Human Chimerism

Human chimerism is a condition characterized by the presence of cells from two or more unique zygotes within an individual, which can occur spontaneously or through artificial means. Natural human chimeras can arise during pregnancy through the exchange of cells between fetus and mother or through genetic fusion between twins. This leads to the emergence of new chimeric genes and phenotypic traits, including skin pigmentation and eye color variation. Identification of natural chimeras involves various techniques such as blood typing, karyotyping, polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH).

Artificial human chimeras result from the introduction of donor cells into the recipient body, typically through tissue or organ transplantations. This can be an intentional process to create man-made allograft or xenograft transplantations, as well as in vitro fertilization (IVF)-related chimeras. Alternatively, chimeras can arise to create iatrogenic chimeras. Detection methods include semi-quantitative molecular DNA tests such as short tandem repeats (STR) analysis.

Both natural and artificial human chimerism give rise to health and ethical implications.

Origins
The term chimera originates from ancient Greek mythology, describing a mythical creature combining features of a lion head, goat body and serpent tail. Chimeras have become part of contemporary scientific and medical discourse, particularly within the fields of biology and genetics, dating back nearly seven decades ago. In the scientific context, human chimeras represent entities composed of cells from different sources. Once considered rare, recent research indicates human chimeras are more prevalent than previously believed. This is due to the challenges in detection when natural human chimeras exhibit entirely normal phenotypes, necessitating genetic testing to reveal underlying differences.

Natural human chimeras
Natural human chimeras are humans who are born with cell populations from more than one zygote. Majority of cases of naturally-occurring chimeras have previously gone undetected, especially for chimeras with zygotes of the same sex. However, greater public attention and recent developments in genomic technologies have contributed to increasing identification of this phenomenon. Natural human chimerism occurs through several means and can be categorized into three groups: microchimerism, fusion chimerism and twin chimerism.

Microchimeras
Microchimerism is the presence of a minor cell population accounting for <1% of the total cell population in an individual. This small number of cells is distinct from the host’s cells and derived from a different individual. Microchimerism has been observed to arise during pregnancy. The process of microchimerism can occur bidirectionally, creating microchimerism in both the mother or child. This is known as fetal microchimerism and maternal microchimerism respectively. The bidirectional transfer of maternal and fetal cells is asymmetrical, where a larger number of fetal cells are transferred from mother to child.

Fetal microchimerism, the most common form of natural human chimerism, involves fetal cells crossing the placenta and integrating into maternal organs. The persistence of fetal cells in the mother's body postpartum has been found to last up to 27 years. Maternal microchimerism is the transfer of maternal cells into the fetus, which persists throughout childhood and adult life.

Fetomaternal microchimerism and the presence of fetal cells in pregnant women have been utilized in the development of the non-invasive prenatal test (NIPT). This diagnostic test utilizes cell-free fetal DNA (cffDNA) in maternal blood to screen for genetic diseases and abnormalities during pregnancy.

Fusion chimeras
Fusion chimerism involves the fusion of two or more zygotes in an early embryonic state. This results in an individual containing cell lineages from both zygotes. Fusion chimerism differs from mosaicism, where multiple distinct cell lines arise from a single zygote due to genetic mutations.

Various mechanisms for fusion chimeras have been proposed, including tetragametic chimerism and parthenogenetic (trigametic) chimerism. Tetragametic chimeras predominately originate from the fusion of two fertilized zygotes. Less commonly, tetragametic chimerism involves the fertilization of a second polar body. Subsequent fusion of the fertilized second polar body with a fertilized ovum gives rise to a tetragametic chimera. Parthenogenetic or trigametic chimerism involves human parthenogenesis, where two daughter cells arise from the parthenogenetic division of an oocyte and are fertilized by two different sperm.

Twin chimeras
Twin chimeras involve the exchange of genetic material and cells between zygotes without undergoing fusion. Blood group chimerism is a type of twin chimerism where an individual carries multiple blood group types. The first case of blood group chimerism was documented in 1953, due to blood group typing discrepancies caused by chimerism. The individual was found to have both A and O blood groups, presumed to be due to twin-related chimerism. This type of chimerism is a result of placental anastomoses or connections of blood vessels between dizygotic twins. These connections enable the exchange of bone marrow and blood cells. The exchanged cells establish themselves in the recipient twin, allowing the chimeric state to occur. Blood chimerism occurs in 8% of dizygotic twins and increases to 21% in triplets. The occurrence of blood chimerism is more likely with in vitro fertilization.

Identification
Phenotypic traits of chimerism can include a variation of skin pigmentation and iris color. However, there is a lack of readily observable phenotypic characteristics in fusion chimeras which would indicate the chimeric nature of the individual.

Various quantitative techniques have been utilized for the identification of natural human chimeras. Many cases of natural chimerism have been serendipitously discovered through various tests such as blood group typing. The primary qualitative methods include blood group typing, karyotyping, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH).

Blood typing
Natural human chimerism was first identified through routine blood typing, which resulted in the discovery of mixed red blood cell populations. Traditional blood group typing involves the determination of blood type based on agglutination reactions between specific antibodies and the antigens present in an individual's blood sample. Multiple agglutination reactions in a blood typing test reveal the presence of more than one blood cell population, indicating a state of chimerism. Column agglutination technology is another standard blood group typing technique that separates agglutinated cells from non-agglutinated cells by filtration. This method standardizes and quantifies red blood cell agglutination by the strength of the reaction.

