It has been shown previously that genetic selection against heterologous cell types generated during the course of spontaneous ES cell differentiation can result in enrichment for the cell types of interest. For example, this approach has been used to obtain purified cardiomyocyte- and neural-like cells from mouse ES cell cultures (21,22). Soria and coworkers used a similar strategy to select insulin-producing cells from spontaneously differentiating mouse ES cells (43). They introduced into the ES cells a plasmid conferring resistance to two antibiotics. The first antibiotic-resistance gene was under control of a constitutive promoter, and the second gene was under control of an insulin promoter. During the first stage of the culture the undifferentiated ES cells were selected for resistance to the first antibiotic. This allowed generation of a stable cell line in which every cell carried the plasmid. After this step, the ES cells were transferred into differentiation medium containing the second antibiotic. Because the second antibiotic resistance gene was under control of insulin promoter, only cells producing insulin survived this round of selection. The authors report that the insulin content of the ES cell-derived progeny obtained with this protocol was approximately 90% of the insulin content of normal mouse islets. When the insulin release in response to glucose and other agonists was measured in vitro, the cells showed stimulated release. Moreover, when implanted into diabetic mice, the insulin-producing cells normalized hyperglycemia. This normalization disappeared, however, after 12 weeks in about 40% of transplanted animals. The comparison of glucose tolerance of the transplanted animals with that of the nondiabetic controls showed that in the transplanted animals the plasma glucose levels were significantly elevated, and the recovery to normal glucose levels was delayed.
The results of this work suggest that spontaneous differentiation protocols combined with genetic selection can enrich ES cell cultures for insulin-producing cells. One can envision these protocols being further improved by placing the second antibiotic resistance gene under control of promoters active in pancreatic progenitor cells such as PDX-1 or ngn3 promoters. A potential advantage of such a strategy is that it would allow selection of a pancreatic cell type that, unlike terminally differentiated insulin producing cells, would have high proliferative potential (44). These pancreatic progenitor cells could then be selectively expanded and induced to differentiate into insulin producing cells.
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