Multilineage potential of homozygous stem cells derived from metaphase II oocytes.

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.

Amplification and overexpression of mutant RET in multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma.

We have previously identified two second hit mechanisms involved in the development of multiple endocrine neoplasia type 2 (MEN 2)-associated tumors: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type RET allele. However, some of the MEN 2-associated tumors investigated did not demonstrate either mechanism. Here, we studied the TT cell line derived from MEN 2-associated medullary thyroid carcinoma with a RET germline mutation in codon 634, for alternative mechanisms of tumorigenesis. Although we observed a 2:1 ratio between mutant and wild-type RET at the genomic DNA level in this cell line, fluorescence in situ hybridization analysis revealed neither trisomy 10 nor loss of the normal chromosome 10. Instead, a tandem duplication event was responsible for amplification of mutant RET. In further studies we could for the first time demonstrate that the genomic chromosome 10 abnormalities in this cell line cause an increased production of mutant RET mRNA. These findings provide evidence for a third second hit mechanism resulting in overrepresentation and overexpression of mutant RET in MEN 2-associated tumors.

Somatic point mutation of the wild-type allele detected in tumors of patients with VHL germline deletion.

The majority of patients with Von Hippel-Lindau (VHL) disease are affected by a VHL germline mutation involving one copy of the VHL gene. Loss of heterozygosity of the second VHL allele can be consistently demonstrated in tumor tissue from these patients, suggesting that allelic deletion is a very early or even initiating event for tumorigenesis. Approximately 20% of VHL disease patients, however, exhibit germline deletion of one entire copy or at least a substantial part of the VHL gene. To investigate the nature of the "second genetic hit" in this patient population, we analysed two renal cell carcinomas and one CNS hemangioblastoma from three unrelated patients for genetic changes of the second copy of the VHL gene. All three tumors showed retention of one VHL allele by FISH. Single-strand conformation polymorphism and mutation analysis of microdissected tumor DNA revealed somatic point mutations of the wild-type VHL copies in each of the three tumors. The results indicate that the "two hit model" is equally applicable to patients with VHL germline mutation and VHL germline deletion. In contrast to tumors from patients with VHL germline mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion.

Somatic VHL gene deletion and point mutation in MEN 2A-associated pheochromocytoma.

Multiple endocrine neoplasia type 2 (MEN 2) is an inherited cancer syndrome that includes pheochromocytoma. Germline mutations in RET are responsible for MEN 2 but the precise pathogenetic mechanisms of tumorigenesis are unknown. We have recently identified possible mechanisms of tumor formation in patients with MEN 2A-related pheochromocytoma. Two of nine tumors investigated, however, did not reveal either of these mechanisms. In the present study, we therefore searched for other possible mechanisms underlying the pathogenesis of MEN 2A-related pheochromocytoma. Hereditary pheochromocytoma also occurs in patients with von Hippel-Lindau (VHL) disease, a syndrome consisting of tumors caused by inactivation of the VHL tumor suppressor gene. A subset of sporadic pheochromocytomas have somatic mutations in RET or VHL, suggesting that both genes contribute to pheochromocytoma pathogenesis in a subset of tumors. It is unknown, however, whether VHL gene alterations would be associated with tumorigenesis in hereditary, MEN 2-related pheochromocytoma. We therefore investigated four pheochromocytomas from patients with MEN 2A and RET germline mutations for the presence of allelic deletion and/or somatic mutation of the VHL gene. LOH analysis using the polymorphic markers D3S1038 and D3S1110 that map to the VHL gene locus 3p25/26, revealed evidence for somatic VHL gene deletion in all four MEN 2A-related pheochromocytomas. Mutation analysis of the VHL gene showed frameshift mutations in two tumors and a splice acceptor mutation in one tumor. The remaining tumor did show LOH but not mutation of the VHL gene. These results suggest that somatic genetic alterations of the VHL gene may play a role in the tumorigenesis of some MEN 2A-related pheochromocytomas.