The leading cause of stress urinary incontinence (SUI) in women is the urethral sphincter muscle deficiency caused by mechanical stress during pregnancy and vaginal delivery. In men, prostate cancer surgery and injury of local nerves and muscles are associated with incontinence. Current treatment often fails to satisfy the patient's needs. Cell therapy may improve the situation. We therefore investigated the regeneration potential of cells in ameliorating sphincter muscle deficiency and UI in a large animal model. Urethral sphincter deficiency was induced surgically in gilts by electrocautery and balloon dilatation. Adipose tissue-derived stromal cells (ADSCs) and myoblasts from Musculus semitendinosus were isolated from male littermates, expanded, characterized in depth for expression of marker genes and in vitro differentiation, and labeled. The cells were injected into the deficient sphincter complex of the incontinent female littermates. Incontinent gilts receiving no cell therapy served as controls. Sphincter deficiency and functional regeneration were recorded by monitoring the urethral wall pressure during follow-up by two independent methods. Cells injected were detected in vivo during follow-up by transurethral fluorimetry, ex vivo by fluorescence imaging, and in cryosections of tissues targeted by immunofluorescence and by polymerase chain reaction of the sex-determining region Y (SRY) gene. Partial spontaneous regeneration of sphincter muscle function was recorded in control gilts, but the sphincter function remained significantly below levels measured before induction of incontinence (67.03% ± 14.00%, n = 6, p < 0.05). Injection of myoblasts yielded an improved sphincter regeneration within 5 weeks of follow-up but did not reach significance compared to control gilts (81.54% ± 25.40%, n = 5). A significant and full recovery of the urethral sphincter function was observed upon injection of ADSCs within 5 weeks of follow-up (100.4% ± 23.13%, n = 6, p < 0.05). Injection of stromal cells provoked slightly stronger infiltration of CD45pos leukocytes compared to myoblasts injections and controls. The data of this exploratory study indicate that ADSCs inherit a significant potential to regenerate the function of the urethral sphincter muscle.
Impact statement
Urinary incontinence is a significant medical challenge and current therapies often do not satisfy patients' needs. This study investigates the potential of two different types of cells, stromal cells versus myoblasts, head-to-head in a robust animal model of incontinence. The stromal cells outperformed the myoblasts. Autologous stromal cells can be isolated from adipose tissue of patients without intolerable side effects in sufficient amounts and produced with much less effort under good manufacturing protocols-compliant conditions. Our results suggest confirmatory preclinical studies to verify the efficacy of stromal cell-mediated sphincter regeneration and recommend such cells for future clinical trials.
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References
1.
BroomeBA.The impact of urinary incontinence on self-efficacy and quality of life. Health Qual Life Outcomes, 2003; 1:35.
2.
LuberKM.The definition, prevalence, and risk factors for stress urinary incontinence. Rev Urol, 2004; 6Suppl 3(Suppl 3):S3–S9.
3.
ShamliyanTA, WymanJF, PingR, et al.Male urinary incontinence: Prevalence, risk factors, and preventive interventions. Rev Urol, 2009; 11(3):145–165.
4.
AbramsP, AnderssonKE, BirderL, et al.Fourth International Consultation on Incontinence Recommendations of the International Scientific Committee: Evaluation and treatment of urinary incontinence, pelvic organ prolapse, and fecal incontinence. Neurourol Urodyn, 2010; 29(1):213–240.
5.
MilsomI, GyhagenM. The prevalence of urinary incontinence. Climacteric, 2019; 22(3):217–222.
6.
CoyneKS, WeinA, NicholsonS, et al.Comorbidities and personal burden of urgency urinary incontinence: A systematic review. Int J Clin Pract, 2013; 67(10):1015–1033.
7.
ChappleCR, OsmanNI. Current issues in managing men with lower urinary tract symptoms in primary care. Int J Clin Pract, 2013; 67(10):931–933.
8.
PatelPD, AmruteKV, BadlaniGH. Pelvic organ prolapse and stress urinary incontinence: A review of etiological factors. Indian J Urol, 2007; 23(2):135–141.
9.
GonzalesAL, BarnesKL, QuallsCR, et al.Prevalence and treatment of postpartum stress urinary incontinence: A systematic review. Urogynecology, 2021; 27(1):e139–e145.
10.
BuckleyBS, LapitanMCM. Prevalence of urinary incontinence in men, women, and children—Current evidence: Findings of the Fourth International Consultation on Incontinence. Urology, 2010; 76(2):265–270.
11.
