A biomechanical assessment of tissue-engineered polymer neo-uteri after orthotopic implantation.

OBJECTIVE: To assess the in vivo biomechanical maturation of tissue-engineered neo-uteri that have previously supported live births in a rabbit model. DESIGN: Nonclinical animal study. SETTING: University-based research laboratory. ANIMALS: Eighteen adult female rabbits. INTERVENTION: Biodegradable poly-DL-lactide-co-glycolide-coated polyglycolic acid scaffolds seeded with autologous uterine-derived endometrial and myometrial cells. Nonseeded scaffolds and seeded, tissue-engineered neo-uteri were implanted into one uterine horn of rabbits for 1, 3, or 6 months, excised, and biomechanically assessed in comparison to native uterine tissue. MAIN OUTCOME MEASURES: Tensile stress-relaxation testing, strain-to-failure testing, and viscoelastic modeling. RESULTS: By evaluating the biomechanical data with several viscoelastic models, it was revealed that tissue-engineered uteri were more mechanically robust than nonseeded scaffolds. For example, the 10% instantaneous stress of the tissue-engineered neo-uteri was 2.1 times higher than the nonseeded scaffolds at the 1-month time point, 1.6 times higher at the 3-month time point, and 1.5 times higher at the 6-month time point. Additionally, as the duration of implantation increased, the engineered constructs became more mechanically robust (e.g., 10% instantaneous stress of the tissue-engineered neo-uteri increased from 22 kPa at 1 month to 42 kPa at 6 months). Compared with native tissue values, tissue-engineered neo-uteri achieved or surpassed native tissue values by the 6-month time point. CONCLUSION: The present study evaluated the mechanical characteristics of novel tissue-engineered neo-uteri that have previously been reported to support live births in the rabbit model. We demonstrate that the biomechanics of these implants closely resemble those of native tissue, giving further credence to their development as a clinical solution to uterine factor infertility.

Effects of Age and Multiple Vaginal Births on Lower Urinary Tract Structure and Function in Nonhuman Primates.

PURPOSE: The relative roles of urinary sphincter damage, aging, and childbirth in stress urinary incontinence (SUI), have not been established. This study was performed to elucidate the roles of these factors. METHODS: The study included: (1) 8 female cynomolgus monkeys (17-19 years of age and 7-8 vaginal births each); (2) six 5-yearold nulliparous monkeys with surgically created chronic urinary sphincter dysfunction; and (3) six 5-year-old, nulliparous, nosurgery controls. Sedated abdominal leak point pressure (ALPP) and maximum urethral sphincter pressures (MUP) were measured. Sphincters, bladders, and pelvic support muscles were quantified for collagen content. Additionally, bladders were analyzed for collagen fiber thickness, length, and angle using CT-FIRE analysis of Picrosirius red-stained tissues. RESULTS: Resting MUP values were similar in the controls and older multiparous monkeys (P>0.05). However, aging and multiple births reduced pudendal nerve-stimulated increases in MUP (P<0.05 vs. controls). ALPP values were lower in the older multiparous versus younger groups of monkeys (P<0.05). Sphincter collagen content was greater, and muscle content less, in the injury model (P<0.05 vs. controls). However, these measures were not affected by age and childbirth (P>0.05 vs. young groups). Bladder collagen content was greater, and muscle content less, in the old multiparous monkeys (P<0.05 vs. younger groups). Additionally, collagen fibers were thicker and more angular in the bladders of the older multiparous monkeys than in the other nonhuman primate groups (P<0.05). Pelvic support muscles had higher collagen and lower muscle content in the older multiparous monkeys than in the younger groups of monkeys (P<0.05). CONCLUSION: SUI, associated with aging and multiple childbirths, appeared to be more strongly associated with bladder dysfunction, reduced pelvic muscle support, and the compensatory response to neural stimulation than with selective urinary sphincter dysfunction.

Treatment of Stress Urinary Incontinence with Muscle Stem Cells and Stem Cell Components: Chances, Challenges and Future Prospects.

