Hological and differentiation inconsistencies were a result of either incomplete reprogramming > 자유게시판

Hological and differentiation inconsistencies were a result of either incomplete reprogramming or the heterogeneity of our iPSC cultures. Recent literature suggests that a prolonged period of proliferation and self-renewal may be necessary to stabilize iPSCs in a pluripotent state [17,26]. Accordingly, we passaged iPSCs at least 10 times prior to repetition of neural induction [26]. At 20-passages, spontaneous differentiation was undetectable in both GG3.1 and miPS-25 cell lines, whereas GFP expression was uniform in the miPS-25 line (Figure 3A). Interestingly, we observed a significant increase in the diameter of EBs ( 90-120 m up to 160-190 m, n = 3) derived from late-passage GG3.1 cells, which was equivalent to the EB size seen in ESC cultures (Figure 3B). Furthermore, relative to early-passage iPSCs, most cells in late-passage GG3.1 cultures expressed Sox2, with few observable differentiated Sox2- cells (Figure 4A and 4B). Real-time qRT-PCR revealed that expression levels of the pluripotency markers Oct4, Sox2, Rex1 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 and Nanog in late-passage cultures were significantly higher than those in early-passage iPSCs and were equivalent to expression levels in ESCs (Figure 4D). Notably, Nanog expression in late-passage cells remained significantly lower than in ESCs, but there was an upward trend (Figure 4D). To assess the transcriptional changes occurring in iPSCs over the course of neural differentiation, we carried out additional qRT-PCR using cDNA generated from undifferentiated cells, cells at EB day 5, and neural induction days 3 (Ni3), and 7 (Ni7). To clearly delineate events of gene up- and down-regulation, we evaluated the expression of immature- and mature-neuronal markers. Expression of pluripotency markers (Rex1, Oct4 and Klf4) in iPSCs declined promptly during the EB Gemcitabine (hydrochloride) stage and subsequent differentiation (Additional file 1, Figs. S2A and S3B). The immature-neural markers, Neurogenin1 (Ngn1), Musashi1 (Msi1), Sox1 and HuC/D are all transiently expressed during in vivo neural development and have been detected in our cultures previously [29,34]. As expected, the mRNA levels of these genes in ESC cultures elevated during early differentiation (Ni3), but declined as neural induction proceeded (Ni7) (Figure 5A). By contrast, the induction of immature-neural marker genes was delayed in early-passage iPSCs (Figure 5A). However, after 20-30 passages, temporal expression patterns and levels of immature-neural markers were not significantly different from ESCs (Figure 5A). We next evaluated the expression of mature neural markers, neuron specific enolase (NSE), Syn (Figure 5A), Calretinin and TrkB (Additional file 1, Fig. S2B). We found consistently that expression of these genes is induced by Ni3, but increases dramatically by Ni7 in ESC cultures (Figure 5A). This pattern of expression was seen in early-passage iPSCs, but was not as robust. As with the other markers, late-passage iPSC-derived cultures exhibited significantly higher levels of NSE and Syn expression than early-passage iPSCs at Ni7 (Figure 5A). To better quantify the efficiency of neural differentiation, we performed flow cytometry analysis for the neural lineage marker CD24 [35-37]. Our data revealed a lower percentage of CD24 + cells in early-passage iPSC-derived cultures ( 30 ) compared to ESC-derivedKoehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-2202/12/Page 5 ofFigure 2 Neurons derived from GG3.1 iPSCs exhibit characteristic neuronal mor.