These experiments suggested that DMSO pretreatment was capab

These experiments suggested that DMSO pretreatment was capable of normalizing the responses of refractory clones. Maximal levels of mRNAs in the 3 clones were similar following incubation with DMSO, while they were quite different without DMSO pretreatment. In order to assess whether the similarity of response following DMSO extended to a larger number of genes, we performed a microarray analysis on the 3 iPS cell clones following optimal DMSO pretreatment and differentiation. We found that the robustness of mRNA increases was a general response extending to several thousand genes (Fig. 4). Cluster analysis showed that the three differentiated samples closely resembled each other and were distinct from the three undifferentiated iPS cell clones. In order to verify that the effect of DMSO would apply to genes not previously selected as part of the differentiation protocol, we selected 6 upregulated and 6 downregulated genes from the microarray results to assess the effect of DMSO. qPCR verified that DMSO pretreatment increased the responses of the 6 upregulated genes in the differentiation protocol (Fig. 5). Some selected genes (LAMA1, CRYAB, ITGA8) showed very little response in the absence of DMSO, but strong responses following DMSO pretreatment. For some other mRNAs weak responses became more robust following DMSO. For the 6 downregulated genes, the patterns were more complex, but in all cases DMSO pretreatment caused the mRNA levels following differentiation to be very simiar, while they were quite dissimilar in the absence of DMSO. As a result of these studies we modified our differentiation protocol by adding a 24h incubation with 0.5–1% DMSO prior to 3days of hanging drop aggregate formation and 3days of incubation in monolayer culture with differentiation factors. To verify that this protocol was useful, we performed immunocytochemistry on the differentiated cells. Fig. 6 shows that there was robust LY335979 cost of NCAD, SOX10, NKX6.1 and NKX2.2 as well as the general neural marker βIII tubulin, whereas there was minimal expression of these proteins in the undifferentiated iPS cells. We therefore concluded that adding a DMSO pretreatment to the differentiation protocol would be of general value in experiments that require rapid and robust differentiation. As noted above, the use of DMSO pretreatment in this modified protocol was based on prior data that suggested that this may be of general use in the differentiation of pluripotent cells (Chetty et al., 2013). In the prior work, it was suggested that DMSO might act by causing the cells to exit from the cell cycle. We tested whether marmoset iPScells are caused to exit from the cell cycle by exposure to DMSO (Fig. 7). Flow cytometric analysis of propidium iodide-stained cells showed no effect of DMSO on cell cycle distribution when used at 0.5%, 1% and 2%.
Discussion In experimental autologous cell therapy in NHPs, rapid and reliable differentiation is essential for the accomplishment of the aims of the procedure in a feasible length of time. Given that biopsies of tissues from a living animal can be used to prepare iPS cells via validated reprogramming methods (Mishra et al., in press), it is then critical to be able to perform directed differentiation of the cells using protocols that can be applied to any iPS cell clone that is generated during reprogramming. Autologous cell therapy would be much more difficult if optimization of differentiation protocols were required to be performed for each iPS cell clone. Nevertheless, the reported variability in response of pluripotent cells to directed differentiation (Chang et al., 2008; Osafune et al., 2008; Hu et al., 2010; Bock et al., 2011) forms an obstacle that must be addressed. Here, we confirmed the variability in responsiveness of pluripotent cells, using marmoset iPS cell clones subjected to a directed neural differentiation protocol. A clone that was used to develop the protocol responded well in terms of expected changes in gene expression, while 2 others responded variably and less well. However, following pretreatment with DMSO for 24hours, all clones responded with large changes in gene expression, and the clone used for the protocol development also showed increased expression of most genes. The uniformity of the changes in gene expression was confirmed using a microarray analysis of the 3 clones following DMSO pretreatment and directed differentiation. A further analysis of 6 genes that were highly upregulated and 6 genes that were highly downregulated showed that DMSO pretreatment caused a high degree of uniformity of response in these 12 genes among the 3 iPS cell clones. Using the pretreatment with DMSO as an added step in an improved protocol, we showed robust expression of several proteins by immunocytochemistry.