Fetomaternal Cell Trafficking and the Stem Cell Debate: Title and subTitle BreakGender Matters

The current ethical, political, and scientific debates on stem cells pitch the benefits and limitations of 2 cell types against each other, adult vs embryonic stem cells. Embryonic stem cells derived from human blastocysts have the key advantage of pluripotency, meaning that they form nearly all cell types but also have the disadvantage of forming tumors in vivo, which may limit clinical application to tissue engineering rather than cell transplantation. In contrast, adult stem cells are derived from mature tissue, typically from bone marrow, but increasingly from most organs. In general, adult stem cells have low plasticity, although reports of differentiation outside traditional lineage boundaries suggest that some adult stem cells may be more pluripotent rather than multipotent.¹

Aside from plasticity, these differing stem cell types have very different ethical issues. Whereas derivation of stem cells from adult tissues is not contentious, the use of embryonic stem cells is complicated by the need to disrupt the early embryo's inner cell mass so that it can never be transferred into a woman's uterus to become a fetus. Although procurement of embryonic stem cells can be achieved through human embryos from in vitro fertilization clinics, a major attraction of the embryonic stem cell approach is the ability to generate autologous tissues for transplantation via therapeutic cloning or nuclear replacement. Scientists and the media have created definitions of adult and embryonic stem cells that provide stark contrasts and easily understood sound bites in a politicized and polarized debate.

These definitions overlook an important aspect of the debate: in considering adult stem cell plasticity, gender matters. That is, stem cells or tissues obtained from a woman who has been pregnant are likely to contain a mixture of her own cells and those of her fetuses. This chimeric cell population may have different biological properties than adult stem cells or an organ obtained from a nulliparous woman or from a man. Surprisingly, this concept has been overlooked in recent publications examining the long-term fate of stem cells transplanted with sex-mismatched bone marrow or solid organs. Pregnancy is a common major health event that influences the composition of the adult stem cell population in most women.

The idea that women become chimeras following pregnancy is not new. The term microchimerism was first used in 1977 by Liégeois et al² to describe the long-term retention of fetal cells in the marrow of postpartum female mice, in which cells that carried a paternally inherited cytogenetic marker were able to survive and proliferate without inducing graft-vs-host disease. In 1996, Bianchi et al³ extended this observation to humans by demonstrating the presence of fetal (male) CD34-positive hematopoietic stem cells in the peripheral blood of healthy, nontransfused women up to 27 years following the birth of their last son. Furthermore, peripheral blood mononuclear cell subsets of fetal origin have been detected in healthy parous women and parous women with scleroderma.⁴ In human pregnancies, fetal CD34-positive progenitors are transferred into the maternal circulation, from which they can be cultured years later.⁵ Male (fetal) CD34-positive cells were detected in the blood products of 48% of healthy parous female donors.⁶ Subsequent research, primarily using fluorescence in situ hybridization analysis with X and Y chromosome–specific probes, demonstrated the presence of fetal cells in paraffin-embedded tissue from the skin,⁷ thyroid,^{8 - 9} intestine,¹⁰ and liver¹¹ of women with and without disease. Thus, transfer and long-term persistence of fetal cells in parous women is a well-validated phenomenon.

To develop microchimerism, it is not necessary to continue a pregnancy and deliver an infant. Using polymerase chain reaction to measure Y-chromosomal sequences, up to 500 000 nucleated fetal cells enter a woman's circulation following routine surgical abortion.¹² Depending on when during pregnancy the termination is performed, a variable proportion of these fetal cells will be stem cells. Furthermore, in a meta-analysis of published articles that included reproductive information, a history of induced or spontaneous fetal loss was the major demographic factor associated with subsequent development of fetal cell microchimerism in the tissues of women.¹³ Blood microchimerism is more frequent and occurs at higher levels in women with a history of induced abortion compared with women with other pregnancy histories.¹⁴ It is also not necessary for the fetus to be male. It is easier to distinguish fetal from maternal cells using the Y chromosome as a biomarker, but any paternally inherited gene will also work. Female fetal cell microchimerism is as significant as male fetal cell microchimerism. In one report, DNA polymorphisms proved that fetal cells from her terminated fetus repopulated the liver of a woman with hepatitis C.¹⁵

