Aims/Introduction Dental pulp stem cells (DPSCs) can be easily obtained from teeth for general orthodontic reasons. We have previously reported the therapeutic effects of DPSC transplantation for diabetic polyneuropathy. As abundant secretomes from DPSCs are considered to play a central role in the improvement of diabetic polyneuropathy, we investigated whether direct injection of DPSC‐conditioned media (DPSC‐CM) into hindlimb skeletal muscles ameliorates diabetic polyneuropathy in diabetic rats. Materials and Methods DPSCs were isolated from the dental pulp of Sprague–Dawley rats. Eight weeks after the induction of diabetes, DPSC‐CM was injected into the unilateral hindlimb skeletal muscles in both normal and diabetic rats. The effects of DPSC‐CM on diabetic polyneuropathy were assessed 4 weeks after DPSC‐CM injection. To confirm the angiogenic effect of DPSC‐CM, the effect of DPSC‐CM on cultured human umbilical vascular endothelial cell proliferation was investigated. Results The administration of DPSC‐CM into the hindlimb skeletal muscles significantly ameliorated sciatic motor/sensory nerve conduction velocity, sciatic nerve blood flow and intraepidermal nerve fiber density in the footpads of diabetic rats. We also showed that DPSC‐CM injection significantly increased the capillary density of the skeletal muscles, and suppressed pro‐inflammatory reactions in the sciatic nerves of diabetic rats. Furthermore, an in vitro study showed that DPSC‐CM significantly increased the proliferation of umbilical vascular endothelial cells. Conclusions We showed that DPSC‐CM injection into hindlimb skeletal muscles has a therapeutic effect on diabetic polyneuropathy through neuroprotective, angiogenic and anti‐inflammatory actions. DPSC‐CM could be a novel cell‐free regenerative medicine treatment for diabetic polyneuropathy.

Background: Dental pulp stem cells (DPSCs) have high proliferation and multi-differentiation capabilities that maintain their functionality after cryopreservation. In our previous study, we demonstrated that cryopreserved rat DPSCs improved diabetic polyneuropathy and that the efficacy of cryopreserved rat DPSCs was equivalent to that of freshly isolated rat DPSCs. The present study was conducted to evaluate whether transplantation of cryopreserved human DPSCs (hDPSCs) is also effective for the treatment of diabetic polyneuropathy. Methods: hDPSCs were isolated from human impacted third molars being extracted for orthodontic reasons. Eight weeks after the induction of diabetes in nude mice, hDPSCs (1 × 10 5 /limb) were unilaterally transplanted into the hindlimb skeletal muscle, and vehicle (saline) was injected into the opposite side as a control. The effects of hDPSCs were analyzed at 4 weeks after transplantation. Results: hDPSC transplantation significantly ameliorated reduced sensory perception thresholds, delayed nerve conduction velocity, and decreased the blood flow to the sciatic nerve in diabetic mice 4 weeks posttransplantation. Cultured hDPSCs secreted the vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) proteins. A subset of the transplanted hDPSCs was localized around the muscle bundles and expressed the human VEGF and NGF genes at the transplanted site. The capillary/muscle bundle ratio was significantly increased on the hDPSC-transplanted side of the gastrocnemius muscles in diabetic mice. Neutralizing antibodies against VEGF and NGF negated the effects of hDPSC transplantation on the nerve conduction velocity in diabetic mice, suggesting that VEGF and NGF may play roles in the effects of hDPSC transplantation on diabetic polyneuropathy.

Stem cell transplantation is a potential novel therapy for diabetic polyneuropathy. Dental pulp stem cells (DPSCs) are attractive stem cell sources because DPSCs can be isolated from extracted teeth and cryopreserved while retaining viability. In this study, we directly compared the efficacy of the transplantation of DPSCs and the administration of the secreted factors from DPSCs (DPSC-SFs) on diabetic polyneuropathy. Eight weeks after streptozotocin injection, DPSCs (1.0 × 106 cells/rat) or DPSC-SFs (1.0 mL/rat) were administered into the unilateral hindlimb skeletal muscles of diabetic Sprague–Dawley rats. DPSC transplantation and DPSC-SF administration did not affect blood glucose levels and body weights in the diabetic rats. Both DPSC transplantation and DPSC-SF administration significantly ameliorated sciatic nerve conduction velocity and sciatic nerve blood flow, accompanied by increases in muscle bundle size, vascular density in the skeletal muscles and intraepidermal nerve fiber density in the diabetic rats, while there was no difference between the results for DPSCs and DPSC-SFs. These results suggest that the efficacy of both DPSC transplantation and DPSC-SF administration for diabetic polyneuropathy four weeks after transplantation/administration was mainly due to the multiple secretomes secreted from transplanted DPSCs or directly injected DPSC-SFs in the early phase of transplantation/administration.

