Combinational Pulsing of TAAs Enforces Dendritic Cell-Based Immunotherapy through T-Cell Proliferation and Interferon-γ Secretion in LLC1 Mouse Model.

NSCLC, the most common type of lung cancer, is often diagnosed late due to minimal early symptoms. Its high risk of recurrence or metastasis post-chemotherapy makes DC-based immunotherapy a promising strategy, offering targeted cancer destruction, low side effects, memory formation, and overcoming the immune evasive ability of cancers. However, the limited response to DCs pulsed with single antigens remains a significant challenge. To overcome this, we enhanced DC antigen presentation by pulsing with TAAs. Our study focused on enhancing DC-mediated immune response specificity and intensity by combinatorial pulsing of TAAs, selected for their prevalence in NSCLC. We selected four types of TAAs expressed in NSCLC and pulsed DCs with the optimal combination. Next, we administered TAAs-pulsed DCs into the LLC1 mouse model to evaluate their anti-tumor efficacy. Our results showed that TAAs-pulsed DCs significantly reduced tumor size and promoted apoptosis in tumor tissue. Moreover, TAAs-pulsed DCs significantly increased total T cells in the spleen compared to the unpulsed DCs. Additionally, in vitro stimulation of splenocytes from the TAAs-pulsed DCs showed notable T-cell proliferation and increased IFN-γ secretion. Our findings demonstrate the potential of multiple TAA pulsing to enhance the antigen-presenting capacity of DCs, thereby strengthening the immune response against tumors.

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

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.

Urine-Derived Stem Cell-Secreted Klotho Plays a Crucial Role in the HK-2 Fibrosis Model by Inhibiting the TGF-ß Signaling Pathway.

Renal fibrosis is an irreversible and progressive process that causes severe dysfunction in chronic kidney disease (CKD). The progression of CKD stages is highly associated with a gradual reduction in serum Klotho levels. We focused on Klotho protein as a key therapeutic factor against CKD. Urine-derived stem cells (UDSCs) have been identified as a novel stem cell source for kidney regeneration and CKD treatment because of their kidney tissue-specific origin. However, the relationship between UDSCs and Klotho in the kidneys is not yet known. In this study, we discovered that UDSCs were stem cells that expressed Klotho protein more strongly than other mesenchymal stem cells (MSCs). UDSCs also suppressed fibrosis by inhibiting transforming growth factor (TGF)-ß in HK-2 human renal proximal tubule cells in an in vitro model. Klotho siRNA silencing reduced the TGF-inhibiting ability of UDSCs. Here, we suggest an alternative cell source that can overcome the limitations of MSCs through the synergetic effect of the origin specificity of UDSCs and the anti-fibrotic effect of Klotho.