*4.3. Changes in mRNA Expression among Early and Late Passage iSGECs by qRT-PCR*

Expression of acinar and other characterization markers was assessed over early and late passages. iSGEC-pSS1 was the most stable cell line where only AMY1A and CST3 expression increased in later passages. Both iSGEC-nSS1 and -nSS2 exhibited pro-acinar changes in expression profiles at later passages where most markers increased (Figure 4A). In iSGEC-nSS1 p-45, pro-acinar markers (AMY1A, AQP5, ANO1, SLC12A2, CST3, and TRPC1) increased in expression compared to early (p-14) cultures (Figure 4A). iSGEC-nSS2 at p-80 expressed AMY1A, ORAI1, STIM1, SLC12A2, CST3, and TRPC1 higher, and only one marker lower (ANO1) compared to p-14. Both AQP5 and STIM2 were stably expressed during extended passaging in iSGEC-nSS2. Overall, AMY1A and CST3 increased during late passages of all three iSGECs lines, which could be important for maintaining pro-acinar characteristics.

**Figure 4.** Changes in mRNA expression of early and late passaged iSGECs by qRT-PCR. **Legend.** mRNA expression of acinar (**A**) and characterization markers (**C**) in monolayer cultures of early and late passaged iSGECs by qRT-PCR (ΔCT). iSGECs increased in AMY1A (**A**), ZO-1 (**C**), and Nanog (**C**) in later passages. Changes in gene expression of iSGEC-nSS2 early (p-14) and late (p-80) are indicated with arrows: increased (**red**), decreased (**green**), and no change (**yellow**). (**B,D**) iSGEC-nSS2 differentially expressed several acinar and characterization markers in later passages. Significance of differences between the means was determined by Student's *t*-test and *p*-values corrected using the Holm–Šidák multiple comparisons post hoc test (alpha = 0.05). Error bars represent mean +/− SEM. \* *p* < 0.05.

Characterization markers were the most differentially expressed and exhibited the greatest changes in late passaged iSGECs. Epithelial marker CDH1 was the only stably expressed gene among all characterization markers, whereas ZO-1 increased in late passages of all three iSGECs (Figure 4C,D). iSGEC-nSS2 maintained an epithelial expression pattern, including a decrease in the mesenchymal marker VIM and a significant increase in CLDN1 expression. Over passages, both KRT5 and KRT19 decreased in expression and the progenitor cell marker NANOG increased and could indicate a shift towards a less-differentiated cell population in iSGEC-nSS2.