RichterHE, BurgioKL, GoodePS, et al.Non-surgical management of stress urinary incontinence: Ambulatory treatments for leakage associated with stress (ATLAS) trial. Clin Trials, 2007; 4(1):92–101.
12.
LucasMG, BoschRJL, BurkhardFC, et al.European Association of Urology guidelines on assessment and nonsurgical management of urinary incontinence. Actas Urológicas Españolas (English Edition), 2013; 37(4):199–213.
HarlandN, WalzS, EberliD, et al.Stress urinary incontinence. An unsolved clinical challenge. Biomedicines, 2023; 11:2486; doi: 10.3390/biomedicines11092486
15.
StrasserH, TiefenthalerM, SteinlechnerM, et al.Urinary incontinence in the elderly and age-dependent apoptosis of rhabdosphincter cells. Lancet, 1999; 354(9182):918–919.
16.
YiouR, LefaucheurJP, AtalaA. The regeneration process of the striated urethral sphincter involves activation of intrinsic satellite cells. Anat Embryol (Berl), 2003; 206(6):429–435.
17.
YiouR, DreyfusP, ChopinDK, et al.Muscle precursor cell autografting in a murine model of urethral sphincter injury. BJU Int, 2002; 89(3):298–302.
18.
MitterbergerM, PinggeraG-M, MarksteinerR, et al.Adult stem cell therapy of female stress urinary incontinence. Eur Urol, 2008; 53(1):169–175.
19.
EberliD, AboushwarebT, SokerS, et al.Muscle precursor cells for the restoration of irreversibly damaged sphincter function. Cell Transplant, 2012; 21(9):2089–2098.
20.
PetersKM, DmochowskiRR, CarrLK, et al.Autologous muscle derived cells for treatment of stress urinary incontinence in women. J Urol, 2014; 192(2):469–476.
21.
SchmidFA, WilliamsJK, KesslerTM, et al.Treatment of stress urinary incontinence with muscle stem cells and stem cell components: Chances, challenges and future prospects. Int J Mol Sci, 2021; 22(8):3981.
22.
BlaganjeM, LukanovićA. The effect of skeletal muscle-derived cells implantation on stress urinary incontinence and functional urethral properties in female patients. Int J Gynecol Obstet, 2022; 157(2):444–451.
23.
DissarananC, CruzMA, KiedrowskiMJ, et al.Rat mesenchymal stem cell secretome promotes elastogenesis and facilitates recovery from simulated childbirth injury. Cell Transplant, 2014; 23(11):1395–1406.
24.
WuR, HuangC, WuQ, et al.Exosomes secreted by urine-derived stem cells improve stress urinary incontinence by promoting repair of pubococcygeus muscle injury in rats. Stem Cell Res Ther, 2019; 10(1):80.
25.
RollandTJ, PetersonTE, SinghRD, et al.Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence. NPJ Regen Med, 2022; 7(1):58.
26.
CorcosJ, LoutochinO, CampeauL, et al.Bone marrow mesenchymal stromal cell therapy for external urethral sphincter restoration in a rat model of stress urinary incontinence. Neurourol Urodyn, 2011; 30(3):447–455.
27.
DengK, LinDL, HanzlicekB, et al.Mesenchymal stem cells and their secretome partially restore nerve and urethral function in a dual muscle and nerve injury stress urinary incontinence model. Am J Physiol Renal Physiol, 2015; 308(2):F92–F100.
28.
HartML, IzetaA, Herrera-ImbrodaBet al.Cell therapy for stress urinary incontinence. Tissue Eng Part B Rev, 2015; 21(4):365–376.
29.
AragónIM, ImbrodaBH, LaraMF. Cell therapy clinical trials for stress urinary incontinence: Current status and perspectives. Int J Med Sci, 2018; 15(3):195–204.
30.
GotohM, YamamotoT, ShimizuS, et al.Treatment of male stress urinary incontinence using autologous adipose-derived regenerative cells: Long-term efficacy and safety. Int J Urol, 2019; 26(3):400–405.
31.
Garcia-ArranzM, Alonso-GregorioS, Fontana-PortellaP, et al.Two phase I/II clinical trials for the treatment of urinary incontinence with autologous mesenchymal stem cells. Stem Cells Transl Med, 2020; 9(12):1500–1508.
32.
Herrera-ImbrodaB, LaraMF, IzetaA, et al.Stress urinary incontinence animal models as a tool to study cell-based regenerative therapies targeting the urethral sphincter. Adv Drug Deliv Rev, 2015; 82–83:106–116.
