Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGROTM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of a third EspA-binding protein that forms part of the type III secretion system of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli utilizes a type III secretion system to deliver virulent effectors into cells. The secretion apparatus comprises a membrane basal body and an external needle complex of which EspA is the major component. An l0050-deletion (DeltaL50) mutation was found to impair type III secretion and bacterial adherence. These phenotypes and the localization of the gene product to the inner membrane support the hypothesis that L0050, renamed EscL, forms part of the secretion apparatus. Furthermore, in DeltaL50, the amount of EspA present within the cell lysate was found to have diminished, whereas the EspA co-cistron-expressed partner protein EspB remained unaffected. The decreased EspA level appeared to result from instability of the newly synthesized EspA protein in DeltaL50 rather than a decrease in EspA mRNA. Using both biochemical co-purification and a bacterial two-hybrid interaction system, we were able to conclude that EscL is a third protein that, in addition to CesAB and CesA2, interacts with EspA and enhances the stability of intracellular EspA.