Abstract
Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P < 0.01). This was associated with significant enhancement of mitochondrial membrane potential (P < 0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P < 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.
Acknowledgments
The authors thank Dr H. F. Lodish (Whitehead Institute, Cambridge, MA, USA), Dr C. C. Zhang (University of Texas Southwestern Medical Centre, TX, USA) and Dr D. M. Virshup (Duke-NUS GMS, Singapore) for their critical reading of the manuscript; and Dr Marc Fivaz and Dr Alexandra Pietersen (Duke-NUS GMS, Singapore) for providing their technical expertise in relation to the confocal microscopy and fluorescent-activated cell sorting, respectively. We would like to thank Zhihong Li (Singapore General Hospital) and Andrea Lim (Singapore General Hospital) for their technical support. The funding support of the project was provided by A*STAR Singapore Stem Cell Consortium (A*STAR SSCC), Biomedical Research Council (BMRC), Singapore, Singapore General Hospital (SGH) and Singapore Cord Blood Bank (SCBB).
Author contributions: PPYC and SB planned the study, performed experiments and wrote the manuscript. XBF and GNCC provided guidance, supervised experiments and contributed to manuscript revisions. FPHG and JMLA conducted critical experiments. SKL provided important guidance on MSC and the revised manuscript. WYKH conceived the original study and planned experiments and made final revisions to the manuscript.
Author disclosure statement: All the authors for this manuscript declare no conflict of interest.