Scientific Resources

The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors,we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 X 10(6) cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry,quantitative reverse-transcriptase polymerase chain reaction,and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures,underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly,physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism,suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. SIGNIFICANCE Human pluripotent stem cells (hPSCs) are a unique source for the,in principle,unlimited production of functional human cell types in vitro,which are of high value for therapeutic and industrial applications. This study applied single-use,clinically compliant bioreactor technology to develop advanced,matrix-free,and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy,unexpected physiological features of hPSCs were discovered. These data allow a more rational process development,providing significant progress in the field of translational stem cell research and medicine. View Publication

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population,broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study,we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach,we identified two endoderm-specific antibodies,HDE1 and HDE2,which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential,whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination,the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. View Publication