Lia Banie

Defining stem and progenitor cells within adipose tissue.

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Freshly isolated ADSC display surface markers that differ from those of cultured ADSC, but both cell preparations are capable of multipotential differentiation. Recent studies have inferred that these progenitors may reside in a perivascular location where they appeared to coexpress CD34 and smooth muscle actin (alpha-SMA) but not CD31. However, these studies provided only limited histological evidence to support such assertions. In the present study, we employed immunohistochemistry and immunofluorescence to define more precisely the location of ADSC within human adipose tissue. Our results show that alpha-SMA and CD31 localized within smooth muscle and endothelial cells, respectively, in all blood vessels examined. CD34 localized to both the intima (endothelium) and adventitia neither of which expressed alpha-SMA. The niche marker Wnt5a was confined exclusively to the vascular wall within mural smooth muscle cells. Surprisingly, the widely accepted mesenchymal stem cell marker STRO-1 was expressed exclusively in the endothelium of capillaries and arterioles but not in the endothelium of arteries. The embryonic stem cell marker SSEA1 localized to a pericytic location in capillaries and in certain smooth muscle cells of arterioles. Cells expressing the embryonic stem cell markers telomerase and OCT4 were rare and observed only in capillaries. Based on these findings and evidence gathered from the existing literature, we propose that ADSC are vascular precursor (stem) cells at various stages of differentiation. In their native tissue, ADSC at early stages of differentiation can differentiate into tissue-specific cells such as adipocytes. Isolated, ADSC can be induced to differentiate into additional cell types such as osteoblasts and chondrocytes.

Injections of Adipose Tissue-Derived Stem Cells and Stem Cell Lysate Improve Recovery of Erectile Function in a Rat Model of Cavernous Nerve Injury

INTRODUCTION Erectile dysfunction (ED) remains a major complication after radical prostatectomy. The use of adipose tissue-derived stem cells (ADSCs) has shown promising results for the treatment of ED. However, the mechanisms of action for stem cell therapy remain controversial, with increasing evidence pointing to paracrine pathways. AIM To determine the effects and to identify the mechanism of action of ADSC and ADSC-derived lysate in a rat model of cavernous nerve (CN) crush injury. METHODS Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: one group underwent sham operation, while three groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of ADSC, lysate, or vehicle only (injured controls). Erectile function was assessed by CN electrostimulation at 4 weeks. Penile tissue was collected for histology. MAIN OUTCOME MEASURES   Intracavernous pressure increase upon CN stimulation; neuronal nitric oxide synthase (nNOS) content in the dorsal penile nerve; smooth muscle content, collagen content, and number of apoptotic cells in the corpus cavernosum. RESULTS Both ADSC and lysate treatments resulted in significant recovery of erectile function, as compared with vehicle treatment. nNOS content was preserved in both the ADSC and lysate group, with significantly higher expression compared with vehicle-treated animals. There was significantly less fibrosis and a significant preservation of smooth muscle content in the ADSC and lysate groups compared with injured controls. The observed functional improvement after lysate injection supports the hypothesis that ADSCs act through release of intracellular preformed substances or by active secretion of certain biomolecules. The underlying mechanism of recovery appears to involve neuron preservation and cytoprotection by inhibition of apoptosis. CONCLUSIONS Penile injection of both ADSC and ADSC-derived lysate can improve recovery of erectile function in a rat model of neurogenic ED.

Treatment of stress urinary incontinence with adipose tissue-derived stem cells.

BACKGROUND AIMS Effective treatment for stress urinary incontinence (SUI) is lacking. This study investigated whether transplantation of adipose tissue-derived stem cells (ADSC) can treat SUI in a rat model. METHODS Rats were induced to develop SUI by postpartum vaginal balloon dilation and bilateral ovariectomy. ADSC were isolated from the peri-ovary fat, examined for stem cell properties, and labeled with thymidine analog BrdU or EdU. Ten rats received urethral injection of saline as a control. Twelve rats received urethral injection of EdU-labeled ADSC and six rats received intravenous injection of BrdU-labeled ADSC through the tail vein. Four weeks later, urinary voiding function was assessed by conscious cystometry. The rats were then killed and their urethras harvested for tracking of ADSC and quantification of elastin, collagen and smooth muscle contents. RESULTS Cystometric analysis showed that eight out 10 rats in the control group had abnormal voiding, whereas four of 12 (33.3%) and two of six (33.3%) rats in the urethra-ADSC and tail vein-ADSC groups, respectively, had abnormal voiding. Histologic analysis showed that the ADSC-treated groups had significantly higher elastin content than the control group and, within the ADSC-treated groups, rats with normal voiding pattern also had significantly higher elastin content than rats with voiding dysfunction. ADSC-treated normal-voiding rats had significantly higher smooth muscle content than control or ADSC-treated rats with voiding dysfunction. CONCLUSIONS Transplantation of ADSC via urethral or intravenous injection is effective in the treatment and/or prevention of SUI in a pre-clinical setting.

Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury

BACKGROUND Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear. OBJECTIVES To investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury. DESIGN, SETTING, AND PARTICIPANTS Sprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest. MEASUREMENTS IC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG. RESULTS AND LIMITATIONS IC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. CONCLUSIONS CN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.

Effects of transplantation of adipose tissue-derived stem cells on prostate tumor

Obesity is a risk factor for prostate cancer development, but the underlying mechanism is unknown. The present study tested the hypothesis that stromal cells of the adipose tissue might be recruited by cancer cells to help tumor growth.

Erectogenic and Neurotrophic Effects of Icariin, a Purified Extract of Horny Goat Weed (Epimedium spp.) In Vitro and In Vivo

INTRODUCTION Epimedium species (aka horny goat weed) have been utilized for the treatment of erectile dysfunction in Traditional Chinese Medicine for many years. Icariin (ICA) is the active moiety of Epimedium species. AIM To evaluate the penile hemodynamic and tissue effects of ICA in cavernous nerve injured rats. We also studied the in vitro effects of ICA on cultured pelvic ganglia. METHODS Rats were subjected to cavernous nerve injury and subsequently treated for 4 weeks with daily gavage feedings of a placebo solution of normal saline and Dimethyl sulfoxide (DMSO) vs. ICA dissolved in DMSO at doses of 1, 5, and 10 mg/kg. A separate group underwent a single dose of ICA 10 mg/kg 2 hours prior to functional testing. Functional testing with cavernous nerve stimulation and real-time assessment of intracavernous pressure (ICP) was performed at 4 weeks. After functional testing, penile tissue was procured for immunohistochemistry and molecular studies. In separate experiments, pelvic ganglia were excised from healthy rats and cultured in the presence of ICA, sildenafil, or placebo culture media. MAIN OUTCOME MEASURE Ratio of ICP and area under the curve (AUC) to mean arterial pressure (MAP) during cavernous nerve stimulation of subject rodents. We also assayed tissue expression of neuronal nitric oxide synthase (nNOS), eNOS: endothelial nitric oxide synthase (eNOS), calponin, and apoptosis via immunohistochemistry and Western blot. Serum testosterone and luteinizing hormone (LH) were assayed using enzyme-linked immunosorbant assay (ELISA). Differential length of neurite outgrowth was assessed in cultured pelvic ganglia. RESULTS Rats treated with low-dose ICA demonstrated significantly higher ICP/MAP and AUC/MAP ratios compared with control and single-dose ICA animals. Immunohistochemistry and Western blot were revealing of significantly greater positivity for nNOS and calponin in penile tissues of all rats treated with ICA. ICA led to significantly greater neurite length in cultured specimens of pelvic ganglia. CONCLUSION ICA may have neurotrophic effects in addition to known phosphodiesterase type 5 inhibiting effects.

Potential of adipose-derived stem cells for treatment of erectile dysfunction.

INTRODUCTION Adipose-derived stem cells (ADSCs) are a somatic stem cell population contained in fat tissue that possess the ability for self-renewal, differentiation into one or more phenotypes, and functional regeneration of damaged tissue, which may benefit the recovery of erectile function by using a stem cell-based therapy. AIM To review available evidence concerning ADSCs availability, differentiation into functional cells, and the potential of these cells for the treatment of erectile dysfunction (ED). METHODS We examined the current data (from 1964 to 2008) associated with the definition, characterization, differentiation, and application of ADSCs, as well as other kinds of stem cells for the cell-based therapies of ED. MAIN OUTCOME MEASURES There is strong evidence supporting the concept that ADSCs may be a potential stem cell therapy source in treating ED. RESULTS The ADSCs are paravascularly localized in the adipose tissue. Under specific induction medium conditions, these cells differentiated into neuron-like cells, smooth muscle cells, and endothelium in vitro. The insulin-like growth factor/insulin-like growth factor receptor (IGF/IGFR) pathway participates in neuronal differentiation while the fibroblast growth factor 2 (FGF2) pathway is involved in endothelium differentiation. In a preliminary in vivo experiment, the ADSCs functionally recovered the damaged erectile function. However, the underlying mechanism needs to be further examined. CONCLUSION The ADSCs are a potential source for stem cell-based therapies, which imply the possibility of an effective clinical therapy for ED in the near future.

