20.500.12556/RUL-185452 Transient HA-100 exposure improves aggregate uniformity and cell-cell contact stability in suspension human pluripotent stem cell cultures Background 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. human pluripotent stem cells suspension culture aggregate uniformity HA-100 manufacturing TJP1 človeške pluripotentne matične celice suspenzijska kultura enakomernost agregatov proizvodnja true false true Angleški jezik Slovenski jezik Članek v reviji 2026-08-06 06:58:02 2026-08-06 06:58:14 2026-08-07 04:08:20 0000-00-00 00:00:00 2026 0 0 7 str. iss. 9, art. 102904 Vol. 28 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 61:602.9:57.086.83 1477-2566 10.1016/j.jcyt.2026.102904 279909891 RAZ_Khurana_Preeti_2026.pdf RAZ_Khurana_Preeti_2026.pdf 1 0F029B916C124F515452D846B01B6B91 eadea0fa35714997b46b7ee989cee632e668b8ccc2f8372a05bf11ad110b73a8 91b8ed69-9155-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243966 https://www.sciencedirect.com/science/article/pii/S1465324926008650 1 186e986e-9153-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243964 Medicinska fakulteta 0 0 0