Publication details

Robust expansion in monolayer reversibly elevates viability of human embryonic stem cells



Year of publication 2012
Type Conference abstract
MU Faculty or unit

Faculty of Medicine

Description The need for a robust, invariable, and cost-effective culture of human embryonic stem cells (hESCs) resulted in several large-scale systems. However, the novelty was often hampered by changes in hESC phenotype, genomic instability, or difficult manipulation. Here we report a comprehensive analysis of pluripotency features by hESCs, which have been propagated for over 100 passages in monolayer (ML) culture. The ML system is based on single-cell dissociation and plating of cells in high densities on a matrix-coated surface, so they become fully confluent within 72 hours. This results in homogenization of the cell population and efficient expansion with 1:20 ratio every 3 days. While the in vitro and in vivo differentiation capacity remained to be the same, successful development of a teratoma required 5-times lower input of hESCs from the ML system then of their feeder-dependent counterparts. This indicates (a) different portion of teratoma initiating cells or (b) different survival rate early after injection. As the presence of SSEA-5- and Oct-4- positive cells was comparable in both culture systems after differentiation, teratoma development from ML hESCs was not driven by an increased pool of differentiation-resistant cells. Also, we found none of the recently described adaptation-related proteins (Bcl-xL, Bcl-2 and survivin) to be overexpressed in ML hESCs. In two viability assays, ML hESCs showed superior survival and participation to assays than colony-dependent culture. Finally, we found that the reverted feeder-dependent culture displayed normal phenotype of the colony growth, gradually developed back the dependence on Y27632 in viability assays, and lowered teratoma efficiency towards the ground state represented by feeder-dependent hESC colonies at the beginning. In addition to the stable karyotype, the reversibility rather points to an unselective process of adaptation and challenges the hypothesis of clonal expansion in ML system. In addition to that, a simple automation and compatibility with xeno-free conditions make the ML system highly suitable for production of vast numbers hESCs necessary in cell-based therapies.

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