*4.1. Primary Isolation, Culture, and Growth of iSGECs*

LSG biopsy fragments remained in SGEC explant medium for approximately 7–14 days to ensure sufficient cell outgrowth. Once the cells reached approximately 70–80% confluency, the culture medium was replaced with SGEC sub-culturing medium. Transitioning to the serum-free low calcium (0.06 mM) medium for 3–5 days prior to trypsinization reduced fibroblast contamination in later passages. Before SV40Lt transduction in all three cell lines, two separate populations of cells were observed and consisted of either larger cells with complex cytoplasm or smaller cells with reduced cytoplasm. The smaller cells were predominant during later passages in all three iSGEC lines. At late passages iSGEC-pSS1 gave rise to colonies appearing that were mixed in size, with polygonal or cobblestone-like cells with differing rates of cytoplasmic complexity (Figure 1A). iSGEC-nSS1 late passages demonstrated a mix of small polygonal and filiform-appearing cells (Figure 1B). Mixed morphology of iSGECs most likely indicates an inhomogeneous population (Table 2) (Figure 1A–D). Therefore, we selected iSGEC-nSS2 for late-passage outgrowth and in-depth characterization over iSGEC-pSS1 and -nSS1. Long term passaged iSGEC-nSS2 exhibited a stable, small cobblestone type of morphology and did not give rise to filiform-appearing cells (Figure 1C,D). iSGEC-nSS2 cultured in 1.2 mM Ca2<sup>+</sup> acquired more complex appearing cytoplasm with granulations and formed tight clusters at both early and late passages (Figure 1E,F).

**Figure 1.** Morphology and proliferation of iSGECs. **Legend.** Representative images of early passage (p-14) monolayer culture (**A**) iSGEC-pSS1 (**B**) iSGEC-nSS1 (**C**) iSGEC-nSS2 and (**D**) late (p-80) iSGEC-nSS2. iSGEC-nSS2 "cobblestone-like" morphology was maintained across multiple passages when grown in SGEC sub-culturing media with low (0.06 mM) Ca2<sup>+</sup>. Cell morphology changes in (**E**) iSGEC-nSS2 p-14 and (**F**) iSGEC-nSS2 p-80 are observed when medium is supplemented with 1.2 mM Ca2<sup>+</sup> (final concentration) for 72 h. (**G**) Proliferation of iSGEC-nSS2 was assessed by the percentage (%) of Ki-67<sup>+</sup> cells using immunocytochemistry. No significant difference in the percentage of Ki-67<sup>+</sup> cells was detected between early (p-14) and late (p-80) iSGEC-nSS2 cultures. Student's *t*-test (\* *p* < 0.05, NS = not significant). Data are means +/− SEM. Magnification ×20, scale bar = 200 μm.



Results of protein expression of characterization markers in iSGECs, SGCLs, and HeLa cells by ICC are shown. Cells were grown on type-1 collagen-coated coverslips for up to 72 h prior fixation. iSGECs were additionally plated on coverslips and grow in SGEC sub-culturing media supplemented with 1.2mM Ca2+ to determine changes in acinar cell marker expression. (+) indicates positive expression of protein. (+/−) indicates low or diffuse expression of protein. (−) indicates no protein expression was detected by methods outlined.

During later passages (iSGEC-nSS2, p-80), colony formations that were different in their overall shape arose from single-cell clonal outgrowths (Supplementary Figure S2A). However, these single-cell outgrowth populations quickly became incorporated into other colonies and were unremarkable at lower confluency.

Proliferation rates of iSGEC-nSS2 did not substantially waver across early (p-14) and late (p-80) passages, as indicated by Ki-67 staining percentage (Figure 1G). No decrease in the % of Ki-67 (+) cells was observed at later passages. Spheroid formation on matrigel was observed after 24 h in all three cell lines (Figure 2, Supplementary Figure S2B–G). iSGEC-nSS2 retained its spheroid formation ability into late passages (>p-80), which could be observed after 24 h of plating. At three days in matrigel, spheroids measured roughly ~75–200 μm in all iSGECs.

**Figure 2.** iSGEC-nSS2 early (p-14) and late (p-80) spheroid formation on matrigel. **Legend.** Cells were initially seeded at 4 <sup>×</sup> 104 per well. Cultures began to form spheroids at 24 h on matrigel (2 mg/mL). Early passages (e.g., p-14) formed larger, but overall fewer spheroids when assessed after 3 days. Spheroids appeared to reach their final size after 3–4 days. Scale bars represent 200 μm.
