Therapeutic Effect of a Xeno-Free Three-Dimensional Stem Cell Mass in a Hind Limb Ischemia Model

Abstract

This study developed a xeno-free (XF) spherical cell cluster of human 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 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 (bFGF)-immobilized polystyrene surface. XF-3DCMs produced comparable amounts of angiogenic factors such as bFGF, vascular endothelial growth factor, and interleukin-8 compared to 3DCMs formed in media containing FBS (fetal bovine serum)-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. Furthermore, 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. Impact Statement We describe an innovative method of culturing a three-dimensional cell mass (3DCM) of human adipose-derived stem cells (hASCs) in the xeno-free (XF) condition for the treatment of severe ischemic diseases. The majority of the mice injected with XF-3DCMs exhibited limb salvaging and displayed similar blood perfusion compared to normal limbs. In vivo tumorigenicity and toxicity analysis showed that XF-3DCMs did not transform into tumor cells and induce toxicity, respectively. Our results strongly suggest that XF-3DCMs can be effectively used for therapeutic applications and eliminate immunological reaction of animal-derived material contamination.