Citric acid buffer

Six-week-old male FVB/N mice were purchased from the Jackson laboratory. Animal studies were performed following relevant guidelines and regulations approved by the University of South Florida under ARRIVE guidelines. Animals were housed at constant RT (20 ± 1 °C) under a controlled 12-h light to 12-h dark cycle and had free access to water and a standard diet. The mice received a single i.p. injection of streptozotocin (100 mg/kg body weight, in 0.1 mol/l citric acid buffer, pH 4.5; Sigma–Aldrich, St Louis, MO, U.S.A.) to induce diabetes, or citric acid buffer vehicle (non-diabetic mice) (19 (link)–21 (link)). Diabetes was confirmed by measuring blood glucose from the saphenous vein using a glucometer (Roche, Basel, Switzerland). Mice with streptozotocin-induced blood glucose levels >300 mg/dl were included in the diabetic groups.

A total of 40 C57BL/6 male mice (ORIENTBIO, SNM, KYG, KR) were included in the study. All experimental animal protocols used were approved by the Institutional Animal Care and Use Committees (IACUC) of EBO cooperation (approval number EBOA-2019-2), and all experimental procedures were performed in accordance with the institutional guidelines. Diabetes was induced by intraperitoneally injecting 12-h fasted mice with 55 mg/kg STZ in citric acid buffer at 4.5 pH (Sigma-Aldrich, St. Louis, MO, USA) for five consecutive days [69 (link)]. Mice were subsequently fasted for another 12 h, and Fasting Blood Glucose (FBG) levels were examined 72 h after STZ administration. Mice with induced T1D were selected using an FBG ≥300 mg/dL as a threshold [70 (link)]. For this experiment, mice were divided into the following four groups: G1, normal control; G2, T1D control, administered STZ (55 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) via intraperitoneal injection once a day (STZ dissolved in 0.1 mol/L citrate buffer, pH 4.5); G3, received STZ and were injected with hUC-MSCs (1 × 10⁶) one week after T1D induction; and G4, received STZ and were injected with differentiated hUC-MSCs into IPCs; hUC-IPCs (5000 IEQ) one week after T1D induction (Table 4). The in vivo experiments were designed according to the protocol depicted in Figure 8a.

The DN rat model was induced according to our previously published study [17 (link)]. DN was induced in the overnight fasted rats by a one-time intraperitoneal injection of streptozotocin (STZ) (50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) dissolved in citric acid buffer 40 mg/mL (pH 4.5, Sigma, St. Louis, USA) [17 (link),24 (link),25 (link)]. Rat blood glucose was maintained at 350 mg/dL by injection with insulin (0.4 unit/rat) and maintained for 12 weeks to establish the DN rat model successfully [17 (link),26 (link),27 (link)].