Figure 2. Panc1 holoclones display enhanced formation of holoclones and Nestin expression compared to paraclones.

(A) Scheme of generation and expansion procedure of different PDEC clones. After 6 day culture as depicted, single PDECs were seeded in a 96-well plate until single colonies were visible and their phenotype was assessed and confirmed by CFA. Only cells with stable phenotype were considered for further expansion and experiments. (B, C) In order to assess self-renewal capacity of expanded Panc1 holo- and paraclone cells, 400 cells were seeded for colony formation which was assessed after crystal violet staining on day 10. Only colonies containing more than 50 cells were counted and (B) the total number of colonies and (C) the proportion of different colony types of total number of colonies was determined. Data are presented as mean and standard deviation or median and quartiles (Q1 as 25% and Q3 as 75%) of 6 to 7 independent experiments. Below, representative images of crystal violet-stained Panc1 para- and holoclones are shown. Scale bar 250 μm. (D) RT-qPCR analysis of Nestin, Nanog and ABCG2 mRNA-expression normalized to GAPDH as housekeeper in Panc1 para- and holoclones. Data are presented as n-fold gene expression of paraclones and as median and quartiles (Q1 as 25% and Q3 as 75%) of 4 independent experiments. (E, F) Immunofluorescence staining of CSC-markers Nanog (red) and Nestin (green) in Panc1 paraclone and holoclone cells after in vitro expansion. Hoechst staining (blue) was performed to mark nuclei. Scale bar 25 μm. (F) Percentage of positively stained cells was determined by considering all cells contacting a horizontal line in 5 randomly chosen fields of view. Proportion of stained cells to total cell number is shown. Data are presented as mean and standard deviation of 3 independent experiments. ^* indicates statistically significant differences (p ≤ 0.05).