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.

Effect of Additional Cytoplasm of Cloned Embryo on In Vitro Developmental Competence and Reprogramming Efficiency in Mice.

Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher (p < 0.05) than that of the SCNT method (96.6% ± 1.1% vs. 86.7% ± 6.0%, 29.5% ± 2.6% vs. 22.1% ± 3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7 ± 0.2 vs. 2.9 ± 0.3, p < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group (p < 0.05), and the relative acH3K56 level in the CICT group was significantly (p < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the in vitro developmental competence and quality of cloned embryos.

Matrix metalloproteinases improves trophoblast invasion and pregnancy potential in mice.

Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson's trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.

Production of cloned cats using additional complimentary cytoplasm.

Somatic cell nuclear transfer (SCNT) is an important technique for producing cloned animals. It, however, is inefficient when there is use of SCNT for cloned animal production. Cytoplasm injection cloning technology (CICT) was developed to overcome the inefficiencies of SCNT use of this purpose. The use of CICT involves additional cytoplasm fusing with enucleated oocytes to restore the cytoplasmic volume, thus improving the in vitro developmental competence and quality of cloned embryos. In this study, there was application of CICT in cats to improve the in vitro developmental competence of cloned embryos, as well as the production of the offspring. The results of this study were that fusion rate of the cloned embryos with use of the CICT method was greater than that with SCNT (80.0 ± 4.8% compared with 67.8 ± 11.3%, respectively), and more blastocysts developed with use of CICT than SCNT (20.0 ± 2.0% compared with 13.5 ± 5.0%, respectively). The 62 cloned embryos that were produced with use of CICT were transferred into five estrous synchronized recipients, and 151 cloned embryos produced using SCNT were transferred to 13 estrous-synchronized recipients. After the embryo transfer, there was birth from surrogate mothers of one live-born kitten that resulted using SCNT compared with three live-born kittens using CICT. The number of CICT-cloned embryos born was greater than that of SCNT-cloned embryos (4.8 ± 2.3% compared with 0.7 ± 1.3%, P < 0.05). These results indicate that the CICT technique can be used to produce cloned kittens, including endangered feline species.

Optimized Clump Culture Methods for Adult Human Multipotent Neural Cells.

Adult human multipotent neural cell (ahMNC) is a candidate for regeneration therapy for neurodegenerative diseases. Here, we developed a primary clump culture method for ahMNCs to increase the efficiency of isolation and in vitro expansion. The same amount of human temporal lobe (1 g) was partially digested and then filtered through strainers with various pore sizes, resulting in four types of clumps: Clump I > 100 µm, 70 µm < Clump II < 100 µm, 40 µm < Clump III < 70 µm, and Clump IV < 40 µm. At 3 and 6 days after culture, Clump II showed significantly higher number of colonies than the other Clumps. Moreover, ahMNCs derived from Clump II (ahMNCs-Clump II) showed stable proliferation, and shortened the time to first passage from 19 to 15 days, and the time to 1 × 108 cells from 42 to 34 days compared with the previous single-cell method. ahMNCs-Clump II had neural differentiation and pro-angiogenic potentials, which are the characteristics of ahMNCs. In conclusion, the novel clump culture method for ahMNCs has significantly higher efficiency than previous techniques. Considering the small amount of available human brain tissue, the clump culture method would promote further clinical applications of ahMNCs.

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.

Significant therapeutic effects of adult human multipotent neural cells on spinal cord injury.

Neural stem cells are emerging as a regenerative therapy for spinal cord injury (SCI), since they differentiate into functional neural cells and secrete beneficial paracrine factors into the damaged microenvironment. Previously, we successfully isolated and cultured adult human multipotent neural cells (ahMNCs) from the temporal lobes of epileptic patients. In this study, we investigated the therapeutic efficacy and treatment mechanism of ahMNCs for SCI using rodent models. When 1 × 106 ahMNCs were transplanted into injured spinal cords at 7 days after contusion, the injection group showed significantly better functional recovery than the control group (media injection after contusion), which was determined by the Basso, Beattie and Bresnahan (BBB) score. Although transplanted ahMNCs disappeared continuously, remained cells expressed differentiated neural cell markers (Tuj1) or astrocyte marker (GFAP) in the injured spinal cords. Moreover, the number of CD31-positive microvessels significantly increased in the injection group than that of the control group. The paracrine pro-angiogenic activities of ahMNCs were confirmed by in vitro tube formation assay and in vivo Matrigel plug assay. Together, these results indicate that ahMNCs have significant therapeutic efficacy in SCI via replacement of damaged neural cells and pro-angiogenic effects on the microenvironment of 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.