Microchimeric fetal cells were first implicated in the etiopathogenesis of autoimmune disease.¹⁶ However, more recent studies with better controls have shown that fetal cell microchimerism occurs in healthy parous women^{14 ,17} as well as in women with nonautoimmune disease.¹⁵ Recently an alternative interpretation has developed: namely, that these pregnancy-associated progenitor cells may have a beneficial role and participate in the repair of maternal disease or injury.¹⁸ Male fetal cells, identified by the presence of an X and Y chromosome, are increased in number in maternal pathological tissue and are capable of expression of tissue-specific antigens, such as cytokeratin in thyroid or cervix and heppar-1 in liver.¹⁸ Furthermore, animal studies using rodent models confirm both the presence of increased fetal cells in maternal diseased tissue^{19 - 20} and the apparent ability of fetal cells to express antigens characteristic of the maternal host organ.^{21 - 22} How and when the fetal cells get to the affected tissues and whether they differentiate from hematopoietic or other stem cells that have crossed the placenta is currently unknown. Also unknown is whether fetal cells that remain in the mother retain fetal characteristics or whether they go on to differentiate, becoming more like adult cells.

O’Donoghue et al²³ cultured pure fetal mesenchymal stem cells from maternal blood after termination of pregnancy and differentiated them into bone and fat. These investigators then identified fetal mesenchymal stem cells in the sternal or rib marrow of all women who had delivered sons 13 to 51 years earlier,²⁴ in which the median ratio of fetal to maternal cells was 1:102 000. This is direct evidence that what would be conventionally considered an adult stem cell population is actually composed of fetal and adult stem cells. Although the ratios seem small, they are not different from those reported for microchimerism after stem cell transplant.²⁵

We tested the hypothesis that recent publications examining the long-term fate of stem cells transplanted into sex-mismatched recipients fail to consider fetal cell microchimerism as a source of sex-mismatched cells. Using the PubMed database, we performed a systematic literature review using the following filter: January 1, 2000, to December 31, 2006, human, English language, and the search terms stem cell or transplant, and gender, sex, or fluorescence in situ hybridization or Y chromosome and 1 or more of cardiac or heart, hepatic or liver, neurons or brain, renal or kidney, pulmonary or lung, and bone marrow. Each author independently reviewed all abstracts and selected pertinent abstracts for further review. The lists were combined and checked for duplication. From a final list of 106 publications, each author scrutinized 53 articles. (A list of these publications is available on request from the authors.)

Of the 106 articles, 48 were considered irrelevant because they either were case reports or reviews or were focused on graft-vs-host disease, tracking donor cells, or both following different bone marrow transplant regimens. Fifty-seven articles were relevant because they studied the fate of sex-mismatched bone marrow transplants (XY donor to XX recipient) and found XY-positive cells in other organs such as heart, kidney, or brain or they examined XX solid organs transplanted into XY recipients and followed the fate of XY-positive differentiated cells in the transplanted organ. A 58th article examined baseline autopsy data in 75 women with known sex history of living children; XY-positive chimeric cells were found in 23.¹⁷ None of the 58 studies reported complete reproductive history information (including termination and miscarriage) of the female donors or recipients. Five (8%) of 58 studies stated that the women had no history of male children, and 9 additional studies acknowledged the possibility of fetal cell microchimerism in their discussion but provided no data on reproductive history. This review of the current literature indicates either a reluctance to incorporate pregnancy history into study design or lack of understanding of its biological significance. Neither the official US National Institutes of Health Web site resource for information on stem cells (http://stemcells.nih.gov/info/basics) nor the International Society for Stem Cell Research Web site (http://www.isscr.org) mention the presence of naturally occurring fetal cells in the adult.

All sources of stem cells, including both embryonic stem and adult cells, should undergo rigorous study. It is completely unknown at present whether fetal stem cells in women who have been pregnant persist in sufficient quantity or retain their unique biological properties to represent a therapeutic alternative to embryonic stem cells. However, from a purely scientific perspective, all studies of adult stem cells that examine their subsequent fate after transplantation should include the complete pregnancy history of the donor, the recipient, or both, as appropriate. Even though obtaining a complete reproductive history, including early miscarriages and terminations, may be difficult, investigators need to consider the possibility that sex-mismatched cells could derive from the donor or recipient's fetus before concluding that fusion or transdifferentiation of transplanted cells has resulted. It is also particularly important in evaluating clinical outcomes of human stem cell trials to determine whether there are biologically significant differences when the stem cell donor or recipient is a woman who has been pregnant. There is no equality of the sexes in stem cell therapy because most women will always be more chimeric than men.