Transient HA-100 exposure improves aggregate uniformity and cell-cell contact stability in suspension human pluripotent stem cell culturesKhurana, Preeti (Avtor) Liović, Mirjana (Avtor) Ilic, Dusko (Avtor) human pluripotent stem cellssuspension cultureaggregate uniformityHA-100manufacturingTJP1Background aims: Human pluripotent stem cell (hPSC) manufacturing workflows frequently rely on suspension aggregation, yet inter-line and batch-to-batch variability in aggregate formation can compromise process consistency and downstream differentiation performance. We evaluated whether a short exposure to HA-100, a small-molecule inhibitor of protein kinase A and protein kinase C signaling, could be used as an upstream process intervention to improve aggregate uniformity without compromising hPSC identity or developmental competence. Methods: Nine hPSC lines, including human embryonic stem cell and induced pluripotent stem cell lines, were examined in suspension culture. HA-100 was applied during the first 24 h of aggregation. Aggregate morphology and size distribution were assessed across lines. To investigate the cellular basis of this effect, we generated an mCherry-TJP1 reporter hESC line, which enabled live visualization of junction dynamics, including responses under calcium-depleted conditions and recovery of transepithelial electrical resistance. Results: HA-100 treatment promoted more compact and spherical aggregates, increased aggregate size into a narrower range across lines, and reduced overall variability relative to medium alone. Across the nine-line panel, HA-100-treated aggregates fell within an empirically definesd size range of 25.37-33.95 x 10^-4 mm^3 after 24 h of suspension culture, providing a practical benchmark for process monitoring. In calcium-depleted conditions, HA-100 delayed disruption of intercellular contacts and accelerated recovery of transepithelial electrical resistance, consistent with improved junctional resilience. Transient exposure to HA-100 did not abolish pluripotency marker expression or tri-lineage differentiation capacity. Conclusions: These data support HA-100 as a practical upstream intervention to reduce aggregate heterogeneity in suspension hPSC cultures and improve reproducibility in manufacturing-oriented workflows requiring consistent aggregation.20262026-08-06 06:58:02Članek v reviji185452sl