rns are consistent with enhanced pancreatic endocrine and b-cell differentiation in the EBs from H7.Px4 cells. Physiological Assessment of Voltage-gated Ca2+Channels in EBs To explore further the nature of the cells induced in the EBs from the Pax4-transfected cells, we examined the dynamics of intracellular Ca2+ signals of H7 and H7.Px4 EBs to depolarization of the cell membrane and voltage-gated Ca2+ channel gene expression. Pancreatic b-cells are electrically active and regulate Ca2+ in response to a membrane depolarization via VGCCs. While glucose results in depolarization and subsequent Beta-Cells from Human ES Cells Ca2+ influx into b-cells, various secretagogues, such as potassium choride, ATP and tolbutamide also trigger a similar response in the absence of glucose. We took advantage of a fluorescent indicator molecule Fura-2 AM, that enabled us to study the changes in intracellular free Ca2+ concentration using dual-wavelength excitation microfluorimetry in Fura-2loaded EBs. In untransfected H7 cells, most EBs71% to 78%, failed to respond to depolarizing concentrations of KCl between 7 and 2597184 21 days. Over the same time points the average rises in the i were 1662 nM at the early time point, 2065 nM in the mid-stage of EB differentiation and 39615 nM in the oldest EBs. The functional responsiveness of the undirected differentiation pathway in H7 cells was therefore associated with a 3-fold increase in VGCCdependent Ca2+ signalling. In comparison, EBs from H7.Px4 cells were already approximately 5-fold more responsive at day 7 when compared to controls and 13 fold more responsive at day 14 and day 21 . Whilst Pax4 led to the induction of marked increases in voltagegated Ca2+ channel operation, ATP-induced changes in i which are mediated via purinergic receptors, independently of a change in the cell membrane potential, were decreased at all timepoints in H7.Px4 cells. To understand the induction of voltagegated Ca2+ signals we examined the expression of VGCC genes associated with the endocrine pancreas. By RT-PCR we found CACNA1S, CACNA1C, CACNA1D, CACNA1A, CACNA1B and CACNA1E, and CACNB1 to 4, mRNAs were all expressed in EBs from H7 and H7.Px4, and also in the human fetal pancreas. Q-PCR was used to 1828342 examine CACNA1C, CACNA1D and CACNA1A over time in H7 and H7.Px4. Pax4 was found to potentiate the expression of CACNA1A, induce a transient increase in CACNA1D and enhanced CACNA1C mRNA at all time points. Finally, to 
confirm that indeed these changes in gene expression are related to the appearance of a putative b-cell phenotype, we found that the EBs from the H7.Px4 clones produced C-peptide, the fragment cleaved from the Proinsulin peptide during its processing in b-cells. Green-positive cells were detected by FACS in EBs from the H7.Px4 HESC than from the untransfected H7 cells. When isolated and analyzed by Q-PCR these cells were enriched in Ins and Pdx1 mRNA expression and depleted in expression of Oct4 relative to presorted and Newport order 6-Methoxy-2-benzoxazolinone Greennegative population. After sorting, the Newport Green-positive cells were further cultivated on collagen-IV-coated plates for an additional 57 days. More than 90% of these cells could be stained with an antibody to proinsulin, indicating de novo insulin synthesis and contained significantly more C-peptide than Newport Greennegative cells. The difference in C-peptide content and the levels of Ins transcripts between the Newport Green-positive and negative cells is consistent with the putative b