Urinary incontinence (UI) is a major problem in health care and more than 400 million people worldwide suffer from involuntary loss of urine. With an increase in the aging population, UI is likely to become even more prominent over the next decades and the economic burden is substantial. Among the different subtypes of UI, stress urinary incontinence (SUI) is the most prevalent and focus of this review. The main underlying causes for SUI are pregnancy and childbirth, accidents with direct trauma to the pelvis or medical treatments that affect the pelvic floor, such as surgery or irradiation. Conservative approaches for the treatment of SUI are pelvic physiotherapy, behavioral and lifestyle changes, and the use of pessaries. Current surgical treatment options include slings, colposuspensions, bulking agents and artificial urinary sphincters. These treatments have limitations with effectiveness and bear the risk of long-term side effects. Furthermore, surgical options do not treat the underlying pathophysiological causes of SUI. Thus, there is an urgent need for alternative treatments, which are effective, minimally invasive and have only a limited risk for adverse effects. Regenerative medicine is an emerging field, focusing on the repair, replacement or regeneration of human tissues and organs using precursor cells and their components. This article critically reviews recent advances in the therapeutic strategies for the management of SUI and outlines future possibilities and challenges.

Chemokine Therapy in Cats With Experimental Renal Fibrosis and in a Kidney Disease Pilot Study.

Background: Chronic tubulointerstitial fibrosis is a common final pathway leading to end stage kidney disease in cats and has no effective treatment. The use of cell-based molecules to treat kidney fibrosis may be a promising approach. The objectives were to test the effects of intra-renal chemokine CXCL12 injection in a pre-clinical cat model of unilateral ischemia/reperfusion (I/R)-induced kidney fibrosis and then, within a clinical pilot study, test the safety/feasibility of CXCL12 injection in cats that might have early chronic kidney disease (CKD). Methods: Pre-clinical: Thirty cats received intra-renal injection of 100, 200, or 400 ng of recombinant human CXCL12, or sterile saline, into the I/R kidney 70 days post-injury, or were non-injured, non-injected controls (n = 6/group). Kidney collagen content was quantified 4 months post-treatment using Masson's Trichrome and Picrosirius Red (PSR) stained tissues. In a separate study (n = 2) exploring short-term effects of CXCL12, 200 ng CXCL12 was injected into I/R kidneys and then harvested either 30 min (n = 1) or 1 month (n = 1) post-injection. Kidney concentrations of CXCL12, matrix metalloproteinase 1 (MMP-1), and lysyl oxidase-like enzyme 2 (LOXL-2) were quantified via ELISA. Clinical Pilot: 14 client-owned cats with potential early kidney disease received a single-treatment, bilateral intra-renal injection of 200 ng CXCL12 (n = 7), or received no injection (n = 7). Blood/urine samples were collected monthly for 9 months to assess renal function and CKD staging. Results: Pre-clinical: I/R increased the affected kidney collagen content, which both mid and high doses of CXCL12 restored to normal (ps < 0.05 vs. untreated). I/R increased collagen fiber width, which both mid and high doses of CXCL12 restored to normal (p < 0.001 vs. untreated). Early changes in kidney MMP-1, associated with collagen breakdown, and subsequent decreases in LOXL-2, associated with collagen cross-linking, in response to CXCL12 treatment may contribute to these findings. Clinical Pilot: Bilateral intra-renal injection of CXCL12 using ultrasound guidance in cats with CKD was feasible and safe in a general practice clinical setting with no obvious side effects noted during the 9-month follow-up period. Conclusions: Intra-renal injection of CXCL12 may prove to be an effective treatment for kidney fibrosis in cats with CKD. Additional mechanistic and clinical evaluations are needed.

A tissue-engineered uterus supports live births in rabbits.

Bioengineered uterine tissue could provide a treatment option for women with uterine factor infertility. In large animal models, reconstruction of the uterus has been demonstrated only with xenogeneic tissue grafts. Here we use biodegradable polymer scaffolds seeded with autologous cells to restore uterine structure and function in rabbits. Rabbits underwent a subtotal uterine excision and were reconstructed with autologous cell-seeded constructs, with nonseeded scaffolds or by suturing. At 6 months postimplantation, only the cell-seeded engineered uteri developed native tissue-like structures, including organized luminal/glandular epithelium, stroma, vascularized mucosa and two-layered myometrium. Only rabbits with cell-seeded constructs had normal pregnancies (four in ten) in the reconstructed segment of the uterus and supported fetal development to term and live birth. With further development, this approach may provide a regenerative medicine solution to uterine factor infertility.

Cell Versus Chemokine Therapy Effects on Cell Mobilization to Chronically Dysfunctional Urinary Sphincters of Nonhuman Primates.

