Transgenic mice produced by retroviral transduction of male germ-line stem cells.

Male germ-line stem cells are the only cell type in postnatal mammals that have the capability to self-renew and to contribute genes to the next generation. Genetic modification of these cells would provide an opportunity to study the biology of their complex self-renewal and differentiation processes, as well as enable the generation of transgenic animals in a wide range of species. Although retroviral vectors have been used as an efficient method to introduce genes into a variety of cell types, postnatal male germ-line stem cells have seemed refractory to direct infection by these viruses. In addition, expression of genes transduced into several types of stem cells, such as embryonic or hematopoietic, is often attenuated or silenced. We demonstrate here that in vitro retroviral-mediated gene delivery into spermatogonial stem cells of both adult and immature mice results in stable integration and expression of a transgene in 2-20% of stem cells. After transplantation of the transduced stem cells into the testes of infertile recipient mice, approximately 4.5% of progeny from these males are transgenic, and the transgene is transmitted to and expressed in subsequent generations. Therefore, there is no intrinsic barrier to retroviral transduction in this stem cell, and transgene expression is not extinguished after transmission to progeny.

Expanded indications for transcervical thymectomy in the management of anterior mediastinal masses.

BACKGROUND: Transcervical thymectomy (TCT) is an accepted though controversial approach for thymectomy in myasthenia gravis (MG). The suggestion of thymoma on computed tomography (CT) has been considered a contraindication to TCT. We sought to determine whether the indications for TCT could be safely expanded to include selected patients with thymomas as well as other types of anterior mediastinal masses. METHODS: Between January 1992 and September 1999, we performed 121 TCTs: 98 in patients with MG and 23 in patients without MG. The patients' records were retrospectively reviewed. RESULTS: Among the 98 MG patients, 28 had CT scans suspicious for thymoma. Of these, 14 had a thymoma pathologically. These were classified as stage I (5), stage II (8), and stage III (1). Five patients required extension of the incision for completion of the procedure. There have been no thymoma recurrences to date with a mean follow-up of 48 months (range 3 to 96 months). In the 23 patients without MG, 12 had new anterior mediastinal masses, 4 had a history of treated lymphoma, 1 had a history of treated germ cell tumor, and 6 had suspected mediastinal parathyroid adenoma. Diagnostic tissue was obtained in all patients undergoing the procedure for diagnosis, and in 4 of 6 patients, a parathyroid adenoma was successfully resected. CONCLUSIONS: Transcervical exploration and thymectomy offers a less invasive approach to the diagnosis and/or definitive treatment of selected anterior mediastinal masses. We suggest that it is appropriate to expand its use to several clinical scenarios beyond the typical indication of thymectomy in MG patients without thymoma.

Culture of mouse spermatogonial stem cells.

Spermatogenesis occurs within the seminiferous tubules of mammals by a complex process that is highly organized, extremely efficient and very productive. At the foundation of this process is the spermatogonial stem cell that is capable of both self-renewal and production of progeny cells, which undergo differentiation over a period of weeks to months in order to generate mature spermatozoa. It had been thought that germ cells survive only a brief period in culture, generally less than a few weeks. However, an accurate assessment of the presence of spermatogonial stem cells in any cell population has only recently become possible with development of the spermatogonial transplantation technique. Using this technique, we have demonstrated that mouse spermatogonial stem cells can be maintained in culture for approximately 4 months and will generate spermatogenesis following transplantation to the seminiferous tubules of an appropriate recipient. Extensive areas of cultured donor cell-derived spermatogenesis are generated in the host, and production of mature spermatozoa occurs. Cultivation of the testis cells on STO feeders is beneficial to stem cell survival. These results provide the first step in establishing a system that will permit spermatogonial stem cells to be cultivated and their number increased in vitro to allow for genetic modification before transplantation to a recipient testis.

Reconstitution of spermatogenesis from frozen spermatogonial stem cells.

Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at -196 degrees C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal.