Iva Kubíková

Adaptation to Robust Monolayer Expansion Produces Human Pluripotent Stem Cells With Improved Viability

The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder‐free, high‐density monolayer system in which hPSCs become SSEA‐3‐high and gradually more viable than their feeder‐dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single‐cell suspension, and colony‐forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long‐term, large‐scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell‐based therapies.

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin

BACKGROUND AIMS Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.

Expression of FGF19 in Human Embryonic Stem Cells

In human embryonic stem cells, continuous stimulation with fibroplast growthfactor 2 is required for self-renewal. inhibition of FGF2 signaling results in rapid differentiation, further underscoring the importance of FGF system in maintenance of hESC undifferentiated state. In addition to FGF2, human FGF family contains 17 members which presence in hESC remains unclear, with exception of FGF4.