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.

Autologous bone marrow-derived MSCs engineered to express oFVIII-FLAG engraft in adult sheep and produce an effective increase in plasma FVIII levels.

Introduction: Hemophilia A (HA) is the most common X-linked bleeding disorder, occurring in 1 in 5,000 live male births and affecting >1 million individuals worldwide. Although advances in protein-based HA therapeutics have improved health outcomes, current standard-of-care requires infusion 2-3 times per week for life, and 30% of patients develop inhibitors, significantly increasing morbidity and mortality. There are thus unmet medical needs requiring novel approaches to treat HA. Methods: We tested, in a highly translational large animal (sheep) model, whether the unique immunological and biological properties of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) could enable them to serve as cellular delivery vehicles to provide long-term expression of FVIII, avoiding the need for frequent infusions. Results: We show that autologous BM-MSCs can be isolated, transduced with a lentivector to produce high levels of ovine (o)FVIII, extensively expanded, and transplanted into adult animals safely. The transplanted cells engraft in multiple organs, and they stably produce and secrete sufficient quantities of FVIII to yield elevated plasma FVIII levels for at least 15 weeks. Discussion: These studies thus highlight the promise of cellular-based gene delivery approaches for treating HA.

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.

Nonhuman primate model of persistent erectile and urinary dysfunction following radical prostatectomy: Feasibility of minimally invasive therapy.

OBJECTIVE: Persistent urinary incontinence (UI) and/or erectile dysfunction (ED) occur in 30-50% of post-radical prostatectomy patients regardless of nerve sparing approaches. Identification of potential treatment options for these patients will require testing in an animal model that develops these chronic conditions. The objective was to characterize a nonhuman primate (NHP) model of persistent post-prostatectomy ED and UI and then test the feasibility of periurethral injection of the chemokine CXCL-12. METHODS: Ten adult male cynomolgus monkeys were used. Two were used for study of normal male nonhuman primate genitourinary anatomy. Five were used for measures of sexual behavior, peak intra-corporal pressure (ICP), abdominal leak point pressures (ALPP) 3 and 6-months post open radical prostatectomy (ORP). Three additional ORP animals received ultrasound-guided peri-urethral injection of chemokine CXCL12 6 weeks after ORP, and UI/ED evaluated for up to 3 months. RESULTS: The anatomy, innervation, and vascular supply to the prostate and surrounding tissues of these male NHPs are substantially similar to those of human beings. ORP resulted in complete removal of the prostate gland along with both neurovascular bundles and seminal vesicles while permitting stable restoration of vesico-urethral patency. ORP produced sustained (6 months) decreases in ALPP, ICP's, and sexual function. Transurethral injection of chemokine CXCL12 was feasible and had beneficial effects on erectile and urinary function. CONCLUSIONS: ORP in NHPs produced persistent erectile and urinary tract dysfunction. Periurethral injection of CXCL-12 was feasible and improved both urinary incontinence and erectile dysfunction and suggests that this model can be used to test new approaches for both conditions.

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.

Efficacy and Initial Safety Profile of CXCL12 Treatment in a Rodent Model of Urinary Sphincter Deficiency.

Disappointing results of skeletal muscle precursor cell (skMPC) therapy for women with intrinsic urinary sphincter deficiency (ISD) associated urinary incontinence has increased interest in alternative sphincter regenerative approaches. This study was to measure the safety and efficacy of the cell homing chemokine CXCL12 versus skMPCs in a rat model of ISD. Thirty-six adult female Sprague Dawley rats were divided into 6 treatment (Tx) conditions: (a) no ISD/noTx [Control]; (b) ISD/noTx; (c) ISD + skMPCs; (d) ISD + 3.5 mg CXCL12; (e) ISD + 7mg CXCL12; and (f) ISD + 14 mg CXCL12. Tx's were injected directly into the sphincter complex 30 days post ISD and rats euthanized 30 days post Tx. Blood samples for measurements of kidney and liver function, white and red blood cell counts, were taken at baseline and at euthanasia. Leak point pressures (LPP) were measured prior to, and sphincter collagen/muscle content measured after, euthanasia. There were no effects of treatments on white or red/white blood cell counts, kidney/liver function tests or histopathology of the urinary sphincter complex or surrounding tissues. ISD lowered LPP 35% and sphincter muscle content by 17% versus control rats. CXCL12, but not skMPC injections, restored both LPP to control values in a dose-dependent fashion. Both skMPCs and CXCL12 restored sphincter muscle content to control values. This chemokine approach may represent a novel therapeutic option for ISD and appears, at least short-term, to produce little clinical or tissue pathology. Stem Cells Translational Medicine 2017;6:1740-1746.

Determinates of muscle precursor cell therapy efficacy in a nonhuman primate model of intrinsic urinary sphincter deficiency.

BACKGROUND: Cell therapy for intrinsic urinary sphincter deficiency (ISD) in women has been moderately effective, and improvements are needed. To improve treatment efficacy, it is important to better understand determinates of cell efficacy in the different patient cohorts. We have reported that in nonhuman primates the chronicity of ISD may affect cell efficacy, but additional factors (age, psychosocial stress, hormone status, body weight) can be associated with many disease/treatment outcomes in women - and these factors are the focus of this study. METHODS: Adult female cynomolgus monkeys were divided into groups: (1) younger (n = 10, 5-8 years of age) versus older (n = 10, 13-18 years of age); (2) age-matched/socially subordinate (n = 15) versus socially dominant (n = 15); and (3) age-matched lower body weight (n = 6) versus higher body weight (n = 6). Autologous skeletal muscle precursor cells (skMPCs, 5 million) were injected into the urinary sphincter 6 weeks after a surgically induced ISD procedure. Resting and pudendal nerve-stimulated maximal urethral pressures (MUP) were measured before, and 3 and 6 months post-skMPC treatment and urinary sphincter muscle/collagen content within the sphincter complex was measured by quantitative histology 6 months posttreatment. RESULTS: Efficacy of skMPCs on MUP and sphincter muscle/collagen ratios are affected by age (average 40% reduction in efficacy, p < 0.05 vs. younger NHPs), social stress (average 30% reduction in efficacy, p < 0.05 vs. socially dominant) and body weight/fasting glucose concentrations (average 35% reduction in efficacy, p < 0.05 vs. lower body weight). CONCLUSION: Multiple factors (age, stress-induced dysmenorrhea, and body weight) affect the efficacy of cell therapy to restore structure and function in the urinary sphincter complex in NHPs with ISD. Consideration of, and alternatives for, these patient cohorts should be considered.

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.

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.