Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs

Abstract

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 10⁵ cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 10⁵ cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K⁺, Cl^- and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Keywords: dogs; endothelial progenitor cells; physiological assessment; stem cells; transplantation.

Fig. 1.

Morphology of EPCs from peripheral blood. (A) After 14 days of culture, EPC colonies appeared and showed cobblestone shape. (B) At passage 2, EPCs had a cobblestone appearance and unnecessary cells were removed by subculture.

Fig. 2.

Characterization of EPCs from peripheral blood. (A) Endothelial progenitor cells determined by cellular uptake of DiI-labeled acetylated LDL. (B) Fluorescent image of endothelial progenitor cells forming vascular networks on ECM matrix after 16 h with calcein.

Fig. 3.

Physical examination outcome. (A) Lead II ECG tracings recorded from the each group of dogs (saline infusion, saline bolus, EPC infusion, and EPC bolus) after transplantation of saline/EPCs. (B) Observation of limbs swelling from each groups of dogs after transplantation of saline/EPCs. (C) Physical examination outcome for heart murmur and neurological symptom. For evaluation of neurological symptom, cranial nerve reflexes and postural reflexes were examined. NE: none exist.

Fig. 4.

Assessment of (A) heart rate (normal range: 100–160 beats/min), (B) body temperature (normal range: 38.0–39.2°C) and (C) respiratory rate (normal range: 10–35 breaths/min) after the saline/EPC transplantation. No significant differences of vital signs among the four groups (p < 0.05) and all groups were within normal ranges. 0, 5, 10, 15, 20, 25, and 30 min: time after the saline/EPC transplantation (infusion rate: 3 ml/min, bolus rate: 60 ml/min). Each bars represent the normal reference intervals.

Fig. 5.

Evaluation of complete blood cell count after the saline/EPC transplantation.^{a, b} Within groups, values with different superscript letters are significantly different (p < 0.05) and all groups were within normal ranges. 1, 7, 14, 21, and 28 days: time after the saline/EPC transplantation (infusion rate: 3 ml/min, bolus rate: 60 ml/min). Each group has three dogs and analyses were performed a total of five times on days 1, 7, 14, 21 and 28 post-transplantation in each dog. (A) WBC; white blood cell, (B) RBC; red blood cell, (C) PLT; platelet, (D) PCV; packed cell volume, (E) HB; hemoglobin, (F) BASO; basophil, (G) EOS; eosinophil, (H) NEUT; neutrophil. Bars represent normal reference intervals.

Fig. 6.

Evaluation of serum chemistry analysis after the saline/EPC transplantation.^{a, b} Within groups, values with different superscript letters are significantly different (p < 0.05) and all groups were within normal ranges. 1, 7, 14, 21, and 28 days: time after the saline/EPC transplantation (infusion rate: 3 ml/min, bolus rate: 60 ml/min). Each group has three dogs and analyses were performed a total of five times on days 1, 7, 14, 21 and 28 post-transplantation in each dog. (A) Na, (B) K, (C) Cl, (D) ALP, (E) ALT, (F) AST, (G) CREA, (H) Albumin, (I) BUN/UREA; blood urea nitrogen/urea, (J) Total protein, (K) Ca, (L) P. Bars represent normal reference intervals.

Fig. 7.

Concentration of (A) TNF-α, (B) VEGF, and (C) IL-10 from serum in each groups after the saline/EPC transplantation. A, B, C Within groups, values with different superscript letters are significantly different (p < 0.05). 1, 7, 14, 21 and 28 days: time after the saline/EPC transplantation (infusion rate: 3 ml/min, bolus rate: 60 ml/min). Each group has three dogs and analyses were performed a total of five times on days 1, 7, 14, 21, and 28 post-transplantation in each dog.