33.
KelpA, AlbrechtA, AmendB, et al.Establishing and monitoring of urethral sphincter deficiency in a large animal model. World J Urol, 2017; 35(12):1977–86.
34.
GengR, KnollJ, HarlandN, et al.Replacing needle injection by a novel waterjet technology grants improved muscle cell delivery in target tissues. Cell Transplant, 2022; 31:9636897221080943.
35.
KnollJ, AmendB, AbruzzeseT, et al. Improved production of proliferation- and differentiation-competent porcine striated muscle-derived myoblasts. Submitted.
36.
DingS, WangF, LiuY, et al.Characterization and isolation of highly purified porcine satellite cells. Cell Death Discov, 2017; 3(1):17003.
37.
MetzgerK, TuchschererA, PalinMF, et al.Establishment and validation of cell pools using primary muscle cells derived from satellite cells of pig skeletal muscle. In Vitro Cell Dev Biol Anim, 2020; 56(3):193–199.
38.
PilzGA, BraunJ, UlrichC, et al.Human mesenchymal stromal cells express CD14 cross-reactive epitopes. Cytometry A, 2011; 79(8):635–645.
39.
LockJT, ParkerI, SmithIF. A comparison of fluorescent Ca(2)(+) indicators for imaging local Ca(2)(+) signals in cultured cells. Cell Calcium, 2015; 58(6):638–648.
40.
ReggianiC.Caffeine as a tool to investigate sarcoplasmic reticulum and intracellular calcium dynamics in human skeletal muscles. J Muscle Res Cell Motil, 2021; 42(2):281–289.
41.
ChenYJ, LiuHY, ChangYT, et al.Isolation and differentiation of adipose-derived stem cells from porcine subcutaneous adipose tissues. J Vis Exp, 2016(109):e53886.
42.
LinzenboldW, JagerL, StollH, et al.Rapid and precise delivery of cells in the urethral sphincter complex by a novel needle-free waterjet technology. BJU Int, 2021; 127(4):463–472.
43.
PilzGA, UlrichC, RuhM, et al.Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells. Stem Cells Dev, 2011; 20(4):635–646.
44.
AmendB, KelpA, VaeglerM, et al.Precise injection of human mesenchymal stromal cells in the urethral sphincter complex of Gottingen minipigs without unspecific bulking effects. Neurourol Urodyn, 2017; 36(7):1723–1733.
45.
JaillardS, Holder-EspinasseM, HubertT, et al.Tracheal replacement by allogenic aorta in the pig. Chest, 2006; 130(5):1397–1404.
46.
NoguchiK, GelYR, BrunnerE, et al.nparLD: An R software package for the nonparametric analysis of longitudinal data in factorial experiments. J Stat Software, 2012; 50:1–23.
47.
TeamRC. R: A language and environment for statistical computing. In: Computing RFfS, editor. v4.2.2 ed. Vienna, Austria. 2022.
MitchellJB, McIntoshK, ZvonicS, et al.Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells, 2006; 24(2):376–385.
50.
JankowskiRJ, TuLM, CarlsonC, et al.A double-blind, randomized, placebo-controlled clinical trial evaluating the safety and efficacy of autologous muscle derived cells in female subjects with stress urinary incontinence. Int Urol Nephrol, 2018; 50(12):2153–2165.
51.
BurdzinskaA, CraytonR, DybowskiB, et al.The effect of endoscopic administration of autologous porcine muscle-derived cells into the urethral sphincter. Urology, 2013; 82(3):743 e1-8.
52.
PokrywczynskaM, AdamowiczJ, CzapiewskaM, et al.Targeted therapy for stress urinary incontinence: A systematic review based on clinical trials. Expert Opin Biol Ther, 2016; 16(2):233–242.
53.
RocheR, FestyF, FritelX. Stem cells for stress urinary incontinence: The adipose promise. J Cell Mol Med, 2010; 14(1–2):135–142.
54.
SauryC, LardenoisA, SchlederC, et al.Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches. Stem Cell Res Ther, 2018; 9(1):128.
55.
DeloDM, EberliD, WilliamsJK, et al.Angiogenic gene modification of skeletal muscle cells to compensate for ageing-induced decline in bioengineered functional muscle tissue. BJU Int, 2008; 102(7):878–884.
56.
BorselliC, StorrieH, Benesch-LeeF, et al.Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors. Proc Natl Acad Sci U S A, 2010; 107(8):3287–3292.
57.
