[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?]. / Embryonale Stammzellen - ein wissenschaftliches Nebenprodukt der assistierten Reproduktionsmedizin?

The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).

Gene expression profiles of similarly derived human embryonic stem cell lines correlate with their distinct propensity to exit stemness and their different differentiation behavior in culture.

Four normal-karyotype human embryonic stem cell (hESC) lines were generated using the same protocol and maintained under identical conditions. Despite these precautions, gene expression patterns were found to be dissimilar among the four lines. The observed differences were typical of each cell line, correlated with their distinct propensity to exit stemness, created heterogeneity among the cells during cell line maintenance, and correlated with their altered capacity as a source of differentiated cells. The capacity of some cell lines to give rise to more, and more mature, neurons within comparable time frames of directed differentiation reflected the distinct proportions of cells already predifferentiated at the onset. These findings demonstrate that the subsequent stages of neural differentiation were altered both in a quantitative and timely fashion. As a consequence, cell lines with apparent better and quicker ability to produce neurons were actually the less capable of reproducing proper differentiation. Previous data suggested that cell lines able to generate more neurons faster would be more suitable to clinical application. Our analysis of the differentiation process strongly suggests the opposite. The spontaneous tendency to predifferentiate of any particular hESC line should be known because it clearly impacts further experimental results.

First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa.

QUESTION UNDER STUDY: The feasibility and the potential advantages of separating X-chromosome bearing spermatozoa for the prevention of a severe X-chromosome linked disorder with the use of intracytoplasmic sperm injection are presented. METHOD: A carrier of muscular dystrophy type Becker was treated with intracytoplasmic sperm injection, using spermatozoa previously stained with the Hoechst dye 33342 and sorted with flow cytometry. RESULTS: After transfer of one single blastocyst, an intrauterine pregnancy arose. In the ninth week of gestation, the female sex of the embryo was confirmed with proof of absence of the SRY gene of the Y-chromosome. After normal pregnancy, the patient delivered a healthy daughter. CONCLUSIONS: The staining of spermatozoa with specific markers and sorting with flow cytometry provides a means of preventing significant disease in the offspring and may help in reducing the number of surplus embryos needed for preimplantation genetic diagnosis.

Evaluation of in vitro cultured rat oocytes, from different strains, by spindle morphology and maturation-promoting-factor activity combined with nuclear-transfer experiments.

Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus.

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport.

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.