Unfertilized Egg Cells Yield Stem Cells Promising for Tissue Transplantations

Parthenogenetic embryonic stem cells (pES)—those derived exclusively from oocytes—may provide a more technically efficient and less ethically challenging method for generating embryonic stem cells to use in tissue transplants. HMS researchers, led by research fellow Kitai Kim and George Daley, HMS associate professor of biological chemistry and molecular pharmacology and associate director of the Children’s Hospital Boston Stem Cell Program, reported the findings online in Science on Dec. 14. Their study yielded pES cells that were both pluripotent and capable of engrafting into immunocompetent mice.

The possibility of generating tissue for transplantation has been one of stem cell research’s greatest promises. To fulfill this need, embryonic stem cells have to meet two requirements: they must be able to differentiate into a vast array of tissue types and to elude detection by the recipient’s immune system. Cells that accomplish the latter—those whose major histocompatibility complex (MHC) genes match the host’s—could mitigate the need to suppress the host’s immune system, a routine aspect of current transplant procedures, required to prevent transplant rejection. So far, only one method can generate stem cells that meet both needs.

As its name implies, somatic cell nuclear transfer (SCNT) involves transferring the nucleus from a somatic cell into an egg from which the nucleus has been removed. Ideally, this gives rise to a blastocyst that is genetically identical to the somatic cell donor. Ethical battles aside, SCNT has proven finicky at best, with some researchers reporting success rates as low as 1 percent. According to the recent study, parthenogenically derived embryonic stem cells could surpass this dismal statistic: Kim reports a success rate of almost 70 percent generating pES cells.

Kim, Daley, and their colleagues derived pES cells from unfertilized egg cells by arresting the first or second meiotic metaphase. After recombination and inheritance of the relevant sister chromatids, 81 percent of the embryonic stem cell genome—including the MHC loci responsible for tissue matching—was genetically identical to the oocyte donor genome. In immunodeficient mice, all transplants were accepted. In immunocompetent strains, MHC-homozygous mice accepted only MHC­homozygous embryonic stem cells, rejecting MHC­homozygous tissue that expressed only half the mice’s MHC antigens. MHC-heterozygous mice, on the other hand, accepted both exact MHC matches and MHC-homozygous donor cells. All pES cells demonstrated levels of multilineage tissue differentiation comparable to that found in embryonic stem cells derived from fertilized embryos.

Because mammalian embryonic development requires expression of a paternal genome, pES cells lack the potential to develop into a full organism. This could alleviate at least some concerns of those opposed to human cloning and those who want to protect any cells with the potential to develop into a full organism.

At the same time, lack of a paternal imprint presents its own set of obstacles. Some paternally imprinted genes, for example, are believed to have tumor suppressor activity, and there is no telling how tissue with two copies of maternally imprinted genes would function in the long term. Another caveat is that because pES cells are derived from oocytes, only females would benefit from any potential therapy, possibly giving rise to a new set of ethical dilemmas. Furthermore, some tissue types, especially bone marrow, could be subject to hybrid resistance, a phenomenon in which the host natural killer (NK) cells of MHC-heterozygous recipients reject transplanted tissues that lack both sets of MHC antigens.

Still, the new technique comes with its own advantages. Unlike embryonic stem cells generated via SCNT, all pES cells retain the mitochondrial genome of their oocyte donor and can therefore avoid immunologic rejection from antigens encoded by the mitochondrial genome. Although the authors point out that the long-term success of tissue engrafted from pES cells remains to be seen, they suggest that a cell bank of pES cells homozygous for the major MHC haplotypes could one day serve as a source of transplantable tissues.

—Jeneen Interlandi

Thymus Renewed When Transplanted from Older to Younger Animals

An aged thymus undergoes rejuvenation when transplanted into a juvenile animal and induces tolerance to organ transplants from the same donor, according to a study by Shuji Nobori, a postdoctoral fellow, and Kazuhiko Yamada, an HMS associate professor of surgery in the Transplantation Biology Research Center at Massachusetts General Hospital. The study was published Dec. 12 in Proceedings of the National Academy of Sciences.

The researchers carried out the transplantation experiments in miniature swine that bear immunological and physiological similarities to humans. When four-month-old animals were transplanted with vascularized thymic lobes from 20-month-old animals, the thymuses underwent structural and functional renewal, causing them to resemble their counterparts from young animals. When the experimental animals underwent a renal transplant after the vascularized thymic grafts were rejuvenated, they accepted the donor-matched grafts without further immunosuppression, indicating tolerance had been induced. The researchers observed these effects across differing levels of major histocompatibility complex (MHC) incompatibility. The vascularization of the thymic lobes prior to transplantation ensured immediate reversion to function inside the host animal.

