Figure 7.

Differences in neuronal cell marker expression levels in dopaminergic neurons generated from iPS cells of different origin. (A) Representative images of morphology of 2D neural differentiation. White scale bars represent 100 μm. (B) Immunofluorescent staining of TUBB and TH in the generated dopaminergic neurons. On day 30, the differentiated cells from iPS-K and iPS-P cells contained TUBB-positive and TH-positive cells. Slightly higher expression of TH was observed in neurons generated from iPS-K cells. Representative images are shown. White scale bars represent 50 μm. (C) Gene expression of neuronal progenitors (FOXA2, LMX1A) and neurons markers (NURR1, TH, TUBB) in dopaminergic neurons generated from iPS-K and iPS-P in each timepoint (day 11, 20 and 30 of differentiation). Statistically significant higher expression of FOXA2 gene was detected on day 20 and 30 in cells generated from iPS-P cells compared to iPS-K cells, ** p < 0.01, * p < 0.05. Higher expression of LMX1A was observed on day 30 in neurons generated from iPS-K, * p < 0.05. On day 20, statistically significant higher expression of the NURR1 gene was observed in cells from iPS-P cells, but on day 30 higher expression of that gene was observed in neurons from iPS-K cells, * p < 0.05, ** p < 0.01. The expression of TUBB was definitely higher in neurons from iPS-K on the last day of differentiation and the differences were statistically significant, **** p < 0.0001. Relative expression of genes was calculated by RT-qPCR using the ΔCt method with GAPDH as constitutive control. Statistically significant differences were analyzed using Student’s t-test. The experiments were performed for two clones from every source of iPS cells. Replicates for each clone were performed twice (n = 4). The data represent the mean ± SEM. TUBB—beta-III-tubulin, TH—tyrosine hydroxylase, NURR1—nuclear receptor related 1 protein, LMX1A—LIM homeobox transcription factor 1 alpha, FOXA2—forkhead box A2.