Ae Ryang Jung

Combination therapy of human adipose-derived stem cells and basic fibroblast growth factor hydrogel in muscle regeneration

Skeletal muscle regeneration after sport injury is inconsistent, and complete healing without fibrosis is very important. In this study, we determined whether the combination therapy using human adipose-derived stem cells (h-ADSCs) and basic fibroblast growth factor (bFGF) incorporated into hydrogel could enhance muscle regeneration in a muscle laceration animal model. The h-ADSCs and/or bFGF hydrogels were applied to the lacerated gastrocnemius muscle. Fast twitch muscle contraction improved significantly and fibrosis decreased significantly in combined h-ADSC and bFGF-hydrogel group compared to other experimental groups. Skeletal muscle differentiation of h-ADSCs was determined by immunohistochemistry (PKH-26/MyHC co-staining) and Western blot. Our data suggested that combination therapy of h-ADSCs and bFGF hydrogel resulted in functional recovery, revascularization and reinnervation with minimal fibrosis in lacerated muscle. A combination of h-ADSCs and bFGF hydrogel can be used as a promising therapy for skeletal muscle regeneration.

Combination Therapy Using Human Adipose-derived Stem Cells on the Cavernous Nerve and Low-energy Shockwaves on the Corpus Cavernosum in a Rat Model of Post-prostatectomy Erectile Dysfunction.

OBJECTIVE To investigate combined therapeutic efficacy of human adipose-derived stem cells (h-ADSCs) application on injured cavernous nerve and low-energy shockwave therapy (SWT) on the corpus cavernosum in a rat model of post-prostatectomy erectile dysfunction. MATERIALS AND METHODS Rats were randomly divided into 5 groups: control, bilateral cavernous nerve injury (BCNI), adipose-derived stem cell (ADSC) (BCNI group with h-ADSCs on the cavernous nerve), SWT (BCNI group with low-energy SWT on the corpus cavernosum), and ADSC/SWT (BCNI group with a combination of h-ADSCs and low-energy SWT). After 4 weeks, erectile function was assessed using intracavernosal pressure. The cavernous nerves and penile tissue were evaluated through immunostaining, Western blotting, and a cyclic guanosine monophosphate assay. RESULTS ADSC/SWT significantly improved intracavernosal pressure compared to the other experimental group. ADSC had significantly increased β-III tubulin expression of the cavernous nerve, and SWT had a markedly enhanced vascular endothelial growth factor expression in corpus cavernosum. The ADSC/SWT group had a significantly increased in alpha smooth muscle actin content (P < .05), neural nitric oxide synthase (nNOS) of the dorsal penile nerve (P < .05), endothelial nitric oxide synthase (eNOS) protein expression (P < .05), and cyclic guanosine monophosphate level (P < .05) compared to the ADSC or SWT alone group. In addition, ADSC/SWT reduces the apoptotic index in the corpus cavernosum. CONCLUSION In this study, h-ADSCs showed an effect on the recovery of injured cavernous nerve and low-energy SWT improved angiogenesis in the corpus cavernosum. The h-ADSCs combined with low-energy SWT showed beneficial effect on the recovery of erectile function in a rat model of postprostatectomy erectile dysfunction.

Prostate-specific extracellular vesicles as a novel biomarker in human prostate cancer.

Extracellular vesicles (EVs) may play an important role in cancer development and progression. We aimed to investigate the prognostic potential of prostate-specific EVs in prostate cancer (PCa) patients. Plasma and prostate tissue were collected from patients who underwent surgery for PCa (n = 82) or benign prostatic hyperplasia (BPH, n = 28). To analyze the quantity of EVs in prostate, we performed transmission electron microscopy (TEM), immuno-TEM with CD63 and prostate-specific membrane antigen (PSMA), and immunofluorescence staining. After EV isolation from plasma, CD63 and PSMA concentration was measured using ELISA kits. PSMA-positive areas in prostate differed in patients with BPH, and low-, intermediate-, and high-risk PCa (2.4, 8.2, 17.5, 26.5%, p < 0.001). Plasma PSMA-positive EV concentration differed in patients with BPH, and low-, intermediate-, and high-risk PCa (21.9, 43.4, 49.2, 59.9 ng/mL, p < 0.001), and ROC curve analysis indicated that plasma PSMA-positive EV concentration differentiated PCa from BPH (AUC 0.943). Patients with lower plasma PSMA-positive EV concentration had greater prostate volume (50.2 vs. 33.4 cc, p < 0.001) and lower pathologic Gleason score (p = 0.025). During the median follow-up of 18 months, patients with lower plasma PSMA-positive EV concentration tended to have a lower risk of biochemical failure than those with higher levels of prostate-specific EVs (p = 0.085).

