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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 neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems,that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR,however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research. View Publication

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless,a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation,oxidative phosphorylation is essential at all stages of differentiation. View Publication

The recent Zika virus (ZIKV) outbreak,which mainly affected Brazil and neighbouring states,demonstrated the paucity of information concerning the epidemiology of several flaviruses,but also highlighted the lack of available agents with which to treat such emerging diseases. Here,we show that heparin,a widely used anticoagulant,while exerting a modest inhibitory effect on Zika Virus replication,fully prevents virus-induced cell death of human neural progenitor cells (NPCs). View Publication