Dataset Information

In vivo tracking on longer retention of transplanted myocardin gene-modified adipose-derived stem cells to improve erectile dysfunction in diabetic rats.

ABSTRACT:

Background

Stem cell therapy has revealed a promising future for treating erectile dysfunction (ED), but the fate and curative mechanism of intracavernosal transplanted stem cells are under further exploration. This study aimed to demonstrate the effects of myocardin gene modification on improving erectile function and prolonging the retention of implanted adipose-derived stem cells (ASCs) using in vivo small animal imaging.

Methods

ASCs were isolated, cultured, and identified by flow cytometry and osteogenic and adipogenic induction. The effects of gene modification on cell proliferation, apoptosis, and contraction were determined by CCK-8, EdU, flow cytometry, and collagen gel lattice contraction assays as well as confocal microscopy. A total of 20 normal and 60 diabetes mellitus ED to (DMED) Sprague-Dawley rats were recruited to the 7?day and 21?day groups. Each group contained subgroups of 10 rats each: the negative control (NC), DMED + ASCs plus Ad-Luc-Myocardin, DMED + ASCs plus Ad-Luc, and DMED + phosphate buffer solution (PBS) groups. Erectile function was evaluated with the intracavernosal pressure/mean arterial pressure (?ICP/MAP) ratio. In vivo small animal imaging and an EdU cell tracking strategy were introduced to detect the transplanted ASCs, and IHC and WB were performed to assess smooth muscle cell protein levels.

Results

The ASCs expressed high CD29 and CD90 and scant CD45, while the multi-induction potential was verified by oil red O and alizarin red staining. Gene transfection of myocardin had no significant influence on ASC apoptosis but inhibited cell proliferation and promoted cell contraction. Myocardin combined with ASCs enhanced the therapeutic potential of ASCs for improving the ?ICP/MAP ratio as well as ?-SMA and calponin expression. In vivo imaging confirmed that ASCs resided within the cavernous body in 21?days, while only a few red EdU dots were detected.

Conclusions

Myocardin induced ASC differentiation towards smooth muscle-like cells and enhanced the therapeutic potential of ASCs for ameliorating ED in STZ-induced diabetic rats. Notably, in vivo small animal tracking was an effective strategy for monitoring the implanted stem cells, and this strategy might have advantages over traditional EdU assays.

SUBMITTER: Zhang HB

PROVIDER: S-EPMC6636019 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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In vivo tracking on longer retention of transplanted myocardin gene-modified adipose-derived stem cells to improve erectile dysfunction in diabetic rats.

Zhang Hai-Bo HB Chen Feng-Zhi FZ He Shu-Hua SH Liang Yan-Bing YB Wang Zhi-Qiang ZQ Wang Li L Chen Ze-Rong ZR Ding Wei W Zhao Shan-Chao SC Wei An-Yang AY

Stem cell research & therapy 20190716 1

<h4>Background</h4>Stem cell therapy has revealed a promising future for treating erectile dysfunction (ED), but the fate and curative mechanism of intracavernosal transplanted stem cells are under further exploration. This study aimed to demonstrate the effects of myocardin gene modification on improving erectile function and prolonging the retention of implanted adipose-derived stem cells (ASCs) using in vivo small animal imaging.<h4>Methods</h4>ASCs were isolated, cultured, and identified by ...[more]

PMID: 31311594

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Project description:AimThe aim of this study was to determine the possibility of improving erectile dysfunction using cell therapy with either human urine-derived stem cells (USCs) or USCs genetically-modified with FGF2 in a type 2 diabetic rat model.MethodsHuman USCs were collected from 3 healthy donors. USCs were transfected with FGF2 (USCs-FGF2). Sixty-five SD male rats were divided into five groups (G). A control group of normal rats (G1, n = 10), and four other test groups of type 2 diabetic erectile dysfunction rats: PBS as a negative control (G2, n = 10), USCs (G3, n = 15), lentivirus-FGF2 (G4, n = 15), and USCs-FGF2 (G5, n = 15). Diabetes was induced in the rats via a high fat diet for 28 days and a subsequent intraperitoneal injection of streptozotocin (35 mg/kg). Erectile dysfunction was screened with apomorphine (100 μg/kg). Cell injections in the test groups (G2-G5) occurred directly into the corpora cavernosa. The implanted cells were tracked at 7 days (n = 5 animals/G) and 28 days (n = 10 animals/G) post injection. Mean arterial pressure (MAP), intracavernosal pressure (ICP), expression of endothelial markers (CD31, VEGF and eNOS), smooth muscle markers (desmin and smoothelin), histological changes and erectile function were assessed for each group.ResultsUSCs expressed mesenchymal stem cell markers, and secreted a number of proangiogenic growth factors. USCs expressed endothelial cell markers (CD31 and vWF) after transfection with FGF2. Implanted USCs or USCs-FGF2 displayed a significantly raised ICP and ICP/MAP ratio (p<0.01) 28 days after intracavernous injection. Although few cell were detected within the implanted sites, histological and western blot analysis demonstrated an increased expression of endothelial and smooth muscle markers within the cavernous tissue following USC or USC-FGF2 injection.ConclusionsThe paracrine effect of USCs or USCs-FGF2 induced improvement of erectile function in type 2 diabetic rats by recruiting resident cells and increasing the endothelial expression and contents of smooth muscle.

Project description:Cavernous nerve injury is the main cause of erectile dysfunction following radical prostatectomy. The recovery of erectile function following radical prostatectomy remains challenging. Our previous studies found that injecting adipose-derived stem cells (ADSCs) into the cavernosa could repair the damaged cavernous nerves, but the erectile function of the treated rats could not be restored to a normal level. We evaluated the efficacy of ADSCs infected with a lentiviral vector encoding rat brain-derived neurotrophic factor (lenti-rBDNF) in a rat model of cavernous nerve injury. The rats were equally and randomly divided into four groups. In the control group, bilateral cavernous nerves were isolated but not injured. In the bilateral cavernous nerve injury group, bilateral cavernous nerves were isolated and injured with a hemostat clamp for 2 minutes. In the ADSCGFP and ADSCrBDNF groups, after injury with a hemostat clamp for 2 minutes, rats were injected with ADSCs infected with lenti-GFP (1 × 106 in 20 ?L) and lenti-rBDNF (1 × 106 in 20 ?L), respectively. Erectile function was assessed 4 weeks after injury by measuring intracavernosal pressures. Then, penile tissues were collected for histological detection and western blot assay. Results demonstrated that compared with the bilateral cavernous nerve injury group, erectile function was significantly recovered in the ADSCGFP and ADSCrBDNF groups, and to a greater degree in the ADSCrBDNF group. Neuronal nitric oxide synthase content in the dorsal nerves and the ratio of smooth muscle/collagen were significantly higher in the ADSCrBDNF and ADSCGFP groups than in the bilateral cavernous nerve injury group. Neuronal nitric oxide synthase expression was obviously higher in the ADSCrBDNF group than in the ADSCGFP group. These findings confirm that intracavernous injection with ADSCs infected with lenti-rBDNF can effectively improve erectile dysfunction caused by cavernous nerve injury. This study was approved by the Medical Animal Care and Welfare Committee of Wuhan University, China (approval No. 2017-1638) on June 20, 2017.

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Project description:Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T(2)*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.

Dataset Information

In vivo tracking on longer retention of transplanted myocardin gene-modified adipose-derived stem cells to improve erectile dysfunction in diabetic rats.

Background

Methods

Results

Conclusions

Publications

In vivo tracking on longer retention of transplanted myocardin gene-modified adipose-derived stem cells to improve erectile dysfunction in diabetic rats.

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