Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/ chicken ␤-actin/rabbit ␤-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells. STEM CELLS 2007;25:1521-1528 Disclosure of potential conflicts of interest is found at the end of this article.

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Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self-renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell-derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES-derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES-derived cells can be contemplated.

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.

The pancreas arises from Pdx1-expressing progenitors in developing foregut endoderm in early embryo. Expression of Ngn3 and NeuroD1 commits the cells to form endocrine pancreas, and to differentiate into subsets of cells that constitute islets of Langerhans. β-cells in the islets transcribe gene-encoding insulin, and subsequently process and secrete insulin, in response to circulating glucose. Dysfunction of β-cells has profound metabolic consequences leading to hyperglycemia and diabetes mellitus. β-cells are destroyed via autoimmune reaction in type 1 diabetes (T1D). Type 2 diabetes (T2D), characterized by impaired β-cell functions and reduced insulin sensitivity, accounts for 90% of all diabetic patients. Islet transplantation is a promising treatment for T1D. Pluripotent stem cells provide an unlimited cell source to generate new β-cells for patients with T1D. Furthermore, derivation of induced pluripotent stem cells (iPSCs) from patients captures "disease-in-a-dish" for autologous cell replacement therapy, disease modeling, and drug screening for both types of diabetes. This review highlights essential steps in pancreas development, and potential stem cell applications in cell regeneration therapy for diabetes mellitus.