Comparison Between Subcutaneous Injection of Basic Fibroblast Growth Factor-Hydrogel and Intracavernous Injection of Adipose-derived Stem Cells in a Rat Model of Cavernous Nerve Injury

OBJECTIVE To compare the effects of subcutaneous penile injection of basic fibroblast growth factor (bFGF)-hydrogel and intracavernous injection of human adipose-derived stem cells (h-ADSCs) on improving erectile function in a rat model of cavernous nerve injury. MATERIALS AND METHODS Adult male Sprague-Dawley rats were randomly divided into 5 groups (n = 10 per group): age-matched control (normal group), bilateral cavernous nerve injury (BCNI group), penile subcutaneous injection of hydrogel after BCNI (hydrogel group), penile subcutaneous injection of bFGF-hydrogel after BCNI (bFGF-hydrogel group) and intracavernous injection of h-ADSCs after BCNI (ADSC group). Four weeks after the treatment, all rats underwent an erectile function test. Then, penile tissue was harvested for immunohistological analysis of bFGF, phalloidin, and cluster of differentiation (CD) 31. The cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum was quantified by cGMP assay. RESULTS From the functional test and immunohistological result, we observed that bFGF-hydrogel and h-ADSCs injection significantly elevated intracavernous pressure. The evaluation of filamentous actin content, CD31 expression, and cGMP concentration in the corpus cavernosum were meaningfully increased in the bFGF-hydrogel and ADSC groups compared with BCNI group. The bFGF released from bFGF-hydrogel prevented smooth muscle atrophy. Moreover, bFGF expression was significantly increased in bFGF-hydrogel group. CONCLUSION The subcutaneous injection of bFGF-hydrogel prevented smooth muscle atrophy, increased the intracavernous pressure, and improved erectile function like an intracavernous injection of h-ADSCs.

The Effect of PnTx2-6 Protein From Phoneutria nigriventer Spider Toxin on Improvement of Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury

OBJECTIVE To explore the therapeutic potential of PnTx2-6 injected 3 times a week for 4 weeks into the intracavernosal tissue in a rat model of bilateral cavernous nerve crush injury (BCNI). METHODS Eight-week-old male Sprague-Dawley rats were randomly divided into the following 6 groups (n = 5 per group): age-matched control (normal group), BCNI (injury group), post-BCNI phosphate-buffered saline injection (PBS group), post-BCNI Sf9 cell-lysate injection (N/C group), post-BCNI injection of cell lysate from S9 cells infected with wild-type recombinant baculovirus (W/T group), and post-BCNI injection of cell lysate from S9 cells infected with recombinant baculovirus containing PnTx2-6 (PnTx2-6 group). Injections were delivered 3 times a week for 4 weeks. After 4 weeks, intracavernosal pressure-to-mean arterial pressure ratio, smooth muscle and collagen content via the Masson trichrome staining, levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase, and cyclic guanosine monophosphate were all measured. RESULTS The PnTx2-6 group showed significantly higher intracavernosal pressure-to-mean arterial pressure ratio (P <.05), smooth muscle-to-collagen ratio (P <.01), expression levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase (P <.05), and cyclic guanosine monophosphate (P <.05) than all other experimental groups. CONCLUSION We conclude that PnTx2-6 improved erectile function and prevented muscle atrophy in a rat model of BCNI via increased synthesis of nitric oxide and cyclic guanosine monophosphate.

Bladder reconstruction using stem cells seeded on multilayered scaffolds in a mucosa preserving partial cystectomy model

Recently, studies have focused more towards using biocompatible scaffolds and stem cells to augment or replace the abnormal bladder. But, due to the lack of biomaterials with appropriate thickness as a suitable scaffold for smooth muscle regeneration, several structural, mechanical, and biocompatibility problems are encountered. Therefore, we aimed to demonstrate whether human muscle-derived stem cells (h-MDSCs) seeded on multilayered polycaprolactone (PCL) nanofiber is an appropriate scaffold for bladder smooth muscle regeneration. h-MDSCs were seeded on a multilayered PCL/collagen nanofiber sheet and implanted in the bladder of a mucosa preserving partial cystectomy rat. From our findings, h-MDSCs seeded on multilayered PCL showed efficient cell seeding and proliferation. In addition, the histological and immunohistochemical analysis showed cell survival in between the multilayered nanofiber sheet, which led to smooth muscle cell regeneration with improved pro-angiogenesis in the regenerated region of the bladder. Therefore, h-MDSCs seeded nanofibers could be a promising tool in treating neurogenic bladder and related diseases.

