Increased Angiogenic Property Of Human Peripheral Blood Monocytes By ex Vivo Culture With c-Mpl Agonists In Hindlimb Ischemia Mouse Model
Authors: Sasajima Junpei , Kawamoto Toru , Sugiyama Yoshiaki , Mizukami Yusuke , Iuchi Yasuyuki , Ashida Toshifumi , Tanabe Hiroki , Torimoto Yoshihiro , Kohgo Yutaka 
Published: October 2019

Abstract
Human peripheral blood mononuclear cells (PB-MNCs) include some populations which have angiogenic properties. Pro-angiogenic monocytes from PB-MNCs are considered as one of candidates for angiogenic therapy in regenerative medicine. Indeed, in a recent German clinical post-infarction remodeling study (TOPCARE-AMI) for ischemic heart disease, the ex vivo culture of PB-MNCs was employed. However, in this trial, there were different therapeutic efficacies in each case, possibly due to the different expansion efficacy of the ex vivo culture of PB-MNCs using autologous serum.
In order to resolve this issue, we developed a new serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Floating spheres obtained by this serum-free culture system were mainly composed of CD11b+ monocytes. Interestingly, the mRNA expression of c-Mpl (thrombopoietin receptor) was markedly elevated compared with PB-MNCs, suggesting c-Mpl agonist could increase angiogenic property of cultured CD11b+ monocytes. Therefore, we assessed the impact of c-Mpl agonists on PB-MNC cultures in our serum-free method composed of X-VIVO15 medium with VEGF and bFGF.
Both recombinant human thrombopoietin (rHuTPO) and romiplostim, a clinical grade second-generation TPO-receptor agonist, successfully increased sphere formations regarding both the number and size. The expressions of angiogenic factors, IL-8, CXCR4, and vasohibin-2, mRNA of CD11b+ monocytes cultured with c-Mpl agonists were up-regulated, indicating that cultivated CD11b+ monocytes have a proangiogenic potential.
Finally, we investigated the proangiogenic potential of PB-MNCs derived CD11b+ monocytes in a hindlimb ischemia model utilizing BALB/c nude mice. Mice were randomly assigned to 7 groups: control mouse group (PBS-injected), freshly isolated CD11b+ monocyte-injected mouse group, cultivated CD11b+ monocyte with 2ng/ml and 20ng/ml rHuTPO -injected mouse groups, cultivated CD11b+ monocyte with 100ng/ml and 1000ng/ml romiplostim -injected mouse groups, cultivated CD11b+ monocyte without rHuTPO and romiplostim -injected mouse group. The intramuscular injection of CD11b+ monocytes cultivated with 20 ng/ml rHuTPO into the ischemic limb completely rescued the limbs from auto-amputations or foot necrosis, while only one (10.0%) of the control mice could be rescued. In addition, the intramuscular injection of both freshly isolated CD11b+ monocytes and CD11b+ monocytes cultivated without rHuTPO and romiplostim had a weak rescue effect on the ischemic limbs (8 and 7 of 10 mice had auto-amputations or foot necrosis, respectively). The salvage rate from necrosis in cultivated CD11b+ monocyte with romiplostim-injected mouse group is also superior to that in cultivated CD11b+ monocyte without rHuTPO-injected mouse group. Analysis of blood perfusion by a laser Doppler perfusion imaging system showed a significantly higher recovery in mice receiving the CD11b+ monocytes cultivated with 2 ng/ml or 20 ng/ml rHuTPO or 100ng/ml romiplostim 1 week after surgery. The functional capillary density and surface area visualized by perfusion with BS-I lectin also significantly increased in the rHuTPO- or romiplostim-treated group.
In conclusion, an ex vivo addition of c-Mpl agonists augmented the pro-angiogenic activity of peripheral CD11b+ monocytes, and this method would be promising for human cell therapy to induce vascular regeneration by activating the angiogenic property in human peripheral blood-derived monocytes.

Disclosures:
Mizukami: The New Energy and Industrial Technology Development Organization of Japan: Research Funding.