No abstract

Abstract. Somatic cell nuclear transfer, the first established technique for producing patient-specific autologous stem cells, inevitably requires the sacrifice of viable embryos. To circumvent the serious ethical issues associated with this use of embryos, researchers have developed several alternative methods for the production of histocompatible stem cells. In our research, we have used two methods to derive histocompatible stem cells from murine ovarian tissue. First, we have established autologous stem cells by culturing degeneration-fated preantral follicles to produce developmentally competent, mature oocytes and then parthenogenetically activating these mature oocytes to acquire genetic homogeneity. Second, we have used cell-to-cell interactions to derive stem cells from ovarian stromal cells without undertaking genetic modification. We have successfully derived autologous murine stem cells by manipulating primary and early secondary follicles in vitro, and this method has proved successful even for follicles retrieved from aged ovaries. Furthermore, we believe that it will be possible to isolate stem cells directly from nongermline ovarian tissue or to derive stem cells by culturing the ovarian cells with other somatic cells. If achieved, these aims will greatly advance the development of induced pluripotent stem cell technology, as well as tissue-specific stem cell research. In this review, we introduce the relevant technologies for establishing histocompatible stem cells from ovarian tissue cells without undertaking genetic manipulation and review the current limitations of, and future research directions in, stem cell biology. Key words: Autologous, Ovarian cells, Parthenogenesis, Preantral follicle, Stem cell (J. Reprod. Dev. 56: [481][482][483][484][485][486][487][488][489][490][491][492][493][494] 2010) luripotent stem cells are widely recognized as critical to the success of certain cutting-edge medical technologies, such as cell replacement, organ regeneration, tissue engineering, and rejuvenation therapy [1][2][3]. However, the potential clinical practicability of stem-cell-based techniques is limited by the absolute requirement for patient-stem cell histocompatibility or patient acquisition of immune tolerance [4]. Another obstacle to the use of stem cells is the current lack of effective techniques for regulating cell-to-cell interactions and for inducing target differentiation of stem cells. The future success of tissue regeneration therapies will also depend on the optimization of stem-cell transplantation techniques and the development of strategies for inducing transplanted stem cells to resume their normal functions. In addition, all stem cell-based procedures must be carried out in an ethically acceptable manner.Efforts to develop stem-cell-based techniques such as those described above have been extensive. In our research, we have consistently attempted to develop novel methodologies for the establishment of histocompatible stem cells. At a very early stage in stem cell research, a ...

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.

This study was conducted to improve efficiency of a follicle culture system without reducing developmental competence of intrafollicular oocytes. Preantral follicles (100 to 125 µm in diameter) of F1 hybrid (B6CBAF1) mice were cultured singly for 216 h in modified α-MEM-glutamax medium, to which 2.5 IU/ml hCG and epidermal growth factor was added 16 h prior to the end of culture. Medium change was either performed three times (54 h interval), twice (72 h interval), once (108 h interval), or not at all (216 h interval). Maturation (progression to the metaphase II stage) of intrafollicular oocytes was detected from 4 days after culture in the three-times change treatment, while all treatments yielded mature oocytes from day 5 of culture. Compared with the three-times change, decreasing the change frequency to once did not reduce the capacity to begin maturation (germinal vesicle breakdown of 82 to 86%), to mature (78 to 79%) and to develop into blastocysts after parthenogenetic activation (29 to 32%). Morphological parameters were similar among these treatments. Except for the no medium change treatment, similar colony-forming activity of inner cell mass cells after culturing of blastocysts in leukemia inhibitory factor-containing medium was detected, while the morphology of the colonyforming cells deteriorated in the change-once treatment compared with the change twice or three-times. In conclusion, the efficiency of the preantral follicle culture system could be improved by reducing frequency of medium change up to a 72 h interval (three times in total 216 h culture) without decreasing developmental competence of oocytes.

No abstract