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. 2021 Sep;24(3):343-350.
doi: 10.1007/s10047-021-01254-8. Epub 2021 Mar 3.

New cell delivery system CellSaic with adipose-derived stromal cells promotes functional angiogenesis in critical limb ischemia model mice

Hideki Tanioka  1 , Shigeru Miyagawa  2 , Daisuke Mori  2 , Ken-Ichi Watanabe  2 , Takayoshi Ueno  2 , Koichi Toda  2 , Takashi Shibuya  2 , Toru Kuratani  2 , Yoshiki Sawa  3
Affiliations

New cell delivery system CellSaic with adipose-derived stromal cells promotes functional angiogenesis in critical limb ischemia model mice

Hideki Tanioka et al. J Artif Organs. 2021 Sep.

Abstract

Current therapies for patients with critical limb ischemia have not reduced amputation risk owing to poor cell engraftment. The recombinant peptide Cellnest increases the engraftment rate of administered cells by forming a complex with the cells (CellSaic). We hypothesized that CellSaic containing adipose-derived stromal cells (ADSCs) could improve lower limb blood flow better than ADSCs alone, resulting in better transplanted cell engraftment. ADSCs were extracted from 8-week-old C57BL/6N mice. Thirty-two critical limb ischemia model mice were established by ligating femoral arteries. They were divided into CellSaic (n = 11), ADSC (n = 10), saline (n = 9), and Cellnest (n = 9) groups. Blood flow rate (affected side blood flow / healthy side blood flow × 100%) was evaluated using a laser Doppler blood flow meter every week. Mice were euthanized on day 28 for histological evaluation. Compared with the ADSC group (54.5 ± 17.2%), treated side blood flow rate of the CellSaic group (78.0 ± 24.9%) showed significant improvement on day 28 after administration (p < 0.05). CD31 staining showed significantly higher number of capillary vessels in the CellSaic group (53.0 ± 8.9 cells/mm3) than in the ADSC group (43.0 ± 6.8 cells/mm3) (p < 0.05). Fluorescent staining showed significantly higher number of arterioles containing both CD31 and αSMA double-positive cells in the CellSaic group than in the ADSC group (p < 0.05). CellSaic containing ADSCs exhibited superiority to ADSC transplantation alone in promoting functional angiogenesis, suggesting its potential in improving clinical outcomes of angiogenic therapy for ischemic limbs.

Keywords: Adipose-derived stromal cell; Angiogenesis; Critical limb ischemia; Engraftment rate.

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Conflict of interest statement

We know of no conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.

Figures

Fig. 1
Fig. 1
Characteristics of ADSCs extracted from mice inguinal adipose tissue. ADSCs were positive for the stromal cell markers, Sca-1 and CD44, and negative for the mesenchymal stem cell markers, CD73 and CD90
Fig. 2
Fig. 2
Synthesis of CellSaic by mixing Cellnest® and ADSCs. CellSaic platforms containing ADSCs were prepared by mixing ADSCs (2.0 × 104 cells) and Cellnest® pieces (0.02 mg) in 200 μL Dulbecco’s modified Eagle’s medium (DMEM)
Fig. 3
Fig. 3
Improvement of blood flow in the affected lower limb. Compared with the control group, the treated side blood flow rate of the ADSC group showed significant improvement on the 28th day after administration. Compared with the ADSC group, the treated side blood flow rate of the CellSaic group showed significant improvement on the 28th day after administration
Fig. 4
Fig. 4
Enumeration of CD31-positive cells via immunostaining. The number of CD31-positive cells was significantly higher in the ADSC group than in the control group and was significantly higher in the CellSaic group than in the ADSC group
Fig. 5
Fig. 5
Evaluation of double-positive (CD31, αSMA) cells determined by fluorescent staining. The number of double-positive cells was significantly higher in the ADSC group than in the control group and was significantly higher in the CellSaic group than in the ADSC group
Fig. 6
Fig. 6
Angiogenic factors such as VEGF and HGF in muscle tissues analyzed using RT-PCR. There was no significant difference in VEGF expression between the CellSaic and ADSC groups. However, relative quantification (RQ) indicated significantly higher HGF expression in the CellSaic group than in the ADSC group
Fig. 7
Fig. 7
Evaluation of engraftment rate of administered cells using GFP-positive cells. No surviving cells were found in the ADSC group. However, in the CellSaic group, it was possible to confirm the presence of some engrafted cells
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