| Abstract: |
Embryonic stem (ES) cells have an indefinite capacity to multiply and are capable of generating all somatic cell lineages. They are thus viewed as a potentially universal and renewable tissue source for transplantation. In addition to ethical concerns, important biological questions remain to be solved, such as isogenicity and fate specification. In this work, we have generated 35 embryonic ES cell lines (ntES) from adult somatic cells (mouse tail fibroblasts or cumulus cells) via nuclear transfer. Thus each ntES line carries the exact genotype of the somatic cell donor. The cell lines could then be propagated in vitro in an undifferentiated state over an extended period of time, in a manner similar to wild-type ES cells. As a measure of their pluripotency, we differentiated them in vitro along endo-, meso- and ectodermal lineages. Cells immunoreactive for Troma-1 and alpha-fetoprotein (endoderm), actin, myosin and fibronectin (mesoderm) and nestin, PSA-NCAM and cytokeratin (ectoderm) could be readily detected. The therapeutic use of stem cells however depends on our ability to direct their fate into specific lineages and cell types lost due to disease. We therefore attempted to differentiate ntES cells into dopamine neurons, a very specialized cell type lost in Parkinson's disease. We obtained significant numbers of dopamine neurons from all seven lines tested, reaching a maximum of over 50% of total cell number. Synaptic dopamine release was confirmed by chromatography (HPLC). By combining ES and cloning technologies, this work demonstrates the feasibility of the first steps required for therapeutic cloning. |
