Topical application of hESC-derived mesenchymal stem cell spheres accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner

It has been reported that mesenchymal stem cells (MSC) derived from adult tissues are effective in promoting wound healing. However, the cell quality varies and cell number is limited as both depend on donations. Moreover, dissociated MSC delivered to an inflammatory lesion are subject to challenges to their survival and functions. Here we demonstrate that dropping of spheres of MSC derived from human embryonic stem cells (EMSC) onto murine dermal wound had much higher survival and efficacy than topical application of dissociated EMSC. RNA sequencing on cells isolated from the wound highlights the CXCL12-CXCR4 signalling in the EMSC sphere-mediated efficacy, which was verified via CXCL12 knockdown in EMSC and CXCR4 inhibition in target cells such as vascular endothelial cells, epithelial keratinocytes, and macrophage. Finally, we enhanced the biosafety of EMSC spheres by engineering the cells with an inducible suicide gene. Together, we propose topical application of EMSC spheres as an unlimited, quality-assured, safety-enhanced, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in the treatment. Overall design: We employed a murine model of excisional splinted skin wounds, in which contraction of skin around a wound is prevented so the wound healing effect of a treatment can be displayed and measured objectively. We directly dropped EMSCSp onto the wound surface, which promoted wound healing much faster than local delivery of EMSCDiss. EMSCSp survived longer, migrated farther, and induced more macrophage accumulation, vascular genesis and re-epithelialization in the wounds than EMSCDiss. Similar results were observed with MSC spheres derived from human bone marrow (BM-MSCSp) versus dissociated control cells. Finally, to reduce tumorigenesis concern, we transduced EMSC with a chemically inducible caspase-9 (iCasp9) and demonstrated that the engineered EMSC could be efficiently killed in vitro and eliminated in vivo post-therapy.