The Question of Donor Cell State

One theoretical uncertainty that besets all cloning studies is the precise phenotypic state of the donor cells from which the rare surviving clones are obtained. Typically, adult or fetal tissues are dissociated into single-cell suspensions. Various enrichment procedures might be employed to favor isolation of a particular cell type. The operator then selects from that suspension cells of a particular type. For example, the operator might select the smallest size cells present, presumably because such cells should be in the G1 stage of the cell cycle, if MII stage oocytes are employed as recipients and polar body extrusion is prevented during oocyte activation. Alternatively, G2/M phase cells may be selected if polar body extrusion is to be permitted. The cell cycle status of cultures can be manipulated (e.g., by serum starvation/ stimulation) to enrich for cells of particular cell cycle stages. If no further enrichment methods are applied, the donor cells are thus selected from a population that is to some degree heterogeneous. Within that population of cells may reside somatic stem cells. Whether such rare stem cells in fact contribute preferentially to live clone births has not been thoroughly studied. There are two circumstances in which the presence of stem cells probably does not contribute to cloning outcome. First, differentiated cells expressing specific molecular markers can be employed. In such cases, cloning success rates are about an order of magnitude lower than observed with primary cell sources (Hochedlinger and Jaenisch, 2002), an outcome that could reflect the differentiated state, absence of stem cells, or simply a differential response of clones made with those particular cell types to the in vitro cloned embryo culture system. The second circumstance wherein stem cell contamination would not affect outcome would be in cases where clonally selected cell lines are established following genetic manipulation (e.g., gene knockout or transgenesis).

Whether stem cells contribute preferentially to the production of clones that develop to term could affect conclusions derived from the study of cloned progeny. Stem cells can differ from differentiated cells in fundamental respects such as the number of past rounds of cell division, telomere length, and telomerase expression. Because reprogramming is slow, these phenotypic differences between stem cells and differentiated cells will likely persist to some degree in cloned embryos. Thus, conclusions regarding somatic mutation load, mitochondrial fitness, and replicative potential of the genome as reflected in telomere length in the context of cloned embryo and cloned stem cell derivation need to account for possible effects of donor cell state.

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Blood Pressure Health

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