PURPOSE: A major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction. METHODS: Either autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment. RESULTS: GFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P<0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex. CONCLUSION: This dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.

Animal Models in Tissue Engineering. Part I.

Animal models play a central and pivotal role in tissue engineering. Although advances in areas such as 3D printing and bioreactor technologies now permit the in vitro development and testing of complex scaffold/cell composites, in vivo testing remains critical not only for refining methods being developed but also for the critical efficacy and safety testing required for regulatory approval. Yet, choosing the appropriate model for a particular application remains a challenge, as each model has its own strengths and weaknesses. In some cases, there are size issues to contend with as scale-up of a 3D structure brings with it considerable challenges with regard to diffusion, infiltration, and structural forces. In others, physiological differences between species make selection of the appropriate animal model that best represents the human disease or injury critical.

Local versus intravenous injections of skeletal muscle precursor cells in nonhuman primates with acute or chronic intrinsic urinary sphincter deficiency.

BACKGROUND: Many factors may influence the efficacy of cell therapy for intrinsic urinary sphincter deficiency (ISD), including the route of administration of the cells and the condition of the sphincter. The goal of this study was to compare local versus intravenous administration of autologous skeletal muscle precursor cells (skMPCs) when administered to nonhuman primates (NHPs) with either acute or chronic ISD. METHODS: Thirty-two adult female monkeys were divided into eight groups (n = 4/group): (1) control; (2) surgically induced ISD/no treatment; (3) acute ISD (6-week duration)/local vehicle only; (4) acute ISD/local skMPC injection; (5) acute ISD/systemic skMPC; (6) chronic ISD (6-month duration)/local vehicle; (7) chronic ISD/local skMPC; (8) chronic ISD/systemic skMPC. Maximal urethral pressures (MUP) were measured prior to ISD, prior to treatment and at 3 and 6 months following treatment. Quantitative histology was used to measure muscle/collagen content, somatic innervation, and vascularity of the sphincter complexes. RESULTS: In NHPs with acute ISD both systemic and local administration of skMPCs increased resting MUP values and sphincter muscle content (p < 0.05 vs. ISD/vehicle). However, the effects of systemic skMPC administration were significantly lower than those of local injection (p > 0.05). In NHPs with chronic ISD local skMPC administration had reduced (compared to NHPs with acute ISD) effects on MUP and sphincter muscle values (p < 0.05 vs. acute ISD/skMPC); systemic administration had no effect. Pudendal nerve-stimulated increases in MUP were significant only in acute ISD NHPs with local skMPC treatment (p < 0.05 vs. resting MUP). The extent of sphincter vascularization and innervation were directly related to MUP and sphincter muscle content. CONCLUSIONS: Both the chronicity of ISD and the route of cell injection influence the efficacy of cell therapy in monkey models of ISD. This may be related to the relative ability of cells to stimulate vascularization and re-innervation in these different treatment conditions.

Regenerative Medicine Therapies for Stress Urinary Incontinence.

PURPOSE: We summarize the current state of knowledge regarding cell therapy for stress urinary incontinence and introduce new approaches of using regenerative pharmacology as an adjunct or replacement for cell therapy. MATERIALS AND METHODS: We reviewed the literature by searching PubMed®, Ovid and Biological Abstracts. The period searched was 1975 to December 2015. The inclusion terms separately or in combination were stress urinary incontinence, cell therapy, chemokine, vascularization, innervation, secretome and/or animal models. Epublished articles were not included. We did not exclude articles based on impact factor. RESULTS: Cell therapy is currently proposed to restore functional muscle cells and aid in closure of the sphincter in women with sphincter associated incontinence. Clinical trials have included small numbers of patients and results have varied depending on the patient cohorts and the cells used. Results of preclinical studies have also varied but show a more favorable outcome. This difference was most likely explained by the fact that animal modeling is not directly translatable to the human condition. However, preclinical studies have identified an exciting new approach to regeneration of the urinary sphincter using the components of cells (secretomes) or chemokines that home reparative cells to sites of injury. CONCLUSIONS: Cell therapy will continue to be explored. However, a regenerative pharmacological approach to the treatment of stress urinary incontinence holds the promise of bypassing the lengthy and expensive process of cell isolation and also increasing the availability of treatment in many clinical settings. This approach requires careful preclinical modeling and attention to its health benefit-to-risk ratio.