In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model.

OBJECTIVE: The purpose of this study was to find an optimal delivery route for clinical trials of intrathecal cell therapy for spinal cord injury in preclinical stage. METHODS: We compared in vivo distribution of Cy5.5 fluorescent dye in the spinal cord region at various time points utilizing in vivo optical imaging techniques, which was injected into the lateral ventricle (LV) or cisterna magna (CM) of rats. RESULTS: Although CM locates nearer to the spinal cord than the LV, significantly higher signal of Cy5.5 was detected in the thoracic and lumbar spinal cord region at all time points tested when Cy5.5 was injected into the LV. In the LV injection Cy5.5 signal in the thoracic and lumbar spinal cord was observed within 12 hours after injection, which was maintained until 72 hours after injection. In contrast, Cy5.5 signal was concentrated at the injection site in the CM injection at all time points. CONCLUSION: These data suggested that the LV might be suitable for preclinical injection route of therapeutics targeting the spinal cord to test their treatment efficacy and biosafety for spinal cord diseases in small animal models.

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.

Effects of phalloidin on hepatic gene expression in mice.

An attempt has been made to identify molecular markers of intrahepatic cholestasis in mice employing phalloidin as a cholestatic agent. Phalloidin was administered to BALB/c mice at three predetermined dose: 250 microg/kg, 500 microg/kg, and 1 mg/kg for 1, 3, and 7 days. Liver function was estimated to confirm cholestasis. Histopathological observations on liver were also made to confirm liver injury. Phalloidin at 1 mg/kg for 7 days was found to induce cholestasis. Therefore gene expression studies were confined to this group only. A total of 88 genes were found to be affected by phalloidin. These were the genes associated with cytoskeleton regulation as well as tight junction, focal adhesion, and ATP-binding cassette transporters. Such proteins obstruct the removal of bile components from hepatocytes to the bile canaliculus or blood. Phalloidin treatment did not affect the proteins responsible for cell maintenance or death. The authors show that phalloidin-induced intrahepatic cholestasis is manifested by disturbing the cytoskeleton. The set of genes up-regulated by phalloidin can be considered as molecular markers of intrahepatic cholestasis. The observations are further expected to be helpful in the management of cholestatic pharmaceuticals and associated problems of liver diseases in humans.

Effects of combined treatment with sildenafil and itraconazole on the cardiovascular system in telemetered conscious dogs.

Sildenafil, a potent PDE5 inhibitor, is widely prescribed as a treatment of erectile dysfunction. Itraconazole is an inhibitor of CYP3A4, a metabolic enzyme of sildenafil. In the current study, we investigated the effects of single treatment with sildenafil and combined treatment with sildenafil and itraconazole on blood pressure, heart rate, and QT interval in conscious beagle dogs. After a transmitter was implanted to beagle dogs for conscious state experiments, a single oral dose of sildenafil was administered to the beagle dogs at dose levels of 3, 15, and 30 mg/kg. Blood pressure, heart rate, and lead II ECG were measured prior to dosing and at 0.5, 1, 2, 4, 6, and 24 h postdosing. In the study of combined treatment with sildenafil and itraconazole, the 100 mg/kg dose of itraconazole was orally administered 1 h prior to oral administration of sildenafil. No changes in blood pressure were observed at any doses in animals receiving either single treatment with sildenafil or combined treatment with sildenafil and itraconazole. Increased heart rate from 0.5 h to 6 h postdosing and decreased QT interval were observed in animals receiving single treatment with sildenafil at 15 or 30 mg/kg. When 30 mg/kg of sildenafil was coadministered with 100 mg/kg of itraconazole, drug-related effects such as increased heart rate and decreased QT interval were significantly enhanced as compared to sildenafil-alone administration at 6 h postadministration. These results demonstrated that increased heart rate and decreased QT interval, the adverse effects of sildenafil, were enhanced and prolonged when sildenafil was coadministered with itraconazole. Therefore, caution should be taken when sildenafil is coadministered with itraconazole, a CYP3A4 inhibitor, or when administered to elderly patients or patients with hepatic or renal impairment who cannot metabolize and excrete sildenafil normally.