P3495Long-term engraftment of human CD271-positive adipose-derived stem cells with pericytic and less-aged gene profile in a mouse model of hindlimb ischemia
Authors: Inoue O , Usui S , Nomura A , Yamaguchi K , Goten C , Hamaoka T , Ootsuji H , Takashima S , Murai H , Iino K , Takemura H , Takamura M 
Published: October 2019

Abstract

Background
Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is an attractive strategy for ischemic cardiovascular diseases. We previously reported that human CD271+ population of adipose-derived stem cells (ADSCs) promoted neovascularization with enhanced engraftment in a mouse model of hindlimb ischemia. However, whether and how CD271+ ADSCs promote the long-term engraftment is still uncertain.

Purpose
We aimed to examine whether the angiogenic effect and cell engraftment capacity of CD271+ ADSCs would be sustained in long-term period. Then, comparative gene profiling between CD271+ and CD271- ADSCs were analyzed. Finally, cell proliferation and endothelial differentiation assays were conducted.

Methods
ADSCs were isolated from subcutaneous adipose tissue of 5 patients received cardiovascular surgery. CD271+ and CD271- ADSCs were sorted from CD45-CD31-CD34+ ADSCs fraction by FACS sorting (Fig. A). Cultured CD271+ and CD271- ADSCs at passage 6 were labeled by PKH26 cell linker dye and used for xenograft experiments. Briefly, athymic nude mice were subjected to hindlimb ischemia and one million of human ADSCs were injected into the ischemic muscles. In control group, PBS was solely injected. At 2 and 5 weeks, neovascularization was evaluated by immunohistochemistry (capillary density using lectin perfusion). Cell engraftment was assessed by counting PKH26-positive cells. Furthermore, we compared gene profiling between CD271+ and CD271- ADSCs by microarray. Proliferative capacity was evaluated by colony-forming unit (CFU) assay with Giemsa staining. In endothelial differentiation assay, CD271+ and CD271- ADSCs were cultured in differentiation induction medium containing vascular endothelial growth factor for 2 weeks and stained with anti-human CD31 antibody.

Results
Cell therapy using CD271+ ADSCs demonstrated approximately 3-fold more enhanced neovascularization than those using CD271- ADSCs or PBS in histological analysis of capillary density at 2 weeks from cell therapy (Fig. B and C). At 5 weeks, mice treated with CD271+ ADSCs were significantly rescued from limb ischemia and this was accompanied by sustained engraftment of ADSCs (Fig. D). In microarray analysis, the differentially expressed 2167 genes were extracted to classify CD271+ and CD271- ADSCs. Pathway analysis demonstrated CD271 expression on ADSCs was associated with the pathways related to stemness and cell differentiation. Indeed, we found that genes related to cell proliferation (PI3K, Cyclin D, and Cyclin D2) were up-regulated in CD271+ ADSCs. Additionally, we found the pericytic marker nestin which was significantly up-regulated in CD271+ ADSCs. Consistent with these findings, CD271+ ADSCs were more proliferative and capable for endothelial differentiation while CD271- ADSCs were not.
FACS and cell therapy experiments

Conclusion
These results suggest that CD271+ ADSCs possess long-term engraftment and angiogenic capacity due to their less-aged and more pericytic gene profile.

Acknowledgement/Funding
Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan) Grant Number JP16H06828