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. 2022 Jul;55(7):336-341.
doi: 10.5483/BMBRep.2022.55.7.003.

Combination stem cell therapy using dental pulp stem cells and human umbilical vein endothelial cells for critical hindlimb ischemia

Chung Kwon Kim  1 , Ji-Yoon Hwang  2 , Tae Hee Hong  3 , Du Man Lee  4 , Kyunghoon Lee  5 , Hyun Nam  6 , Kyeung Min Joo  7
Affiliations

Combination stem cell therapy using dental pulp stem cells and human umbilical vein endothelial cells for critical hindlimb ischemia

Chung Kwon Kim et al. BMB Rep. 2022 Jul.

Abstract

Narrowing of arteries supplying blood to the limbs provokes critical hindlimb ischemia (CLI). Although CLI results in irreversible sequelae, such as amputation, few therapeutic options induce the formation of new functional blood vessels. Based on the proangiogenic potentials of stem cells, in this study, it was examined whether a combination of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) could result in enhanced therapeutic effects of stem cells for CLI compared with those of DPSCs or HUVECs alone. The DPSCs+ HUVECs combination therapy resulted in significantly higher blood flow and lower ischemia damage than DPSCs or HUVECs alone. The improved therapeutic effects in the DPSCs+ HUVECs group were accompanied by a significantly higher number of microvessels in the ischemic tissue than in the other groups. In vitro proliferation and tube formation assay showed that VEGF in the conditioned media of DPSCs induced proliferation and vessel-like tube formation of HUVECs. Altogether, our results demonstrated that the combination of DPSCs and HUVECs had significantly better therapeutic effects on CLI via VEGF-mediated crosstalk. This combinational strategy could be used to develop novel clinical protocols for CLI proangiogenic regenerative treatments. [BMB Reports 2022; 55(7): 336-341].

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Therapeutic effects of DPSCs and/or HUVECs transplantation for CLI. (A) Experimental schedule. (B) Leg images of CLI animal models. (C) The degree of the legs damages were analyzed and compared. n = 10 for each group. *P < 0.05. (D) Blood flow in the legs of CLI animal models was measured by LDI. (E) The blood flow was analyzed and compared. n = 10 for each group. *P < 0.05.
Fig. 2
Fig. 2
Histological analysis of CLI animal models. Ischemic hind limb muscles were retrieved at 14 days post-injection for histological analysis. (A) In the H&E staining, the severity of muscle degeneration and infiltration of immune cells were analyzed and compared. (B) The degree of fibrosis was assessed by Masson’s trichrome staining. (C) The fibrosis area was quantified from Masson’s trichrome staining and compared. (D) The number of vessels was assessed by immunohistochemistry against CD31. (E) The number of vessels was quantified and compared. n = 10 for each group. *P < 0.05.
Fig. 3
Fig. 3
Angiogenic paracrine effects of DPSCs CM. (A) Effects of the DPSCs CM on the HUVECs differentiation were analyzed by the in vitro tube formation assay. (B) Effects of the DPSCs CM on the proliferation of HUVECs were analyzed. (C) Concentration of VEGF, HGF, and TNF-α was quantified by ELISA. n = 10 for each group. *P < 0.05. n.s., not significant. LLOQ = lower limit of quantification. (D) Effects of the DPSCs CM on the HUVECs signaling path-ways were analyzed by western blot analysis.
Fig. 4
Fig. 4
Proangiogenic effects of DPSCs mediated by VEGF. Effects of the DPSCs CM on differentiation (A), proliferation (B), and signaling pathways (C, D) of HUVECs with or without bevacizumab, a VEGF neutralizing antibody, were analyzed and compared. *P < 0.05.
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