Clayton J. Brinster

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.

Maintenance of Mouse Male Germ Line Stem Cells In Vitro

Abstract The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. We analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. Allthough the number of adult and immature SSCs decreased in a time-dependent manner, nearly one in four stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or utilization of specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines and addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained when cultured with TM4 cells or activin A, and 6% remained when cultured with SF7 cells or BMP4. These results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.

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.

Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation

Abstract Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.

Quantitative mitral valve modeling using real-time three-dimensional echocardiography: technique and repeatability.

BACKGROUND Real-time three-dimensional (3D) echocardiography has the ability to construct quantitative models of the mitral valve (MV). Imaging and modeling algorithms rely on operator interpretation of raw images and may be subject to observer-dependent variability. We describe a comprehensive analysis technique to generate high-resolution 3D MV models and examine interoperator and intraoperator repeatability in humans. METHODS Patients with normal MVs were imaged using intraoperative transesophageal real-time 3D echocardiography. The annulus and leaflets were manually segmented using a TomTec Echo-View workstation. The resultant annular and leaflet point cloud was used to generate fully quantitative 3D MV models using custom Matlab algorithms. Eight images were subjected to analysis by two independent observers. Two sequential images were acquired for 6 patients and analyzed by the same observer. Each pair of annular tracings was compared with respect to conventional variables and by calculating the mean absolute distance between paired renderings. To compare leaflets, MV models were aligned so as to minimize their sum of squares difference, and their mean absolute difference was measured. RESULTS Mean absolute annular and leaflet distance was 2.4±0.8 and 0.6±0.2 mm for the interobserver and 1.5±0.6 and 0.5±0.2 mm for the intraobserver comparisons, respectively. There was less than 10% variation in annular variables between comparisons. CONCLUSIONS These techniques generate high-resolution, quantitative 3D models of the MV and can be used consistently to image the human MV with very small interoperator and intraoperator variability. These data lay the framework for reliable and comprehensive noninvasive modeling of the normal and diseased MV.