Implantation of human mesenchymal stromal cell pellet for therapeutic angiogenesis.

Abstract

Although mesenchymal stromal cell (MSC) therapy holds great potential, therapeutic efficacy, which is predicted by pre-clinical studies, has fallen short of expectations in most clinical studies. This discrepancy may arise from inadequate standardization of cell preparations, unstable cell engraftment and insufficient understanding of cellular fitness in vivo due to the lack of a relevant experimental system in which human MSCs stably engraft and the behavior of the MSCs can be systemically analyzed. MSCs stimulate angiogenesis and accelerate tissue/organ regeneration by releasing various bioactive factors (secretome). In this study, we established a system in which an implanted human MSC pellet stably engrafts and the angiogenic ability of the MSCs can be systematically analyzed in vivo. Surgically excised human subcutaneous white adipose tissue (4 g) was enzymatically digested and MSCs were successfully isolated from 237 patients (19-91 years old). MSCs isolated from donors (13 clones) less than 65 years old were used for the experiments. MSCs stimulated sprouting of human adipose endothelial cells in endothelial cell-organoids in vitro. Various angiogenesis-related factors were detected in cell lysate and conditioned medium from cultured non-frozen human MSCs in vitro. Within 21 days after initial cell isolation, an implantable MSC pellet was created by culturing human MSCs (5 × 10⁶ live cells) over a polycarbonate membrane at the air-liquid interface for 16 h without exogenous biomaterials. The subcutaneously implanted MSC pellet stably engrafted in immunodeficient NOD scid gamma (NSG) mice for up to 4 weeks (28 days). The implanted MSC pellet kept expressing relevant factors that were profiled in vitro, and specific factors were also detected in the plasma of recipient mice after implantation. The MSC pellet locally created a dense host-derived capillary network in which MSCs closely interacted with the capillaries. Implantation of the human MSC pellet increased capillary density in the local skin, pancreas, subcutaneous white adipose tissue and quadriceps of the recipient mice 4 weeks (28 days) after implantation. Furthermore, implanting MSC pellets restored capillary density and reduced tissue hypoxia in the distal muscles of mice 4 weeks (28 days) after femoral artery ligation. Fabrication and implantation of an MSC pellet can be leveraged not only to pre-clinically explore fitness of human MSCs in vivo but also to potentiate efficacy of MSC therapy in humans.