Therapeutic Effects of Clustered Human Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells for Chronic Hind Limb Ischemia

Introduction: Human induced pluripotent stem cell-derived mesenchymal stem cell (iPS-MSC) is a promising cell source of regeneration therapy for hind limb ischemia (HLI). To date, we have already demonstrated the clustered cell administration has better therapeutic effects for HLI than single-cell administration using autologous myoblast cells. In this study, we administered clustered iPS-MSCs into the chronic HLI mouse and evaluated the regeneration effect. Methods: Human iPS cells were cultured in a serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542 for the differentiation to MSC in vitro . These iPS-MSCs were expanded to form cell patches (10 6 cells/sheet). These iPS-MSC patches were mechanically fragmented and were then administered into HLI mice as clustered cells (CC group, n=10). As control groups, non-clustered single iPS-MSC (SC, n=12) or Hanks’ balanced salt solution (HBSS, n=9) were administered. HLI was induced to the NOD/Shi-scid mouse by resecting the femoral artery and vein. The mice of which blood perfusion, being evaluated with Laser Doppler Perfusion Imaging (LDPI) at day 7, was under 40 % as compared with contralateral leg were included in the further study as the “chronic HLI model”. Serial change of LDPI and histological change of leg muscles were analyzed regarding angiogenesis and skeletal muscle regeneration. Results: Blood perfusion was significantly improved in CC (56.16±20.03%) as compared with HBSS (34.09±12.80%, P=0.0140) and SC (29.04±6.770%, P=0.0010) at day 14. Also, histological analysis showed the number of CD31 + endothelial cells is significantly larger in CC (353.6±59.97/mm 2 ) as compared with HBSS (126.2±22.85/mm 2 , P=0.0004) and SC (103.6±23.81/mm 2 , P=0.0001). The mean diameter of skeletal muscle cells is significantly larger in CC (42.28±4.113μm) as compared with SC (29.02±2.535μm, P=0.0001) and HBSS (29.61±3.103μm, P=0.0007). Conclusions: The clustered iPS-MSC significantly augmented angiogenesis and muscle regeneration in chronic HLI as compared with single-cell administration.