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

Abstract

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.

Keywords: Maximal urethral pressure; Pathology; Skeletal muscle; Stem cells; Urinary incontinence.

Figure 1.

Overall study design. “Injury” indicates the skeletal muscle stripping of urinary sphincter complex. “Cells” indicates autologous skeletal muscle-derived precursor cells collected from a muscle biopsy. “Carrier” indicates vehicle (Dulbecco’s modified Eagle’s medium without added serum). Maximal urethral pressures and blood samples were taken before the injury procedure, immediately before the sphincter injections, and once each month for 9 months. Euthanasia and tissue collection were performed 9 months after the sphincter injections. Abbreviation: H&E, hematoxylin and eosin.

Figure 2.

Body weights. The baseline values for the animal weights were similar at the onset of the experiment (control: 9.2 ± 0.7 kg, n = 4; vs. 25 million cells per milliliter: 8.5 ± 0.3 kg, n = 4; 50 million cells per milliliter: 8.3 ± 0.4 kg, n = 5; and 100 million cells per milliliter: 8.1 ± 0.5 kg, n = 5; p = .531). Furthermore, no significant change was seen in the body weights of the dogs throughout the study, as assessed by the within-group analysis (control, n = 4, p = .996; 25 million cells per milliliter, n = 4, p = .986; 50 million cells per milliliter, n = 5, p = .999; and 100 million cells per milliliter, n = 5, p = .819). Abbreviation: M, million.

Figure 3.

Serum markers. BUN and creatinine values were similar among the experimental groups throughout the study (BUN: p = .254; creatinine: p = .601, n = 4–5). AST showed a modest difference between the 100 million cells per milliliter and control groups and the 50 million cells per milliliter (∗) (p = .039, n = 4–5; and p = .048, n = 5, respectively). No change was seen in ALT (p = .061, n = 4–5). The hemoglobin (Hgb), red blood cell (RBC) count, total white blood cell (WBC) count, and lymphocyte counts were similar among the groups throughout the study (Hgb: p = .927; RBC count: p = .569; WBC count: p = .089; and lymphocyte count: p = .100; n = 4–5). Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; M, million.

Figure 4.

Efficacy. (A): Desmin staining (nova red) of the urethral sphincter complex in the CON, 25, 50, and 100. Arrows point to the desmin staining in these cross-sections of the sphincter. Scale bars = 200 µm. (B, C): Sphincter pressures. The actual MUP values (B) and the percentage of change in MUP (C) taken before sphincter injury (point a), immediately after sphincter injury (point b), 4 weeks later immediately before cell injection (point c), and then monthly for 9 months (points 1–9). The mean MUP values for all experimental groups decreased from point a to point b (∗) (CON: p = .037, n = 4; 25: p = .036, n = 4; 50: p = .007, n = 5; 100: p = .020, n = 5). No difference was found in postoperative MUPs among the experimental groups (B) at point b (p = .576, n = 4–5). The MUP values in the dogs treated with 50 million cells per milliliter were significantly greater than those in the CON group (∗∗) (p = .0004) or 25 (p = .0014) and 100 group (p < .0001) (C). However, the MUP values of the other 2 skMPC groups (25 and 100) were not different from the CON values (p = .956 and p = .871, respectively). Abbreviations: 25, 25 million cells per milliliter; 50, 50 million cells per milliliter; 100, 100 million cells per milliliter; CON, control; M, million; MUP, maximal resting urethral sphincter pressure.