Abstract Body: Introduction: Peripheral artery disease (PAD) is a significant comorbidity in aging population regarding diminished quality of life and increased mortality rates. There are urgent needs for developing advanced therapeutics for functional limb salvage beyond conventional revascularization. We hypothesized that 3D spheroids composed by induced Skeletal Muscle Progenitor Cells (iSKMPs) and Mesenchymal Stem Cells (iMSCs) could recapitulate embryonic niches and provide promising regenerative potentials in hindlimb ischemia (HLI) models.
Methods: We manufactured scaffold-free, self-aggregated 3D spheroids using microcavity plates with ultra-low attachment surface. 3D spheroids were designed into 3 groups according to the induced cell types derived from Human Pluripotent Stem Cells (hPSCs); 1) iSKMPs only, 2) iMSCs only, 3) iSKMPs + iMSCs mixed. We transplanted 3D spheroids into the ischemic area and compared their therapeutic potentials using Laser Doppler Perfusion Imaging (LDPI) and immunohistochemical staining analysis.
Results: iSKMPs secreted various angiogenic factors including IGF, HGF and FGF and facilitated tube formation and migration of endothelial cells (ECs). iSKMPs also showed the differentiation potential into myofibers in defined 2D culture condition. During the 28-day follow-up period, the transplantation of 3D mixed spheroids not only increased blood perfusion in ischemia limb, but also significantly reduced amputation rate compared with those of control and other 3D single spheroid groups. Notably, in vivo capacity of muscle regeneration was more potent in 3D mixed spheroid group with higher rate of engraftment, myotube formation, and vascularization. These findings suggested that iSKMPs played a significant role in limb salvage via myogenesis and angiogenesis, and their cell-to-cell interaction with iMSCs within 3D spheroids provided a favorable microenvironment to enhance the engraftment and maturation of iSKMPs in ischemic hindlimb.
Conclusion: We demonstrated that the transplantation of 3D spheroids composed by iSKMPs and iMSCs could recapitulate developmental embryonic niches and provide potent regenerative potentials in HLI models. These findings provide compelling evidence that this injectable platform of 3D mixed spheroids can be a promising tool to rescue limb amputation in patients with PAD.