In vivo safety and biodistribution profile of Klotho-enhanced human urine-derived stem cells for clinical application

Abstract

Background: Urine-derived stem cells (UDSCs) can be easily isolated from urine and possess excellent stem cell characteristics, making them a promising source for cell therapeutics. Due to their kidney origin specificity, UDSCs are considered a superior therapeutic alternative for kidney diseases compared to other stem cells. To enhance the therapeutic potential of UDSCs, we developed a culture method that effectively boosts the expression of Klotho, a kidney-protective therapeutic factor. We also optimized the Good Manufacturing Practice (GMP) system to ensure stable and large-scale production of clinical-grade UDSCs from patient urine. In this study, we evaluated the in vivo safety and distribution of Klotho-enhanced UDSCs after intravenous administration in accordance with Good Laboratory Practice (GLP) regulations.

Methods: Mortality and general symptoms were continuously monitored throughout the entire examination period. We evaluated the potential toxicity of UDSCs according to the administration dosage and frequency using clinical pathological and histopathological analyses. We quantitatively assessed the in vivo distribution and retention period of UDSCs in major organs after single and repeated administration using human Alu-based qPCR analysis. We also conducted long-term monitoring for 26 weeks to assess the potential tumorigenicity.

Results: Klotho-enhanced UDSCs exhibited excellent homing potential, and recovered Klotho expression in injured renal tissue. Toxicologically harmful effects were not observed in all mice after a single administration of UDSCs. It was also verified that repeated administration of UDSCs did not induce significant toxicological or immunological adverse effects in all mice. Single and repeated administrated UDSCs persisted in the blood and major organs for approximately 3 days and cleared in most organs, except the lungs, within 2 weeks. UDSCs that remained in the lungs were cleared out in approximately 4-5 weeks. There were no significant differences according to the variation of sex and administration frequency. The tumors were found in the intravenous administration group but they were confirmed to be non-human origin. Based on these results, it was clarified that UDSCs have no tumorigenic potential.

Conclusions: Our results demonstrate that Klotho-enhanced UDSCs can be manufactured as cell therapeutics through an optimized GMP procedure, and they can be safely administered without causing toxicity and tumorigenicity.

Keywords: Distribution; GMP; Klotho; Safety; Toxicity; Tumorigenicity; Urine-derived stem cells (UDSCs).

Fig. 1

Characterization of Klotho-enhanced UDSCs. In these experiments, all UDSCs from different donors were manufactured in a GMP facility. A Multi-differentiation capability was confirmed by Oil Red O, Alizarin Red S, and Alcian Blue staining. B Cell surface marker expression was analyzed by flow cytometry at passage 5. C An increase in Klotho protein expression after culturing with KL-medium was confirmed by western blotting. Full-length original blot images are presented in Additional file 2: Figure S1. Results were shown as the mean ± SEM. *p < 0.05, ***p < 0.001 vs. 5% KSFM (p5). KSFM, keratinocyte serum-free medium; IRI, Ischemia–reperfusion injury

Fig. 2

Therapeutic potentials of Klotho-enhanced UDSCs. A Klotho-enhanced UDSCs (PKH26: Red) were intravenously administered to both wild-type (Sham) and ischemia–reperfusion injury (IRI) groups. B Klotho expression (Klotho: Green) was detected in the renal cortex region after Klotho-enhanced UDSCs administration. C The images of A and B have been quantitatively analyzed. D Tissue injury was assessed using H&E staining. Renal fibrosis was measured using MT staining and type IV Collagen expression. E Kidney injury was represented by urine NGAL levels and was normalized using urine creatinine levels. Results were shown as the mean ± SEM. **p < 0.01, ****p < 0.0001. H&E, hematoxylin and eosin; MT, Masson’s trichrome; NGAL, neutrophil gelatinase associated lipocalin

Fig. 3

The body weight and food consumption changes following a single and repeated administration. A, B Body weight changes after a single and repeated intravenous administration were assessed. C Food consumption after repeated intravenous administration was assessed. An additional 2-week recovery period in the vehicle control (G1) and high-dose group (G4) was investigated. Results were shown as the mean ± SEM

Fig. 4

Lymphocyte phenotype and T cell subpopulation analysis following repeated administration of UDSCs. A Representative gating strategy: Lymphocytes were first gated based on forward scatter (FSC) versus side scatter (SSC), and CD3⁺, CD4⁺, CD8⁺, CD45R⁺, and CD49b⁺ cells were identified from the lymphocyte subset. B Both male and female mice showed no significant changes in lymphocyte and T cell subpopulation. Results were shown as the mean ± SEM

Fig. 5

In vivo distribution following single and repeated administration of UDSCs. A, B After single and repeated administration of UDSCs, the hAlu-based qPCR analysis was conducted on major organs at various time points. The concentration of UDSCs was estimated using a validated quantification formula (data were not shown). Results were shown as the mean ± SEM. LOD = 1 pg. LLOQ = 10 pg. LN, lymph node

Fig. 6

Long-term monitoring of tumorigenicity following repeated administration of UDSCs. A Tumorigenic potential was examined during 26 weeks after repeated subcutaneous and intravenous administration of UDSCs. B UDSCs-administered groups (G3, G4) exhibited no significant changes in mortality rate compared to the vehicle control group (G1). C Tumor formation was assessed in all groups during the final necropsy. D Immunohistochemistry analysis on tumor tissues from the intravenous administration group (G4) was conducted to determine the origin of tumors. **p < 0.01. NOS, not otherwise specified