Tissue Distribution of Mesenchymal Stem Cell Marker Stro-1

Stro-1 is the best-known mesenchymal stem cell marker. However, despite its bone marrow origin, its localization in bone marrow has never been demonstrated. By immunofluorescence staining, we show here that ∼ 0.74% of nucleated bone marrow cells expressed Stro-1. We also found that ∼ 8.7% of CD34-expressing cells expressed Stro-1, and more than 20% of Stro-1-expressing cells did not express CD34. In adipose tissue Stro-1 expression was identified in the endothelium of arterioles and capillaries. Stro-1 was also localized in the endothelium of some but not all adipose tissue veins. Endothelial expression of Stro-1 was also identified in blood vessels in penis and in leg muscles, but not in other tested tissues. In these other tissues, Stro-1 was scantly expressed near but not in blood vessels. These variable and endothelial expression patterns of Stro-1 point to a need to re-examine published data that relied on Stro-1 as a mesenchymal stem cell marker.

Pentoxifylline Attenuates Transforming Growth Factor-β1-Stimulated Collagen Deposition and Elastogenesis in Human Tunica Albuginea-Derived Fibroblasts Part 1: Impact on Extracellular Matrix

INTRODUCTION Transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of Peyronies disease (PD) and also plays a role in collagen and elastin metabolism. Pentoxifylline (PTX) antagonizes the effects of TGF-β1 and has been utilized in our clinic for the management of PD. AIM We studied the effects of TGF-β1 and PTX on collagen metabolism and elastogenesis in tunica albuginea-derived fibroblasts (TADFs). METHODS TADFs from men with and without PD were cultured and treated with TGF-β1 and PTX as monotherapy at differing concentrations and time points. Combination treatment (TGF-β1 followed by PTX and vice versa) was also investigated. MAIN OUTCOME MEASURES Cell proliferation assay, enzyme-linked immunosorbent assay, and immunohistochemistry were utilized to assess the impact of TGF-β1 and PTX on TADF with respect to elastin and collagen I metabolism. RESULTS PTX inhibited fibroblast proliferation at doses of 100 µM. TGF-β1 stimulated elastogenesis and collagen I fiber deposition in TADF in a dose- and time-dependent fashion. Pretreatment with PTX dramatically attenuated TGF-β1-mediated elastogenesis and collagen fiber deposition in TADF from men with and without PD. Interestingly, production of collagen I was higher in untreated Peyronies tunica (PT) cells relative to normal tunica (NT) cells; furthermore, PTX attenuated collagen production to levels similar to untreated control TADF in PT cells but not in NT cells, suggesting important intrinsic differences between PT and NT cells. CONCLUSION Both elastin and collagen are upregulated by TGF-β1 in TADF. This likely contributes to the PD phenotype. Pretreatment with PTX attenuates both collagen fiber deposition and elastogenesis in TADF exposed to TGF-β1; these effects suggest a useful role for PTX in the management of PD.

Labeling and tracking of mesenchymal stromal cells with EdU.

BACKGROUND AIMS The thymidine analog bromodeoxyuridine (5-bromo-2-deoxyuridine; BrdU) has been used widely to label cells in culture and in tissue. The labeled cells can also be tracked when transplanted into a suitable host. In the present study we tested a new thymidine analog, 5-ethynyl-2-deoxyuridine (EdU), for labeling and tracking of mesenchymal stromal cells (MSC), specifically adipose tissue-derived stem cells (ADSC). METHODS Labeling of ADSC was examined for the dosage effect of EdU and stability of label by Alexa-594 staining followed by fluorescence microscopy. Labeling of various organs/tissues was done by intraperitoneal injection of EdU and examined by histology and fluorescence microscopy. Tracking of ADSC was done by intratissue or intravenous transplantation of EdU-labeled ADSC into various tissues and examined by histology and fluorescence microscopy. RESULTS EdU was incorporated specifically into the nucleus in approximately 50% of ADSC and the percentage of cells that remained fully labeled declined with time. Peritoneal injection of EdU resulted in the appearance of EdU-positive cells in most organs and tissues. In the intestine, EdU-positive cells were found in both the epithelium and connective tissues 7 h after injection. Long-term (2-6 week) follow-ups found EdU-positive cells only in the connective tissue. Tracking of ADSC was successful in tissues 10 weeks after intratissue or intravenous transplantation. CONCLUSIONS Cell labeling with EdU in culture or living animals can be performed easily. The detection of EdU label requires no harsh treatment or immunologic reaction, as detection of BrdU label does. EdU can be used for long-term tracking of ADSC.