Transient HA-100 exposure improves aggregate uniformity and cell-cell contact stability in suspension human pluripotent stem cell cultures

Khurana, Preeti (avtor), Liović, Mirjana (avtor), Ilic, Dusko (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: human pluripotent stem cells, suspension culture, aggregate uniformity, HA-100, manufacturing, TJP1
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: MF - Medicinska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2026
• Št. strani: 7 str.
• Številčenje: Vol. 28, iss. 9, art. 102904
• PID: 20.500.12556/RUL-185452
• UDK: 61:602.9:57.086.83
• ISSN pri članku: 1477-2566
• DOI: 10.1016/j.jcyt.2026.102904
• COBISS.SI-ID: 279909891
• Datum objave v RUL: 06.08.2026

Gradivo je del revije

• Naslov: Cytotherapy
• Skrajšan naslov: Cytotherapy
• Založnik: Elsevier, International Society for Cell & Gene Therapy
• ISSN: 1477-2566
• COBISS.SI-ID: 521762329

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: človeške pluripotentne matične celice, suspenzijska kultura, enakomernost agregatov, proizvodnja