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Updated: Aug 6, 2026
Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Published on: July 24, 2014
Transient HA-100 exposure improves aggregate uniformity and cell-cell contact stability in suspension human
Preeti Khurana1, Amar J Azad2, Nikola Kolundzic3
1Department of Women and Children's Health, School of Life Course and Population Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK; Unit for Paediatric & Population-Based Dermatology Research, St John's Institute of Dermatology, Faculty of Life Sciences and Medicine, King's College London, London, UK.
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.
