Therapeutic effect of a xeno-free three-dimensional stem cell mass in a hind limb ischemia model.

This study developed a xeno-free (XF) spherical cell cluster of adipose-derived stem cells (hASCs) for the treatment of severe ischemic diseases. hASCs are an attractive cell source for therapeutic angiogenesis. Herein, we describe an innovative method of culturing a three-dimensional stem cell mass (3DCM) in the XF condition and demonstrate the therapeutic potential and safety of XF-3DCMs in ischemic mice and in SCID/ BALB/c Slc-nu/nu nude mice, respectively. XF-3DCMs were generated by culturing hASCs on a maltose-binding protein-linked basic fibroblast growth factor-immobilized polystyrene surface. XF-3DCMs produced comparable amounts of angiogenic factors such as basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8 compared to 3DCMs formed in media containing fetal bovine serum (FBS-3DCMs). The majority of the mice injected with XF-3DCMs exhibited limb salvaging and displayed similar blood perfusion compared to normal limbs. To a similar degree as for FBS-3DCMs, XF-3DCMs enhanced therapeutic efficacy by increasing blood vessel regeneration, attenuating muscle degeneration and fibrosis, and facilitating the implantation of injected cells into host vessels. Further, the soft agar colony forming assay and in vivo tumorigenicity analysis showed that XF-3DCMs did not transform into tumor cells. No side effects were observed, including hematological and immunological toxicity, in mice injected with XF-3DCMs. These results suggest that XF-3DCMs show angiogenic and therapeutic efficacy in critical limb ischemia in mice and eliminate the safety problem of animal-derived material contamination, which is a prerequisite for clinical applications.