Bone marrow-derived mesenchymal stromal cell therapy in a rat model of cavernous nerve injury: Preclinical study for approval.

BACKGROUND AIMS: Although clinical studies using stem cells to treat erectile dysfunction have been performed or are ongoing, there is little consensus on the optimal protocol. We aimed to develop a protocol optimizing human bone marrow-derived mesenchymal stromal cell (hBMSC) therapy in a rat model of cavernous nerve injury. METHODS: We performed, in order, a dose-finding study, a toxicokinetic study of hBMSCs, and a study to determine the timing and number of cell injections. RESULTS: From the dose-finding study, 1 × 10(6) cells were selected as the dose per hBMSC injection. From the toxicokinetic study, 14 days was selected as the interval between repeat treatments. In the final study, the ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the control group than in the sham group (23.4% vs. 55.1%, P <0.001). An immediate single injection of hBMSCs significantly improved erectile function compared with the control group (39.8%, P = 0.035), whereas a delayed single injection showed improvement with a marginal trend (38.1%, P = 0.079). All histomorphometric changes were significantly more improved in the immediate or delayed single injection groups than in the control group. Repeat treatments did not provide any benefit for the recovery of erectile function and histomorphometric changes. CONCLUSIONS: Intracavernous injection of 1 × 10(6) hBMSCs results in a recovery of penile erection and histomorphometric changes in a rat model of cavernous nerve injury, even when treatment was delayed until 4 weeks after cavernous nerve injury.

Establishment of NOAEL for intracavernous injections of human bone marrow-derived mesenchymal stem cells in rats.

Purpose: To assess the possible negative health effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on fertility and early embryonic development following intracavernous injections in rats. Materials and Methods: A total of 88 Crl:CD(SD) male and female rats were equally divided into 4 groups in a random manner: control group (normal saline), low-dose group (2×105 hBMSCs), moderate-dose group (1×106 hBMSCs), and high-dose group (2×106 hBMSCs). hBMSCs or normal saline was injected into the penis of the rats 3 times at 2-week-intervals prior to mating. We compared each group with respect to parameters of reproduction and histopathology. Results: For male rats, various degrees of flushing and swelling were observed at the penile injection site in all the groups, although the severity increased in a dose-dependent manner in the hBMSC injection groups. There were no statistically significant differences in mean body weights and food consumption among all the groups of both sexes. There were no statistically significant differences in reproductive parameters among all the groups of both sexes. The absolute and relative organ weights did not significantly differ among the groups. At the time of necropsy, no remarkable findings were observed in gross examinations in all groups. On histopathological analysis, minimal mononuclear cell infiltration was observed in the right epididymis of each rat in the moderate- and high-dose groups. Conclusions: The non-toxic amount of hBMSCs for male fertility and early embryogenesis in rats under the test conditions was determined to be 2×106 cells/head.

Induction of immunogenic cell death of tumors by newly synthesized heterocyclic quinone derivative.

Many cancer types are serious diseases causing mortality, and new therapeutics with improved efficacy and safety are required. Immuno-(cell)-therapy is considered as one of the promising therapeutic strategies for curing intractable cancer. In this study, we tested R2016, a newly developed heterocyclic quinone derivative, for induction of immunogenic tumor cell death and as a possible novel immunochemotherapeutic. We studied the anti-cancer effects of R2016 against LLC, a lung cancer cell line and B16F10, a melanoma cell line. LLC (non-immunogenic) and B16F10 (immunogenic) cells were killed by R2016 in dose-dependent manner. R2016 reduced the viability of both LLC and B16F10 tumor cells by inducing apoptosis and necrosis, while it demonstrated no cytotoxicity against normal splenocytes. Expression of immunogenic death markers on the cell surface of R2016 treated tumor cells including calreticulin (CRT) and heat shock proteins (HSPs) was increased along with the induction of their genes. Increased CRT expression correlated with dendritic cell (DC) uptake of dying tumor cells: the proportion of CRT+CD11c+cells was increased in the R2016-treated group. The gene transcription of Calr3, Hspb1, and Tnfaip6, which are related to immunogenicity induction of dead cells, was up-regulated in the R2016 treated tumor cells. On the other hand, ANGPT1, FGF7, and URGCP gene levels were down-regulated by R2016 treatment. This data suggests that R2016 induced immunogenic tumor cell death, and suggests R2016 as an effective anti-tumor immunochemotherapeutic modality.

Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been studied for their therapeutic potential. However, evaluating the quality of hBM-MSCs before transplantation remains a challenge. We addressed this issue in the present study by investigating deformation, the expression of genes related to reactive oxygen species (ROS) generation, changes in amino acid profiles, and membrane fluidity in hBM-MSCs. Deformability and cell size were decreased after storage for 6 and 12 h, respectively, in phosphate-buffered saline. Intracellular ROS levels also increased over time, which was associated with altered expression of genes related to ROS generation and amino acid metabolism. Membrane fluidity measurements revealed higher Laurdan generalized polarization values at 6 and 12 h; however, this effect was reversed by N-acetyl-L-cysteine-treatment. These findings indicate that the quality and freshness of hBM-MSCs is lost over time after dissociation from the culture dish for transplantation, highlighting the importance of using freshly trypsinized cells in clinical applications.

Immunogenic Cell Death Induced by Ginsenoside Rg3: Significance in Dendritic Cell-based Anti-tumor Immunotherapy.

Cancer is one of the leading causes of morbidity and mortality worldwide; therefore there is a need to discover new therapeutic modules with improved efficacy and safety. Immune-(cell) therapy is a promising therapeutic strategy for the treatment of intractable cancers. The effectiveness of certain chemotherapeutics in inducing immunogenic tumor cell death thus promoting cancer eradication has been reported. Ginsenoside Rg3 is a ginseng saponin that has antitumor and immunomodulatory activity. In this study, we treated tumor cells with Rg3 to verify the significance of inducing immunogenic tumor cell death in antitumor therapy, especially in DC-based immunotherapy. Rg3 killed the both immunogenic (B16F10 melanoma cells) and non-immunogenic (LLC: Lewis Lung Carcinoma cells) tumor cells by inducing apoptosis. Surface expression of immunogenic death markers including calreticulin and heat shock proteins and the transcription of relevant genes were increased in the Rg3-dying tumor. Increased calreticulin expression was directly related to the uptake of dying tumor cells by dendritic cells (DCs): the proportion of CRT(+) CD11c(+) cells was increased in the Rg3-treated group. Interestingly, tumor cells dying by immunogenic cell death secreted IFN-γ, an effector molecule for antitumor activity in T cells. Along with the Rg3-induced suppression of pro-angiogenic (TNF-α) and immunosuppressive cytokine (TGF-ß) secretion, IFN-γ production from the Rg3-treated tumor cells may also indicate Rg3 as an effective anticancer immunotherapeutic strategy. The data clearly suggests that Rg3-induced immunogenic tumor cell death due its cytotoxic effect and its ability to induce DC function. This indicates that Rg3 may be an effective immunotherapeutic strategy.