Maurice Garcia

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.

Adipose derived stem cells ameliorate hyperlipidemia associated detrusor overactivity in a rat model.

PURPOSE Adipose tissue derived stem cells can differentiate into muscle and neuron-like cells in vitro. We investigate the usefulness of adipose tissue derived stem cells for overactive bladder in obese hyperlipidemic rats. MATERIALS AND METHODS Hyperlipidemia was induced in healthy rats by a high fat diet. The resulting obese hyperlipidemic rats were treated with bladder injection of saline, adipose tissue derived stem cells or tail vein injection of adipose tissue derived stem cells. Bladder function was assessed by 24-hour voiding behavior study and conscious cystometry. Bladder histology was assessed using immunostaining and trichrome staining, followed by image analysis. RESULTS Serum total cholesterol and low density lipoprotein were significantly higher in obese hyperlipidemic rats than in normal rats (p <0.01). The micturition interval was shorter in saline treated obese hyperlipidemic rats than in normal rats, obese hyperlipidemic rats that received adipose tissue derived stem cells via the tail vein and obese hyperlipidemic rats that received adipose tissue derived stem cells by bladder injection (mean +/- SEM 143 +/- 28.7 vs 407 +/- 77.9, 281 +/- 43.9 and 368 +/- 66.7 seconds, respectively, p = 0.0084). Bladder wall smooth muscle content was significantly lower in obese hyperlipidemic rats than in normal animals (p = 0.0061) while there was no significant difference between obese hyperlipidemic groups. Nerve content and blood vessel density were lower in controls than in obese hyperlipidemic rats treated with adipose tissue derived stem cells. CONCLUSIONS Hyperlipidemia is associated with increased urinary frequency, and decreased bladder blood vessel and nerve density in rats. Adipose tissue derived stem cell treatment ameliorates these adverse effects and holds promise as a potential new therapy for overactive bladder.

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.

Brain‐Derived Neurotrophic Factor (BDNF) Acts Primarily via the JAK/STAT Pathway to Promote Neurite Growth in the Major Pelvic Ganglion of the Rat: Part 2

INTRODUCTION Identification of the molecular mechanism of cavernous nerve regeneration is essential for future development of neuroprotective and regenerative strategies. AIM To identify specific signal transduction pathway(s) associated with brain-derived neurotrophic factor (BDNF) enhanced cavernous nerve regeneration in an in vitro model. MATERIALS AND METHODS Using 6-month-old male Fisher rats, inhibitors of four candidate signaling pathways were added to BDNF-treated explant cultures of major pelvic ganglia with attached cavernous nerve fragments. Study groups comprised of controls, BDNF alone at 50 ng/mL, or BDNF 50 ng/mL and inhibitors against MEK, PI3-K, PKA, and JAK/STAT pathways at increasing concentrations. MAIN OUTCOME MEASURE The maximal neurite length for each tissue culture was measured and the mean maximal length +/- standard deviation was determined for all groups at 24, 36, and 48 hours. RESULTS The JAK/STAT specific inhibitor AG490 significantly reduced BDNF-enhanced neurite growth. Maximum neurite lengths at 24, 36, and 48 hours for BDNF 50 ng/mL treated groups were 182.3, 348.1, and 528.1 microm, compared with AG490 at 25 microM (86.4, 165.1, 278.3 microm), 50 microM (78.8, 151.7, 235.3 microm), and 100 microM (71.83, 107.0, 219.6 microm) (P < 0.05). Neurite measures for BDNF with 25 and 50 microM U0126 (MEK pathway) were reduced to 402.0 and 424.3 microm at 48 hours, respectively (P < 0.05), likely reflecting an accessory molecular pathway. A similar observation was made for 100 uM LY294002 (PI3-K). No difference was observed for PKA inhibition. CONCLUSION The JAK/STAT pathway is the major signal-transduction pathway of BDNF-enhanced cavernous nerve growth in an in vitro rat model.