Exercise improves not only glycemic control, but also other metabolic disorders in diabetic patients. Furthermore, exercise also stimulate muscle-derived secreted factors, myokines, via muscle contraction. The receptors of myokines expressed in the whole body and myokines affect autocrine, paracrine, and systemic functions for all people and patients with diabetes. In this study, we have demonstrated that there are exercise-like impacts of conditioned media from cultured dental pulp stem cells (DPSCs) on myokine expressions in skeletal muscles. DPSCs were isolated and cultured from the mandibular incisors of 6-week-old male Sprague-Dawley (SD) rats. Conditioned media from DPSCs (DPSC-CM) was collected after 24-hour culture of DPSCs in the serum-free medium and concentrated by a factor of 10. We firstly investigated the effects of DPSC-CM on exercise-induced myokines, follistatin-like 1 (Fstl1), Fractalkine, and fibroblast growth factor 21 (FGF-21) in C2C12 myotubes. DPSC-CM significantly increased the gene expressions of Fstl1 (209±16%, P<0.01), Fractalkine(182±41%, P<0.05), and FGF-21 (1021±39%, P<0.01) in C2C12 myotubes. To confirm these effects in vivo, we administered DPSC-CM (1.0 ml/rat) into 10 sites of unilateral hindlimb skeletal muscles of male SD rats, and examined the effects on myokine gene and protein expression. DPSC-CM significantly increased Fstl1 gene expression in the gastrocnemius muscles (477±140%, P<0.05). Immunohistochemical staining revealed that the expression of Fstl1 was increased in the DPSC-CM-injected gastrocnemius muscles. These results suggest that there is a novel exercise-like impact of DPSC-CM on skeletal muscles. Disclosure S. Kanada: None. E. Makino: None. N. Nakamura: None. M. Miyabe: None. M. Ito: None. M. Hata: None. T. Saiki: None. T. Minato: None. K. Miyazawa: None. S. Goto: None. T. Matsubara: None. K. Naruse: None.

Transplantation of cryopreserved DPSCs ameliorated diabetic neuropathy equal to freshly isolated DPSCs. Human DPSCs (hDPSCs) can be isolated from teeth extracted at a young age and can be cryopreserved until use. The aim of this study is to clarify the therapeutic mechanism of hDPSCs transplantation on diabetic polyneuropathy. We collected human impacted third molars from adult at Aichi-Gakuin University hospital. Written informed consent was obtained from each donor. Identification of hDPSCs was analyzed by FACS and differentiation capabilities. Diabetes was induced by injection of STZ in 6 week-old BALB/cAJcl-nu/nu mice. Eight weeks after STZ injection, hDPSCs were transplanted into unilateral hindlimb skeletal muscles of normal and diabetic mice. Four weeks after transplantation, sciatic blood flow (SNBF), sciatic motor/sensory nerve conduction velocity (MNCV/SNCV) and current perception threshold (CPT) were assessed. To elucidate the therapeutic effects of hDPSCs, diabetic mice were treated with neutralizing antibody soon after transplantation. Immunohistological and gene expression analysis of hindlimb skeletal muscles were also performed at the end of the experiments. Diabetic mice showed significant reductions in SNBF, MNCV and SNCV and increase in CPTs in the control side compared with normal mice. Transplantation of hDPSCs significantly ameliorated the impaired SNBF, MNCV, SNCV and CPTs in the hDPSCs-injected side. The transplanted hDPSCs were located around the muscle bundles and expressed the human VEGF and NGF genes. Capillary/muscle bundle ratio was also increased in the hDPSCs-injected side. Furthremore, the effects of hDPSCs transplantation were cancelled by neutralizing antibodies of VEGF and NGF. These results suggest that cell therapy using hDPSCs may be useful for treatment of diabetic neuropathy via the angiogenic and neurotrophic factor by hDPSCs secretion. Disclosure M. Hata: None. M. Omi: None. N. Nakamura: None. M. Miyabe: None. M. Ito: None. E. Makino: None. S. Kanada: None. T. Ono: None. Y. Imanishi: None. T. Himeno: None. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehlinger Ingelheim Japan Co., Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott Japan Co., Ltd., ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co., Ltd.,, Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Mylan, Novartis Pharma K.K., Novo Nordisk Pharma Ltd, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. H. Kamiya: Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim K.K., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. S. Ozawa: None. J. Takebe: None. T. Matsubara: None. K. Naruse: None.