, CD11a-, CD49b-, CD49dlow, SH2+, SH3+, Vimentin+, CD29+, CD49e+, CD106+, HLA-Class II-; determined by immunocytochemistry and flow cytometry), by their self-renewal in vitro and by osteogenic and adipogenic differentiation (figure, F–H). Combining immunocytochemistry and FISH,4 we identified cells with a Y chromosome with MSC markers in MSC cultures from all women with sons (figure, E). Male cells were identified in rib sections from the three women with male pregnancies (figure, J–L) and in the patient with three miscarriages, but not from two women with no sons (figure, I). Male cells (0–5 per section) were seen in similar locations in contiguous sections, mostly in the lamellae of cortical bone, and www.thelancet.com Vol 364 July 10, 2004
were osteocyte-like: small angular cells with eccentric nuclei, arranged within pericellular lacunae (figure, J–L). We also showed male cells in periosteum and in connective tissue adjacent to trabecular bone (figure, K). Although limited by technical difficulties in combining immunostaining and FISH in bone, we identified several vimentin-positive and laminin-positive cells with a Y chromosome (figure, M). We showed that male cells of putative fetal origin can be identified years after pregnancy in all women who had sons. This finding implies that fetal cells transferred into maternal blood during pregnancy engraft marrow and bone, where they persist for decades. The cell type responsible for microchimerism has not been established, although transplacental passage of fetal stem cells has been suggested.5 Our data confirm that fetal cells entering maternal blood after early fetal loss might be primitive progenitors, and show that the progenitor type persisting in women is mesenchymal in origin. The observation of single male cells encased in bone, like true osteocytes, from patients with known male pregnancies, but not in controls, suggests that fetal MSC differentiate in vivo and integrate into bone. Clinical variables are important in interpretation of microchimerism, since male cells could originate from other sources: trafficking at delivery, twin pregnancy, blood transfusion, or stem cell or organ transplantation.1 We obtained histories by interview, which is a more accurate method than retrospective chart survey or questionnaire. In patients with male children (n=9), three had had a blood transfusion, one of whom had a renal transplant. Compared with those with no alternative source of chimerism (n=6), these women had similar numbers of male MSC. Miscarriage is a source of microchimeric fetal cells and might allow more fetal cells to enter maternal blood than in a term pregnancy.5 We used stringent measures to minimise contamination as a source of false positive results, and controls were consistently negative. We relied on FISH for male cell detection, which is less subject to contamination than PCR, and also allows quantification and morphologic assessment. Since a repeating unit of the DYZ1 family (pHY10), often used in microchimerism studies, has varying male specificity, and because some Y chromosome sequences cross-react with autosomes, we used different Y chromosome sequences (DYZ1, DYZ3, SRY). We suggest that fetal microchimerism might be more common than estimated, and propose that fetal MSC are responsible. Microchimerism could be an incidental result of normal pregnancy without biological significance or could have long-term consequences. Persisting fetal stem cells in marrow and other tissues might explain why women make poor donors for organ and marrow transplants. Whether fetal MSC can, as suggested experimentally, be activated by chemical stimuli or by further pregnancy, or induce disease by 181
Research Letters
altering the maternal immune system remains unknown. However, fetal stem cells in maternal marrow could also act as a long-term reservoir of stem cells and might even explain why women live longer than men and why pregnancy protects against susceptibility to some diseases. Contributors K O’Donoghue designed the study, interviewed patients and collected the samples, developed the FISH and immunocytochemistry methods, undertook the laboratory analyses, and drafted the report. J Chan assisted with cell culture and MSC differentiation, and did the masked repeat scoring of slides. J de la Fuente performed flow cytometry of MSC. N Kennea assisted in developing the methods and with cell culture and epifluorescence microscopy. A Sandison prepared and provided the rib sections. J R Anderson managed the patients and provided the samples intraoperatively. I A G Roberts and N M Fisk secured grant funding, supervised the project and helped design the study, analyse data, and prepare the report. Role of the funding source The sponsors of the study had no role in study design, collection or analysis of data, interpretation of results, or in writing of the report.
182
Conflict of interest statement None declared. Acknowledgments KOD’s salary was funded by the Institute of Obstetrics & Gynaecology Trust (Registered Charity No. 292518) and consumables by a grant from the Hammersmith Hospital Trust Research Committee. 1 2
3
4
5
Nelson JL. Pregnancy and Microchimerism in Autoimmune Disease: Protector or Insurgent? Arthritis Rheum 2002; 46: 291–97. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996; 93: 705–08. Srivatsa B, Srivatsa S, Johnson KL, Samura O, Lee SL, Bianchi DW. Microchimerism of presumed fetal origin in thyroid specimens from women: a case-control study. Lancet 2001; 358: 2034–38. O’Donoghue K, Choolani M, Chan J, de la Fuente J, Kumar S, Campagnoli C, Bennett PR, Roberts IA, Fisk NM. Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis. Mol Hum Reprod 2003; 9: 497–502. Khosrotehrani K, Bianchi DW. Fetal cell microchimerism: helpful or harmful to the parous woman? Curr Opin Obstet Gynecol 2003; 15: 195–99.
www.thelancet.com Vol 364 July 10, 2004