T-Shaped Shunt and Intracavernous Tunneling for Prolonged Ischemic Priapism

PURPOSE Conservative management of prolonged ischemic priapism is rarely effective. Interventions include corporal aspiration/irrigation, injection of vasoconstrictive agents or surgical procedures. We describe a technique that fulfills several important criteria in the surgical management of ischemic priapism in that immediate resolution of ischemic pain is achieved, a wide area, reliably patent shunt is created, the procedure is technically simple and it may be performed with the patient under a local anesthetic. MATERIALS AND METHODS We reviewed the records of 13 patients treated with the T-shunt for whom followup, including erectile function, was available. RESULTS Records were available for review for 13 men who underwent the T-shunt procedure from April 2006 to January 2008. In most cases priapism had lasted for more than 24 hours and previous irrigation/intracorporal administration of sympathomimetics had been unsuccessful. Of these 13 men 6 had undergone unsuccessful distal or proximal shunt procedures before presentation to our service. All procedures were performed using local anesthetic only. Cavernous blood flow was restored in all but 1 patient and another required a second procedure. T-shunts resulted in resolution of penile pain in all patients and all but 2 had recovery of erectile function. CONCLUSIONS The T-shunt technique results in immediate resolution of ischemic penile pain and rigidity. Ultrasonography confirms that blood flow is usually restored to the previously ischemic corpora cavernosa after the procedure. The T-shaped shunt is simple and reliable, and access also allows for proximal trans-shunt dilation. We observed surprisingly excellent recovery of erectile function. This procedure may facilitate recanalization of corporal circulation and could make proximal shunts obsolete.

Motile and non-motile sperm diagnostic manipulation using optoelectronic tweezers

Optoelectronic tweezers was used to manipulate human spermatozoa to determine whether their response to OET predicts sperm viability among non-motile sperm. We review the electro-physical basis for how live and dead human spermatozoa respond to OET. The maximal velocity that non-motile spermatozoa could be induced to move by attraction or repulsion to a moving OET field was measured. Viable sperm are attracted to OET fields and can be induced to move at an average maximal velocity of 8.8 ± 4.2 µm s(-1), while non-viable sperm are repelled to OET, and are induced to move at an average maximal velocity of -0.8 ± 1.0 µm s(-1). Manipulation of the sperm using OET does not appear to result in increased DNA fragmentation, making this a potential method by which to identify viable non-motile sperm for assisted reproductive technologies.

Preimplantation Mouse Embryo Selection Guided by Light-Induced Dielectrophoresis

Selection of optimal quality embryos for in vitro fertilization (IVF) transfer is critical to successful live birth outcomes. Currently, embryos are chosen based on subjective assessment of morphologic developmental maturity. A non-invasive means to quantitatively measure an embryos developmental maturity would reduce the variability introduced by the current standard. We present a method that exploits the scaling electrical properties of pre-transfer embryos to quantitatively discern embryo developmental maturity using light-induced dielectrophoresis (DEP). We show that an embryos DEP response is highly correlated with its developmental stage. Uniquely, this technique allows one to select, in sequence and under blinded conditions, the most developmentally mature embryos among a mixed cohort of morphologically indistinguishable embryos cultured in optimized and sub-optimal culture media. Following assay, embryos continue to develop normally in vitro. Light-induced dielectrophoresis provides a non-invasive, quantitative, and reproducible means to select embryos for applications including IVF transfer and embryonic stem cell harvest.

Identification of an aberrant cell line among human adipose tissue-derived stem cell isolates

Adipose tissue-derived stem cells (ADSC) are isolated from the stromal vascular fraction (SVF) of adipose tissue and considered an excellent cell source for regenerative medicine. During the isolation and propagation of several human ADSC cell lines, we observed the emergence of an unusual cell line designated HADSC-6. Although initially fibroblast-like as typical ADSC are, HADSC-6 cells became homogeneously cuboid in shape, had very little cytoplasm, and formed aggregates with capsule-like boundary. Proliferation assay showed that HADSC-6 grew much faster than typical HADSC cell lines, such as HADSC-20. Immunocytochemistry showed that HADSC-6 did not express endothelial markers CD31 and vWF, and matrigel tube formation assay showed that it was unable to form endothelial-like tube structures. However, LDL uptake, a reliable endothelial marker, was positively identified. Chromosomal analysis showed that HADSC-6 cells were hypertriploid, and soft agar colony formation assay showed that they were able to proliferate and form large colonies in an anchorage-independent manner. However, tumorigenicity test showed that HADSC-6 was unable to form tumors in athymic mice. RT-PCR analysis showed that both HADSC-6 and HADSC-20 expressed VEGF-A, VEGF-B, VEGF-D, and VEGFR1 but not VEGFR2 or VEGFR3. VEGF-C, however, was expressed at a high level in HADSC-20 but undetectable in HADSC-6. In the IGF system, IGF-1 was abundantly expressed in HADSC-20 but marginally detectable in HADSC-6, and IGF-1R was abundantly expressed in HADSC-6 but not detectable in HADSC-20. In the FGF system, bFGF was abundantly expressed in HADSC-20 but marginally detectable in HADSC-6, and FGFR1 was abundantly expressed in both. Taken together, these results suggested that HADSC-6 cells were spontaneously transformed from the endothelium; therefore, they were further compared to previously published data of four naturally occurring human angiosarcoma cell lines. The results showed that the established angiosarcoma cell lines exhibit considerable variations among themselves and HADSC-6 displayed most of these variable characteristics.

