Combination stem cell therapy using dental pulp stem cells and human umbilical vein endothelial cells for critical hindlimb ischemia.

Narrowing of arteries supplying blood to the limbs provokes critical hindlimb ischemia (CLI). Although CLI results in irreversible sequelae, such as amputation, few therapeutic options induce the formation of new functional blood vessels. Based on the proangiogenic potentials of stem cells, in this study, it was examined whether a combination of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) could result in enhanced therapeutic effects of stem cells for CLI compared with those of DPSCs or HUVECs alone. The DPSCs+ HUVECs combination therapy resulted in significantly higher blood flow and lower ischemia damage than DPSCs or HUVECs alone. The improved therapeutic effects in the DPSCs+ HUVECs group were accompanied by a significantly higher number of microvessels in the ischemic tissue than in the other groups. In vitro proliferation and tube formation assay showed that VEGF in the conditioned media of DPSCs induced proliferation and vessel-like tube formation of HUVECs. Altogether, our results demonstrated that the combination of DPSCs and HUVECs had significantly better therapeutic effects on CLI via VEGF-mediated crosstalk. This combinational strategy could be used to develop novel clinical protocols for CLI proangiogenic regenerative treatments. [BMB Reports 2022; 55(7): 336-341].

Optimal Preclinical Conditions for Using Adult Human Multipotent Neural Cells in the Treatment of Spinal Cord Injury.

Stem cell-based therapeutics are amongst the most promising next-generation therapeutic approaches for the treatment of spinal cord injury (SCI), as they may promote the repair or regeneration of damaged spinal cord tissues. However, preclinical optimization should be performed before clinical application to guarantee safety and therapeutic effect. Here, we investigated the optimal injection route and dose for adult human multipotent neural cells (ahMNCs) from patients with hemorrhagic stroke using an SCI animal model. ahMNCs demonstrate several characteristics associated with neural stem cells (NSCs), including the expression of NSC-specific markers, self-renewal, and multi neural cell lineage differentiation potential. When ahMNCs were transplanted into the lateral ventricle of the SCI animal model, they specifically migrated within 24 h of injection to the damaged spinal cord, where they survived for at least 5 weeks after injection. Although ahMNC transplantation promoted significant locomotor recovery, the injection dose was shown to influence treatment outcomes, with a 1 × 106 (medium) dose of ahMNCs producing significantly better functional recovery than a 3 × 105 (low) dose. There was no significant gain in effect with the 3 × 106 ahMNCs dose. Histological analysis suggested that ahMNCs exert their effects by modulating glial scar formation, neuroprotection, and/or angiogenesis. These data indicate that ahMNCs from patients with hemorrhagic stroke could be used to develop stem cell therapies for SCI and that the indirect injection route could be clinically relevant. Moreover, the optimal transplantation dose of ahMNCs defined in this preclinical study might be helpful in calculating its optimal injection dose for patients with SCI in the future.

In vivo distribution of U87MG cells injected into the lateral ventricle of rats with spinal cord injury.

Stem cells could be the next generation therapeutic option for neurodegenerative diseases including spinal cord injury (SCI). However, several critical factors such as delivery method should be determined before their clinical applications. Previously, we have demonstrated that lateral ventricle (LV) injection as preclinical simulation could be used for intrathecal administration in clinical trials using rodent animal models. In this study, we further analyzed in vivo distribution of cells that were injected into LVs of rats with SCI at thoracic level using in vivo imaging techniques. When 5 × 106 U87MG cells labelled with fluorescent magnetic nanoparticle (FMNP-labelled U87MG) were administrated into LVs at 7 days after SCI, FMNP-labelled U87MG cells were observed in all regions of the spinal cord at 24 hours after the injection. Compared to water-soluble Cy5.5 fluorescent dye or rats without SCI, in vivo distribution pattern of FMNP-labelled U87MG cells was not different, although migration to the spinal cord was significantly reduced in both Cy5.5 fluorescent dye and FMNP-labelled U87MG cells caused by the injury. The presence of FMNP-labelled U87MG cells in the spinal cord was confirmed by quantitative PCR for human specific sequence and immunohistochemistry staining using antibody against human specific antigen. These data indicate that LV injection could recapitulate intrathecal administration of stem cells for SCI patients. Results of this study might be applied further to the planning of optimal preclinical and clinical trials of stem cell therapeutics for SCI.

Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures.

Patients with head and neck cancer are treated with therapeutic irradiation, which can result in irreversible salivary gland dysfunction. Because there is no complete cure for such patients, stem cell therapy is an emerging alternative for functional restoration of salivary glands. In this study, we investigated in vitro characteristics of primarily isolated epithelial cells from human salivary gland (Epi-SGs) and in vivo formation of acini-like structures by Epi-SGs. Primarily isolated Epi-SGs showed typical epithelial cell-like morphology and expressed E-cadherin but not N-cadherin. Epi-SGs expressed epithelial stem cell (EpiSC) and embryonic stem cell (ESC) markers. During long-term culture, the expression of EpiSC and ESC markers was highly detected and maintained within the core population with small size and low cytoplasmic complexity. The core population expressed cytokeratin 7 and cytokeratin 14, known as duct markers indicating that Epi-SGs might be originated from the duct. When Epi-SGs were transplanted in vivo with Matrigel, acini-like structures were readily formed at 4 days after transplantation and they were maintained at 7 days after transplantation. Taken together, our data suggested that Epi-SGs might contain stem cells which were positive for EpiSC and ESC markers, and Epi-SGs might contribute to the regeneration of acini-like structures in vivo. We expect that Epi-SGs will be useful source for the functional restoration of damaged salivary gland.

Silver Nanoparticles Potentiates Cytotoxicity and Apoptotic Potential of Camptothecin in Human Cervical Cancer Cells.

Silver nanoparticles (AgNPs) are widely used metal nanoparticles in health care industries, particularly due to its unique physical, chemical, optical, and biological properties. It is used as an antibacterial, antiviral, antifungal, and anticancer agent. Camptothecin (CPT) and its derivatives function as inhibitors of topoisomerase and as potent anticancer agents against a variety of cancers. Nevertheless, the combined actions of CPT and AgNPs in apoptosis in human cervical cancer cells (HeLa) have not been elucidated. Hence, we investigated the synergistic combinatorial effect of CPT and AgNPs in human cervical cancer cells. We synthesized AgNPs using sinigrin as a reducing and stabilizing agent. The synthesized AgNPs were characterized using various analytical techniques. The anticancer effects of a combined treatment with CPT and AgNPs were evaluated using a series of cellular and biochemical assays. The expression of pro- and antiapoptotic genes was measured using real-time reverse transcription polymerase chain reaction. The findings from this study revealed that the combination of CPT and AgNPs treatment significantly inhibited cell viability and proliferation of HeLa cells. Moreover, the combination effect significantly increases the levels of oxidative stress markers and decreases antioxidative stress markers compared to single treatment. Further, the combined treatment upregulate various proapoptotic gene expression and downregulate antiapoptotic gene expression. Interestingly, the combined treatment modulates various cellular signaling molecules involved in cell survival, cytotoxicity, and apoptosis. Overall, these results suggest that CPT and AgNPs cause cell death by inducing the mitochondrial membrane permeability change and activation of caspase 9, 6, and 3. The synergistic cytotoxicity and apoptosis effect seems to be associated with increased ROS formation and depletion of antioxidant. Certainly, a combination of CPT and AgNPs could provide a beneficial effect in the treatment of cervical cancer compared with monotherapy.