From left to right: Nestor Vicente-Salar, Beatriz Paredes, Juan Antonio Reig, María Isabel Arribas, Encarna Fuster and
Enrique Roche.

Undifferentiated Mouse Embryonic Stem Cells

The practically unlimited potential of embryonic stem (ES) cells in regenerative medicine is causing an important increase in the number of in vitro differentiation protocols being published, each promising an efficient and viable cell-replacement therapy. However, at present, there is no conclusive data concerning the composition and stage of differentiation of such cell populations, and some of them may be potentially dangerous after transplantation. To this end, the Oct4 promoter, a gene expressed in undifferentiated ES cells, was used in order to track residual undifferentiated ES cells that may survive in the cultures after standard procedures of differentiation in absence of leukaemia inhibitory factor (LIF). Mouse ES cells were stably transfected with a genetic construction containing the promoter, which regulated the expression of the enhanced green fluorescent protein. Green fluorescent “embryoid bodies” were cultured and residual fluorescent cells were further sorted and characterized. The possibility that these green fluorescent cells were differentiated primordial germ cells, which also express Oct4, was discarded since they expressed only slight levels of germ-specific markers, such as VASA or DAZL. The residual undifferentiated cell population can grow in vitro in absence of LIF, displaying high levels of Oct4. When transplanted into nude mice, teratomas were apparent after three months. Under microscopic analysis, ectoderm, mesoderm and endoderm cell types could be distinguished in the tumours, demonstrating the pluripotentiality of these residual cells in vivo. Karyotyping showed an additional copy of chromosomes 8 and 9 in the majority of the cells. In conclusion, this study demonstrates that during standard culture protocols that lead to differentiated cells, a residual population of undifferentiated ES cells can spontaneously acquire a recurrent abnormal karyotype, which may promote an undifferentiated stage in vitro but with the potential to induce teratomas in vivo. A control of ES cell cultures after standard differentiation protocols must be considered after these results. Roberto Ensenat-Waser, Alfredo Santana, Nestor Vicente-Salar, Juan C. Cigudosa, Enrique Roche, Bernat Soria, and Juan A. Reig. Isolation and Characterization of Residual Undifferentiated Mouse Embryonic Stem Cells from Embryoid Body Cultures by Fluorescence Tracking, In Vitro Cellular & Developmental Biology – Animal, 42:115-123, 2006.

Michel A. Horisberger

Prolonged Survival of Primary Cell Lines

Primary cell lines are of great interest for research, for drug discovery and development; they are used in a range of quality control and analytical assays. The present study was initially a part of a larger project of Aivogen focused on the identification of external stimuli capable to maintain prolonged survival of primary cell cultures. An obvious advantage of this approach is the avoidance of transfection with viral carriers or the use of mutagens, as used in standard immortalisation procedures. It is notorious that primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. We have found that addition of a complex of growth promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. One of these cell lines will soon be available from ECACC. The finding has been extended to other types of cells. It is important to mention that the survival effect is reversible since cells degenerate when the supplement is removed. We wish to reveal the full unexploited potential of processed follicular fluid as an alternative or as a complement to animal serum for optimising culture conditions for cell survival and proliferation.Michel A. Horisberger. A Method for Prolonged Survival of Primary Cell Lines, In Vitro Cellular & Developmental Biology – Animal,42:143-148, 2006.