Cavernous nerve repair with allogenic adipose matrix and autologous adipose-derived stem cells

OBJECTIVES To investigate whether adipose-derived matrix seeded with adipose-derived stem cells (ADSC) can facilitate the repair of injured cavernous nerves (CNs). METHODS Human and rat adipose tissues were decellularized and fabricated into various forms, including adipose tissue-derived acellular matrix thread (ADMT). ADMT seeded with ADSC were transplanted into subcutaneous space and examined for signs of inflammation. ADSC-seeded ADMTs were then used to repair CN injury in rats, followed by assessment of histology and erectile function. RESULTS Adipose tissue can be fabricated into acellular matrices of various shapes and sizes, including threads and sheets. Seeding of ADMT occurred rapidly: within 24 hours, 55% of the surface was covered with ADSC and within 1 week, 90% was covered. Transplantation of the seeded ADMT into the subcutaneous space of an allogenic host showed no signs of inflammatory reaction. At 3 months after grafting into CN injury rats, approximately twice as many cells were found on seeded ADMT as on unseeded ADMT. The seeded ADMT also had various degrees of S100 and neuronal nitric oxide synthase expression, suggesting CN axonal ingrowth. Rats grafted with seeded ADMT overall had the best erectile function recovery when compared with those grafted with unseeded ADMT and those ungrafted. However, as a result of large variations, the differences did not reach statistic significance (P = .07). CONCLUSIONS Grafting of ADSC-seeded matrix resulted in a substantial recovery of erectile function and improvement of histology. However, further refinement of the matrix architecture is needed to improve the success rate.

National Incidence and Impact of Noninfectious Urethral Catheter Related Complications on the Surgical Care Improvement Project

PURPOSE We defined the incidence and health outcomes related impact of noninfectious urethral catheter related complications for the 7 surgical procedures monitored by the Joint Commission as part of the Surgical Care Improvement Project. MATERIALS AND METHODS We performed a cross-sectional analysis of the 2007 National Inpatient Sample (a 20% stratified sampling of nonfederal United States hospitals) using ICD-9-CM procedure and diagnostic codes to identify the incidence of catheter related complications for coronary artery bypass graft, and noncoronary artery bypass graft cardiac surgery, hysterectomy, colon, hip, knee and major vascular surgery. Univariate and multivariate analysis (with a significance level of less than 0.05) was performed to determine if these complications were associated with length of stay, urinary tract infections and/or deaths. RESULTS A total of 1,420 cases of catheter related complications were identified nationally. The incidence of catheter related complications varied by surgical procedure (average 1 in 528 men and 1 in 5,217 women for all procedures). Univariate analysis revealed that in the presence of catheter related complications, mean length of stay (6 of 7 procedures, range 1.5 to 3.0 days, p <0.05) and urinary tract infection (5 of 7 procedures, absolute range 6.9% to 11.8%, p <0.05) were statistically increased for most procedures. Multivariate analysis demonstrated a significant association between catheter related complications, and increased length of stay (range 1.5 to 3.5 days, p <0.05) and urinary tract infection (OR 2.4-6.8, p <0.05) for 5 and 6 of 7 procedure types, respectively, but not mortality rate (0 of 7 procedures). CONCLUSIONS Catheter related complications are reported rarely, but are associated with increased length of stay and urinary tract infection rates for patients in the Surgical Care Improvement Project.