PD33-06 EFFECTS OF CONTROLLED OXYGEN RELEASE FROM HOLLOW MICROPARTICLES FOR PROLONGED STEM CELL SURVIVAL AND IMPROVED ERECTILE FUNCTION

with a fluorescent cell linker, PKH26. Spheroids were formed in 96-well plates (4 104 cells/well/100ml). Spheroids were assembled into 3D shape by a robotic bio-fabrication system according to the pre-designed 3D data. The fabricated structures were perfusion cultured for 7 days to promote self-organization of the cells. Autologous 3D-printed AdMSCs structures were implanted around the cryo-injured urethra, immediately after injury, created by spraying liquid nitrogen for 20s. Sham operations were performed. Two weeks after implantation, the urethras were harvested for analyses. RESULTS: Results: The cultured cells expressed the mesenchymal cell marker STRO1, but not muscle cell markers. In HE staining, there was presence of distinct and developed muscle structure. In immunohistochemical analysis, the reconstructed skeletal and smooth muscle areas in AdMSCs structures implanted group were more developed than those of controls. Implanted PKH26 labeled AdMSCs within the bio-fabricated structures were positive for myoglobin, SMA, S100, and vWF antibodies. In addition, these implanted cells were positive for transforming growth factor b1 and nerve growth factor. CONCLUSIONS: Conclusions: Our results demonstrated that the bio-fabricated structures might have potential to treat for various urethral injuries and dysfunctions by differentiating into smooth muscle, skeletal muscle, nerve, and endothelial cells, and by secreting growth factors.

GV1001 Induces Apoptosis by Reducing Angiogenesis in Renal Cell Carcinoma Cells Both In Vitro and In Vivo

OBJECTIVE To investigate the anticancer effects of GV1001 and its biological mechanism of action in renal cell carcinoma (RCC). METHODS The effects of GV1001 on cell survival and apoptosis in RCC cells were examined in vitro using cell viability assay, fluorescence-activated cell sorting, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To evaluate the effect of GV1001 on migration, invasion, and angiogenesis, we used wound healing, invasion, endothelial cell tube formation assay, and western blot analysis. Furthermore, we used an RCC xenograft model with either phosphate buffered saline or GV1001 to confirm the anticancer effect of GV1001 in vivo. Tumor volume was monitored during treatment, and tumor weight was measured after animals were killed. Apoptosis and angiogenesis of the tumor tissue were assessed using hematoxylin and eosin staining, immunohistochemistry, and western blot analysis. RESULTS GV1001 reduced cell viability and induced apoptosis in RCC cells in vitro. Furthermore, GV1001 suppressed the migration and invasion of RCC cells through regulation of matrix metalloproteinases and tissue inhibitors of metalloproteinases. In addition, GV1001 reduced angiogenesis via regulation of hypoxia-inducible factor 1α. In xenograft mouse model experiment, GV1001 reduced tumor growth and induced apoptosis. As in the in vitro results, GV1001 significantly reduced angiogenesis through regulation of hypoxia-inducible factor 1α in vivo. CONCLUSION Our data demonstrated that GV1001 induced apoptosis through suppression of angiogenesis in RCCs both in vitro and in vivo, which suggests that GV1001 may be a potential therapeutic target for RCC.

MP28-20 EXPRESSION OF HMGB1 IN PROSTATE CANCER: CLINICAL AND BIOLOGICAL CORRELATIONS

the yield/print mean, median (SD) for DNA is 1611 ng, 942 ng (1191) and for RNA is 550 ng, 481 ng (506); from cores with no PCa the yield/print for DNA is 1020 ng, 926 ng (744) and for RNA is 351 ng, 250 ng (418). Prostate biopsy tissue print RNA and DNA is snap-frozen quality and has been successfully utilized for gene expression profiling, genotyping, DNA methylation and sequencing analyses. CONCLUSIONS: Tissue print technologies provide a practical approach to biopsy-based biomarker analyses that preserves the tissue core for pathology diagnosis and other FFPE based testing. For research studies, prospective collection of biopsy tissue prints is feasible in both academic and private practice settings. Because tissue prints provide high quality RNA and DNA suitable for a wide range of molecular biomarker tests, the technology may also be useful in situations where there is insufficient FFPE biopsy tissue to satisfy clinical molecular testing requirements.