An important observation of this study is that thymic rejuvenation is dependent on external stimuli within the host animal and not on the local environment within thymic tissue. In an effort to extend this finding, the researchers are involved in elucidating the stimuli from the host environment that induce rejuvenation.

The thymus has a central role in teaching thymocytes to differentiate between self- and nonself-antigens. The organ presents a vast repertoire of antigens to the immature cells, which arrive from the bone marrow. Through positive selection, T cells that recognize nonself-antigens continue to mature and pass into peripheral circulation; those that react with self-antigens are deleted through negative selection. Yamada explains that recognition of donor antigens as self-antigens within the thymus may facilitate their subsequent acceptance by host T cells, ensuring survival of the transplant.

This study suggests advantages over current methods of transplantation. Patients who now receive organ transplants need to be on immunosuppressive drugs for life. But transplantation of the donor thymus prior to the organ transplant may obviate this need. Furthermore, xenogeneic transplants, which traditionally are harder for the recipient to accept than allogeneic procedures, may also be more easily accepted if accompanied by thymic transplants from the donor animals. This would also circumvent the shortage of transplant organs from humans.

—Amita Joshi

Mutation Linked to Genetic Disorder Sheds Light on Heart and Blood Pathologies

Roughly 20 percent of Noonan syndrome cases have now been linked to missense mutations in the SOS1 gene, which encodes a critical component of the RAS/ERK pathway. The findings, reported in the January Nature Genetics, are helping tease apart differences in symptomatology, the range and severity of which can vary greatly among individual patients.

Noonan syndrome—a genetic disease characterized by facial abnormalities, short stature, learning disabilities, and congenital heart malformation—occurs in one out of every 1,000 to 2,500 live births. The condition predisposes its sufferers to leukemia. Previous studies linked 50 percent of syndrome cases to mutations in PTPN11, the gene encoding SHP2, a tyrosine phosphatase required to activate the RAS/ERK cascade. This gain-of-function mutation leads to enhanced activation of ERK, a widely expressed kinase involved in innumerable cellular functions, including mitosis and meiosis.

Although causes of the remaining 50 percent of cases were unknown, evidence suggested that other contributing mutations might also be found in proteins of the MAPK/ERK signaling pathway. Benjamin Neel, the William Bosworth Castle professor of medicine at at Beth Israel Deaconess Medical Center; Amy Roberts, HMS instructor in pediatrics at Children’s Hospital Boston and the HMS–Partners Center for Genetics and Genomics; Raju Kucherlapati, the Paul C. Cabot professor of genetics at HMS and the scientific director of the HMS–Partners center; and colleagues investigated 91 patients with a confirmed diagnosis of Noonan syndrome. Thirteen patients who did not have the known PTPN11 mutation expressed one of nine novel missense mutations in SOS1. None of these autosomal dominant alleles were found in the 188 chromosomes the group examined from non-affected individuals.

SOS1 encodes RAS-GEF, the protein that catalyzes the exchange of GDP with GTP on RAS proteins, enabling them to activate several downstream effectors of the MAPK/ERK pathway. Like the previously identified PTPN11 mutation, Noonan syndrome–associated SOS1 mutations are hypermorphs whose products enhance RAS and ERK activation. The SOS1 mutations, however, provide the first example of activating GEF mutations tied to human disease. The results also implicate SOS1 as a potential human proto-oncogene, given the increased risk of certain leukemias, especially juvenile myelomonocytic leukemia, faced by Noonan syndrome patients. Somatic mutations of PTPN11 have been found in 60 percent of sporadic cases of this childhood disease, and researchers speculate that a similar correlation will be revealed for the newly discovered mutations.

Since uncovering the PTPN11 mutation in 2001, researchers have worked to understand the process by which this genetic defect incites the pathology associated with Noonan syndrome. Although they point out that a larger cohort needs to be analyzed, the authors suggest that the level of ERK hyperactivation may be a key determinant of at least one major symptom. Atrial septal defect—a congenital heart disease that allows blood to travel from the left to the right side of the heart, mixing atrial and venous blood—increased with ERK overexpression and was more common in Noonan syndrome caused by PTPN11 mutations. At the same time, pulmonic stenosis—a condition in which blood flow from the right ventricle to the lungs is obstructed and another common feature of Noonan syndrome—was more frequent in syndrome patients with SOS1 mutations than in those without SOS1 or PTPN11 mutations.

Four Noonan syndrome cases associated with SOS1 mutations were believed to be familial and nine sporadic. Researchers hope this find will aid in prenatal diagnosis and genetic counseling for the disorder.

—Jeneen Interlandi

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