A preclinical study-systemic evaluation of safety on mesenchymal stem cells derived from human gingiva tissue

Abstract

Background: Mounting evidence has shown that a novel subset of mesenchymal stem cells (MSCs) derived from human gingiva referred to as gingival mesenchymal stem cells (GMSCs) displays a greater immunotherapeutic potential and regenerative repair expression than MSCs obtained from other tissues. However, the safety of the use of transplanted GMSCs in humans remains unclear.

Methods: In this study, we evaluated the safety of GMSCs transplanted into mouse, rat, rabbit, beagle dog, and monkey as well as two animal models of autoimmune diseases.

Results: In short- and long-term toxicity tests, infused GMSCs had no remarkable adverse effects on hematologic and biochemical indexes, particularly on the major organs such as heart, liver, spleen, and kidney in recipient animals. It was also shown that GMSCs were well tolerated in other assays including hemolysis, vascular, and muscular stimulation, as well as systemic anaphylaxis and passive skin Arthus reaction in animal models. GSMC infusion did not cause any notable side effects on animal models of either autoimmune arthritis or lupus. Significantly, GMSCs most likely play no role in genotoxicity and tumorigenesis. The biological features remained stable for an extended period after cell transfer.

Conclusions: GMSCs are safe in various animal models of autoimmunity, even during active disease episodes, especially in monkeys. This study paves a solid road for future clinical trials of GMSCs in patients with autoimmune and inflammatory diseases.

Keywords: Gingival mesenchymal stem cells (GMSCs); Mesenchymal stromal cell; Safety.

Fig. 1

GMSCs do not induce inflammatory responses in blood vessels or muscles and do not induce hemolysis in vitro. a Visual inspection of rabbit ear marginal vessel. b H&E staining of ear marginal veins in the rabbit. 1.2 × 10⁶/kg of GMSCs were infused into rabbits in 1 ml/side of 0.9% NaCl solution. The same solution was used as a control, N ≥ 3 rabbits/group. c H&E staining of ear marginal muscle tissues of rabbits infused with 1.2 × 10⁵/ml of GMSCs as before N ≥ 6 rabbits/group. d Comparison of erythrocytes lysis for each group. A negative control, 0.9% NaCl solution; positive control, distilled water. N ≥ 3/group

Fig. 2

GMSCs have no effect on the passive skin allergy test in rats. BSA (100 mg/kg) was used as a positive control, and 0.9% NaCl solution (5 ml/kg) was used as a negative control. Low dose (LD) of GMSCs was 3.0 × 10⁶/kg. High dose (HD) of GMSCs was 6.0 × 10⁶/kg. N ≥ 6 rats/group

Fig. 3

GMSCs have no side effects in long-term toxicity test in rats. a GSMC influence on RBC related indexes, including hemoglobin concentration (HGB), hematocrit (HCT), erythrocyte mean corpuscular volume (MCV), mean corpuscular-hemoglobin concentration (MCHC), red blood cell distribution width (RDW), and (b) the percentage (%) of immune cells, including white blood cells (WBC), lymphocyte, monocytes (MONO), neutrophilic granulocyte (NEUT), basophilic granulocyte (BASO), eosinophilic granulocyte (EOS) by infusing GMSCs into rats in the first dose period (FP), withdrawal period (WP), and convalescence period (CP) respectively. GMSC effect on biochemical indexes (c) and H&E staining (d–e) of parts of important organ of rats. The data indicate the mean ± SD of three separated experiments. *p < 0.05, **p ≤ 0.01. N ≥ 6 rats/group

Fig. 4

GMSCs did not cause reverse mutation of S. typhimurium and chromosome aberration of CHL strain. a, b Under conditions of metabolic activation (presence of S9), positive control (50 μg/dish Dexon was for TA97, TA98, and TA102 strains, 2.0 μg/dish NaN₃ was for TA100 and TA1535 strains), c, d non-metabolic activation (absence of S9), positive control (50 μg/dish Dexon was for TA97, TA98, and TA102 stains: 2.0 μg/dish NaN3 was for TA100 and TA1535 stains). Meanwhile, 0.1 ml/dish 0.9% NaCl was as negative control. e Chromosome aberration rates under metabolic activation (with S9 mix) and f non-metabolic activation (without S9 mix) with 6-h GMSC treatment. CP and mitomycin C served as a positive control with and without S9 mix, and 0.9% NaCl solution was used as a negative control. The data represent the mean ± SD of three separated experiments. NS means no significance; *p < 0.05, **p ≤ 0.01). N ≥ 3/group

Fig. 5

The distribution and time course of GMSCs in organs of rats. a The distribution and duration in the spleen, b liver, and c lung of rats at 10-, 30-, and 58-day time points. Red fluorescence represents CM-DI pre-treated GMSCs (arrowhead). 0.9% NaCl solution-treated rats served as control. N ≥ 6 rats/group

Fig. 6

GMSCs do not influence long-term toxicity in rhesus monkeys. a Variety of blood indexes including RBC, HGB, HCT, MCV, MCHC, and RDW, and b the percentage (%) of immune cells, including WBC, MONO, NEUT, BASO, and EOS, in monkeys during the first-dose period (FP), withdrawal period (WP), and convalescence period (CP), respectively. c GMSCs effect on biochemical indexes and H&E staining (d, e) in an important organ of rhesus monkeys. The data represent the mean ± SD of three separated experiments. *p < 0.05, **p ≤ 0.01. N ≥ 10 animals/group