Cell versus Chemokine Therapy in a Nonhuman Primate Model of Chronic Intrinsic Urinary Sphincter Deficiency.

PURPOSE: Mixed efficacy results of autologous skeletal muscle precursor cell therapy in women with chronic intrinsic urinary sphincter deficiency have increased interest in the therapeutic value of alternative regenerative medicine approaches. The goal of this study was to compare the effects of the cell homing chemokine CXCL12 (C-X-C motif chemokine 12) and skeletal muscle precursor cells on chronic urinary sphincter regeneration in chronic intrinsic urinary sphincter deficiency. MATERIALS AND METHODS: Five million autologous skeletal muscle precursor cells or 100 ng CXCL12 were injected in the urinary sphincter complex of adult female cynomolgus monkeys with chronic (6-month history) intrinsic urinary sphincter deficiency. These treatment groups of 3 monkeys per group were compared to a group of 3 with no intrinsic urinary sphincter deficiency and no injection, and a group of 3 with intrinsic urinary sphincter deficiency plus vehicle injection. Maximal urethral pressure was measured at rest, during stimulation of the urinary sphincter pudendal nerves at baseline and again 6 months after treatment. The monkeys were then necropsied. The urinary sphincters were collected for tissue analysis of muscle and collagen content, vascularization and motor endplates. RESULTS: CXCL12 but not skeletal muscle precursor cells increased resting maximal urethral pressure in nonhuman primates with chronic intrinsic urinary sphincter deficiency compared to that in monkeys with intrinsic urinary sphincter plus vehicle injection (p >0.05). Skeletal muscle precursor cells and CXCL12 only partially restored pudendal nerve stimulated increases in maximal urethral pressure (p >0.05), sphincter vascularization and motor endplate expression in monkeys with chronic intrinsic urinary sphincter deficiency. Additionally, CXCL12 but not skeletal muscle precursor cell injections decreased collagen and increased the muscle content of urinary sphincter complex in monkeys with chronic intrinsic urinary sphincter deficiency compared to those with intrinsic urinary sphincter plus vehicle injection and no intrinsic urinary sphincter plus no injection (p <0.05 and >0.05, respectively). CONCLUSIONS: These results raise questions about cell therapy for chronic intrinsic urinary sphincter deficiency and identify a chemokine treatment (CXCL12) as a potential alternative treatment of chronic intrinsic urinary sphincter deficiency.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Characterization of CD133 Antibody-Directed Recellularized Heart Valves.

CD133mAb conjugation (CD133-C) hastens in vivo recellularization of decellularized porcine heart valve scaffolds when placed in the pulmonary position of sheep. We now characterize this early cellularization process 4 h, 3, 7, 14, 30, or 90 days post-implantation. Quantitative immunohistochemistry identified cell types as well as changes in cell markers and developmental cues. CD133(+)/CD31(-) cells adhered to the leaflet surface of CD133-C leaflets by 3 days and transitioned to native leaflet-like CD133(-)/CD31(+) cells by 30 days. Leaflet interstitium became increasingly populated with both alpha-smooth muscle actin (αSMA) and vimentin(+) cells from 14 to 90 days post-implantation. Wnt3a, and beta-catenin proteins were expressed at early (3-14 days) but not later (30-90 days) time points. In contrast, matrix metalloproteinase-2 and periostin proteins were increasingly expressed over 90 days. Thus, early development of CD133-C constructs includes a fairly rapid transition from a precursor cell adhesion/migration/transdifferentiation phenotype to a more mature cell/native valve-like matrix metabolism phenotype.

The dose-effect safety profile of skeletal muscle precursor cell therapy in a dog model of intrinsic urinary sphincter deficiency.