BazeedMA, ThuroffJW, SchmidtRA, et al.Effect of chronic electrostimulation of the sacral roots on the striated urethral sphincter. J Urol, 1982; 128(6):1357–1362.
58.
SmithC, KrugerMJ, SmithRM, et al.The inflammatory response to skeletal muscle injury: Illuminating complexities. Sports Med, 2008; 38(11):947–969.
59.
GillBC, SunDZ, DamaserMS. Stem cells for urinary incontinence: Functional differentiation or cytokine effects?. Urology, 2018; 117:9–17.
60.
MiyoshiN, FujinoS, TakahashiY, et al.Implantation of human adipose-derived stromal cells for the functional recovery of a murine heat-damaged muscle model. Surg Today, 2020; 50(12):1699–1706.
PeyromaureM, SebeP, PraudC, et al.Fate of implanted syngenic muscle precursor cells in striated urethral sphincter of female rats: Perspectives for treatment of urinary incontinence. Urology, 2004; 64(5):1037–1041.
63.
GorodetskyR, AicherWK. Allogenic use of human placenta-derived stromal cells as a highly active subtype of mesenchymal stromal cells for cell-based therapies. Int J Mol Sci, 2021; 22(10).
64.
SantosAdL, SilvaCGD, BarretoLSDS, et al.Treatment of muscle injury with stem cells—Experimental study in rabbits. Rev Bras Ortop, 2022; 57(5):788–794.
65.
AmendB, HarlandN, KnollJ, et al.Large animal models for investigating cell therapies of stress urinary incontinence. Int J Mol Sci, 2021; 22(11):6092.
66.
KnollJ, HarlandN, AmendB, et al. Göttingen minipigs present with significant regeneration kinetics after sphincter injury compared to German landrace gilts. Submitted.
67.
DominiciM, Le BlancK, MuellerI, et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006; 8(4):315–317.
68.
McIntoshK, ZvonicS, GarrettS, et al.The immunogenicity of human adipose-derived cells: Temporal changes in vitro. Stem Cells, 2006; 24(5):1246–1253.
69.
KimMJ, ShinKS, JeonJH, et al.Human chorionic-plate-derived mesenchymal stem cells and Wharton's jelly-derived mesenchymal stem cells: A comparative analysis of their potential as placenta-derived stem cells. Cell Tissue Res, 2011; 346(1):53–64.
70.
WinklerT, PerkaC, von RothP, et al.Immunomodulatory placental-expanded, mesenchymal stromal cells improve muscle function following hip arthroplasty. J Cachexia Sarcopenia Muscle, 2018; 9(5):880–897.
71.
Kobayashi-KinoshitaS, YamakoshiY, OnumaK, et al.TGF-β1 autocrine signalling and enamel matrix components. Sci Rep, 2016; 6(1):33644.
72.
KalbeC, MauM, RehfeldtC. Developmental changes and the impact of isoflavones on mRNA expression of IGF-I receptor, EGF receptor and related growth factors in porcine skeletal muscle cell cultures. Growth Horm IGF Res, 2008; 18(5):424–433.
73.
ZhangS, ChenX, HuangZ, et al.Leucine promotes differentiation of porcine myoblasts through the protein kinase B (Akt)/Forkhead box O1 signalling pathway. Br J Nutr, 2018; 119(7):727–733.
74.
MaakS, WickeM, SwalveHH. Analysis of gene expression in specific muscle of swine and turkey. Arch Tierzucht, 2005; 48:135–140.
75.
HausmanGJ, BarbCR, DeanRG. Patterns of gene expression in pig adipose tissue: Insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors. Domest Anim Endocrinol, 2008; 35(1):24–34.
76.
LiuK, JiG, MaoJ, et al.Generation of porcine-induced pluripotent stem cells by using OCT4 and KLF4 porcine factors. Cell Reprogram, 2012; 14(6):505–513.
77.
CasadoJG, Gomez-MauricioG, AlvarezV, et al.Comparative phenotypic and molecular characterization of porcine mesenchymal stem cells from different sources for translational studies in a large animal model. Vet Immunol Immunopathol, 2012; 147(1–2):104–112.
78.
TetzlaffS, MuraniE, SchellanderK, et al.Differential expression of growth factors and their receptors indicates their involvement in the inverted teat defect in pigs1. J Anim Sci, 2009; 87(11):3451–3457.
79.
HanJ, JeongW, GuMJ, et al.Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal—conceptus interface during early pregnancy in pigs. Biol Reproduct, 2018; 99(6):1137–1148.
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