Transplantation of nonhematopoietic adult bone marrow stem/progenitor cells isolated by p75 nerve growth factor receptor into the penis rescues erectile function in a rat model of cavernous nerve injury

Abstract

Purpose: Radical prostatectomy for prostate cancer frequently results in erectile dysfunction and decreased quality of life. We investigated the effects of transplanting nonhematopoietic adult bone marrow stem/progenitor cells (multipotent stromal cells) into the corpus cavernosum in a rat model of bilateral cavernous nerve crush injury.

Materials and methods: Multipotent stromal cells were isolated from the bone marrow of transgenic green fluorescent protein rats by plastic adherence (rat multipotent stromal cells) or magnetic activated cell sorting using antibodies against p75 low affinity nerve growth factor receptor (p75 derived multipotent stromal cells). Bilateral cavernous nerve crush injury was induced in adult male Sprague-Dawley rats. Immediately after injury 8 rats each were injected intracavernously with phosphate buffered saline (vehicle control), fibroblasts (cell control), rat multipotent stromal cells (cell treatment) or p75 derived multipotent stromal cells (cell treatment). Another 8 rats underwent sham operation (phosphate buffered saline injection). Four weeks after the procedures we assessed erectile function by measuring the intracavernous-to-mean arterial pressure ratio and total intracavernous pressure during cavernous nerve stimulation.

Results: Intracavernous injection of p75 derived multipotent stromal cells after bilateral cavernous nerve crush injury resulted in a significantly higher mean intracavernous-to-mean arterial pressure ratio and total intracavernous pressure compared with all other groups except the sham operated group (p <0.05). Rats injected with typical multipotent stromal cells had partial erectile function rescue compared with animals that received p75 derived multipotent stromal cells. Fibroblast (cell control) and phosphate buffered saline (vehicle control) injection did not improve erectile function. Enzyme-linked immunosorbent assay suggested that basic fibroblast growth factor secreted by p75 derived multipotent stromal cells protected the cavernous nerve after bilateral cavernous nerve crush injury.

Conclusions: Transplantation of adult stem/progenitor cells may provide an effective treatment for erectile dysfunction after radical prostatectomy.

Figure 1

Morphology of cultured GFP rat fibroblasts (Fibro), GFP rat MSCs (rMSC), and GFP rat p75-derived MSCs (p75dMSC). The left column is phase contrast and the right column is epifluorescence microscopy for GFP (FITC channel). Scale bars= 100 micrometers.

Figure 2

Determination of intracavernous-to-mean arterial pressure ratio (ICP/MAP) and total ICP values to assess erectile function at 4 weeks after injury and treatment. A, Magnitude of the mean ICP/MAP. B, Mean total ICP. Cavernous nerve stimulation (CNS) at a frequency of 15 Hz. and pulse width of 30 milliseconds was performed in each rat. CNS at 2.5, 5.0 and 7.5 V was performed in the current protocol to achieve significant and consistent erectile responses. The duration of stimulation was 1 minute with a rest period of 3 to 5 minutes between subsequent CNS episodes. The total erectile response or total ICP was determined by the area under the curve (AUC) in mmHg/second from the beginning of CNS until the ICP returned to baseline or pre-stimulation pressure. The mean ICP-to-blood pressure ratio (ICP/MAP) at the peak erectile response was determined to control for variations in systemic blood pressure. C, Diagram of cavernous nerve anatomy and area of NCI. * p <0.05. Note: in all cases, PBS and fibro are significantly less than sham (p <0.05) and p75dMSC is significantly greater than PBS and fibro (p <0.05). NCI: Bilateral cavernous nerve-crush injury. N=8 animals per group.

Figure 3

The survival of MSCs in the penile sections using differential interference contrast (DIC) to define tissue morphology and epifluorescence to localize GFP. A, DIC image from edge of penis section. B, DIC image from edge of penis section. C, DIC image merged with staining by anti-GFP antibody (ALEXA 594, red, TRITC channel) and DAPI to visualize cell nuclei (blue). D, Epifluoresence alone from image in (C) showing signals for GFP and DAPI. E, Epifluoresence image from a different p75dMSC-injected rat in which the engrafted cells were located in close proximity to a blood vessel. BV: Blood vessel. Scale bars= 100 micrometers.

Figure 4

Phenotypic characteristics of cultured p75dMSCs. A–C, By immunohistochemistry, similar to rMSCs and fibroblasts, all of the p75dMSCs expressed the intermediate filament protein vimentin (ALEXA 594, red, TRITC channel). D–F, Also similar to rMSCs and fibroblasts, some of the p75dMSCs expressed the myofibroblast and smooth muscle marker, alpha smooth muscle actin (ALEXA 594, red, TRITC channel). G–I, In contrast to rMSCs and fibroblasts, a portion of the p75dMSCs expressed alpha sarcomeric actin, a marker of cardiac or skeletal muscle cells. Arrows: GFP-positive cells (FITC channel) that are negative for the immunostains (TRITC channel). Scale bars= 100 micrometers.

Figure 5

ELISA data for secreted bFGF, β-NGF, BDNF, VEGF, and IGF-1 from fibroblasts, rMSCs, and p75dMSCs. All growth factor levels are shown as expressed per volume (pg/ml) on the left and per total protein (pg/mg total protein) on the right. * p <0.05, ** p <0.01, † p <0.05 vs. p75dMSC, # p <0.01 vs. p75dMSC. N=3 samples per cell type.