Locally injected skeletal muscle precursor cells (skMPCs) integrate into and restore the muscle layers, innervation, vasculature, and function of the sphincter complex in animal models of intrinsic urinary sphincter deficiency (ISD). The goal of the present study was to test the dose-effect safety profile of skMPC therapy in a dog model of ISD. Sphincter deficiency was created in 20 adult female dogs by surgically removing the skeletal muscle layer of the urinary sphincter complex. skMPCs isolated from the hind leg were expanded in culture and injected 4 weeks later into the sphincter complex at a dose of 25 million cells (n = 5), 50 million cells (n = 5), or 100 million cells (n = 5) per milliliter in a 2-ml volume. Five dogs received no sphincter injection. The measures of maximal sphincter pressure, complete blood count, and blood chemistry were performed monthly until their sacrifice at 9 months. At that point, full necropsy was performed to assess the safety of the skMPC injections. Injection of different doses of cells had no effects on the body weight, blood cell count, or kidney or liver function test results (p > .05 among the skMPC doses). Some incidental pathologic features were found in the lower urinary tract in all groups and were most likely associated with repeat catheterization. The maximal urinary sphincter pressure was higher in the 50 million cells per milliliter treatment group than in the other experimental groups (p < .05). The findings of the present study have confirmed that urinary sphincter injection of skMPCs results in no significant local or systemic pathologic features within the dose range that improves sphincter pressures.

Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy.

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Long-term structural and functional effects of autologous muscle precursor cell therapy in a nonhuman primate model of urinary sphincter deficiency.

PURPOSE: We measured the long-term efficacy of autologous muscle precursor cell therapy in premenopausal female nonhuman primates with sustained urinary sphincter deficiency. MATERIALS AND METHODS: Urinary sphincter deficiency was created in adult premenopausal female cynomolgus monkeys by selectively cauterizing and then transecting the pudendal innervation to the sphincter complex. The monkeys were then treated (18) or not treated (18) with intra-urinary sphincter injections of 5 million autologous green fluorescent protein labeled skeletal muscle precursor cells. Four untreated, uninjured monkeys served as controls. Maximal urethral pressure measurement and corresponding histological analysis of the structural and cellular components of the sphincter complex were performed up to 12 months after injection. RESULTS: Cell treatment produced sustained (12 months) increases in resting, somatic nerve stimulated and adrenergic nerve stimulated maximal urethral pressure, and a greater percent of sphincter area occupied by muscle as well as a decrease in the sphincter area occupied by collagen compared to the untreated group (each p>0.05). These results were within control values (each p>0.05). By 3 months after injection green fluorescent protein positive cells were found in the skeletal muscle layer, expressing desmin and connexin-43, and in the smooth muscle layer, expressing α-smooth muscle actin and connexin-43, and they were incorporated into the subendothelial vasculature, expressing Von Willebrand factor. Cell injected sphincter tissue contained a mixture of green fluorescent protein positive cells and predominantly green fluorescent protein negative cells. CONCLUSIONS: Injected skeletal muscle progenitor cells incorporated into the injured sphincter complex resulted in long-term structural and functional restoration of the injured sphincter complex in this nonhuman primate model.

Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro.

Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for ß-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a ß-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage.

A nonhuman primate model of stable urinary sphincter deficiency.

PURPOSE: The pathophysiology of urinary sphincter deficiency in women remains incompletely understood and current treatment options have limitations. Female nonhuman primates may represent a relevant animal model for studies of pathophysiology and treatment interventions because of their human-like reproductive and age associated stages of life (premenopause, perimenopause and postmenopause), lower urinary tract structure and bipedal posture. We developed and characterized a nonhuman primate model of defined injury to the urethral sphincter complex. MATERIALS AND METHODS: We used 22 adult female cynomolgus monkeys in which injury to the sphincter complex was created by cauterizing and then transecting its pudendal innervation. Urodynamic studies were performed before and during pudendal and hypogastric nerve stimulation at baseline, and 3, 6 and 12 months after injury. We also analyzed sphincter structure in vivo by cystourethrography, and ex vivo by quantitative histology and immunohistochemistry at these time points. RESULTS: Injury produced a 47% to 50% decrease in maximal urethral pressure (vs baseline p <0.05). It also abolished the increase in maximal urethral pressure in response to pudendal and hypogastric nerve stimulation (vs baseline p >0.05), which persisted more than 12 months after injury. Urodynamic changes were consistent with decreased skeletal and smooth muscle content, decreased nerve responses and an associated decrease in somatic and adrenergic innervation in the sphincter complex. CONCLUSIONS: These structural and urodynamic changes are consistent with those in patients with stress urinary incontinence. They support the usefulness of nonhuman primates as translatable surrogates for pathophysiological studies of urinary sphincter deficiency and testing novel therapies for that condition.

Life stage differences in mammary gland gene expression profile in non-human primates.

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.