Karyotyping
Karyotyping is a form of cytogenetic analysis used to detect numerical and structural chromosomal abnormalities. Karyotyping as a method for chimera identification is limited to chimerism with major differences between the karyotypes of individual cells. This includes variations in the number and type of sex chromosomes. Karyotyping can be employed to detect sex-discordant chimerism, where a 46,XX/46,XY karyotype is observed. However, this technique fails to detect chimerism in cases where major chromosomal variation is absent. In such cases, more sophisticated molecular testing methods such as PCR and FISH are required for accurate assessment of chimerism.

Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) utilizes the principle of DNA replication to produce specific DNA regions of interest, enabling the detection by techniques such as gel electrophoresis. This widely used method can effectively identify chimerism with human genomic DNA due to cost-effectiveness, particularly for detecting microchimerism. The critical step in PCR involves examining unique target genes within the chimera cell population or differences in polymorphisms between minor cell populations and host cells. For instance, the Y-chromosome gene TSPY1 is commonly used to target microchimerism in women. Another PCR approach for chimerism detection involves the use of polymorphic short tandem repeats (STR).

Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a cytogenetics technique that localizes donor cells at the individual cell level in the background of host cells. This technique employs labeled nucleotide probes that hybridize with specific genomic sequences, allowing the detection of fluorescent signals. FISH is used in chimerism biology to identify the localization of non-host cells within tissue samples.

Man-made chimeras
Man-made or “therapeutic” human chimeras arise from tissue or organ transplants, leading to the presence of two or more blood or tissue donor populations in the recipient's body. This condition creates an environment where donor and recipient cells from transplantation compete to survive, potentially resulting in one cell population outcompeting another over time.

Xenotransplant chimeras
Xenogeneic transplant is a procedure that involves the infusion of non-human animal sources into live human cells, tissues or organs, creating a human-animal xenotransplant chimera. For example, an interspecies chimera such as a human-pig chimera can be created by generating humanized hearts in humans from gene-edited pigs. This same technology would apply to other organ sources for transplantation.

Iatrogenic transplant chimeras
Iatrogenic transplant chimeras arise from an unintended allogeneic transplantation. It is commonly observed in multiply-transfused trauma patients who require a significant amount of blood transfusion. Research indicates that these types of patients would accept donor granulocytes and lymphocytes despite leukoreduction. The donated cells are thus able to persist for decades and become “transfusion-associated microchimeras”.

IVF chimeras
IVF-associated chimeras are created through assisted reproductive technologies known as in vitro fertilization (IVF). During IVF, two or more embryos are transferred into one uterus to increase the chance of successful pregnancy.

During IVF, the human oocyte is carefully selected from the follicle in the ovary to be fertilized. Next, the chosen oocyte is fertilized with sperms that have been prepared. Finally, two or more fertilized embryos are implanted into the uterus endometrium within five days post-fertilization.

The consequence of IVF is the increased likelihood of monozygotic and dizygotic twinning, leading to IVF chimeras also known as twin chimeras. Another potential chimeric outcome is “singletons”, which refers to fusion chimeras or surviving twin chimeras that acquire genetic material from a sibling that perished in the uterus. This is known as the “vanishing twin syndrome”, where the vanished twin disappears in utero. This phenomenon can also be examined in postpartum with the placenta as evidence of the “vanished twin”.

Identification
The methods for the identification of artificial human chimeras are similar to those used for natural human chimeras, though qualitative methods are insufficient. Early detection of artificial human chimerism is important for transplantation procedures, allowing for timely intervention towards potential complications. Patients can undergo monitoring using semiquantitative molecular DNA tests of their blood short tandem repeat (STR) loci. These tests can estimate the proportion of donor and recipient white blood cell populations. The logic lies in the equivalence of DNA quantity between that of a patient and their donor cells. Therefore, the patient-to-donor cell ratio mirrors their respective DNA ratio, a relationship maintained after post-PCR amplification. The resulting amplified DNA (STR alleles) give insights into the patient’s cell proportion relative to donor cells.

Health implications
There are potential risks and benefits associated with various types of human chimerism. Microchimerism has been extensively investigated for its potential role in human diseases. Fetal microchimerism is associated with autoimmune diseases including scleroderma, but may also enhance maternal health through involvement in the repair of maternal injuries. Studies have also associated the “vanishing twin syndrome” with an increased risk of congenital malformations in the surviving twin.

Ethical implications
Ethical considerations surrounding human chimerism predominantly revolve around cases of artificial human chimerism. The earliest discussions of bioethical issues stem from xenotransplantation practices. This procedure involves the transplantation of non-human animal cells, tissues or organs into humans. Concerns include the harm inflicted upon the donor and the potential health risks posed to the recipient. Proponents argue from a moral standpoint that such practices are beneficial, offering a means to alleviate human organ shortage by utilizing non-human animals as organ sources to save human lives. However, ethical dilemmas arise due to the risks to human health posed by the xenogenic transplant procedures, alongside concerns for animal welfare and challenges related to obtaining informed consent.