Transplantation of mature adipocyte-derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model

Objectives: To examine the effects of mature adipocyte‐derived dedifferentiated fat (DFAT) cell transplantation on urethral tissue regeneration and sphincter function. Methods: Sixteen female Sprague–Dawley rats underwent vaginal distension (VD) for 3 h. Subsequently, green fluorescence protein (GFP)‐labeled DFAT cells (1 × 10⁶ in 20 µL saline, DFAT group, n = 8) or saline (20 µL, control group, n = 8) were injected into paraurethral connective tissue. Two weeks following VD, leak point pressure (LPP) was measured and an immunohistochemical analysis of the urethra was performed to evaluate urethral sphincter regeneration. Results: The VD model was characterized by atrophy of the urethral sphincter and showed a decrease in LPP. DFAT cell transplantation resulted in a significant improvement of LPP (DFAT group: 37.3 ± 6.4 vs control group: 21.7 ± 5.7 mmHg, P < 0.01). Immunohistochemistry revealed that the striated muscle thickness and smooth muscle α?actin‐positive area were significantly (P < 0.05) larger in the DFAT group than in the control group. DFAT cell transplantation enhanced macrophage accumulation followed by an increased number of cells in the proliferative state. Transplanted DFAT cells were observed in the damaged smooth muscle layer and showed positive staining for smooth muscle α?actin, suggesting conversion into the smooth muscle cell phenotype. Conclusions: DFAT cell transplantation promotes sphincter muscle regeneration and improves LPP in the rat VD model.     

Applicability of regenerative medicine and tissue engineering for the treatment of stress urinary incontinence in female patients

Stress urinary incontinence (SUI) is an age health‐related issue that generates interest due to its considerable public health burden and the controversies surrounding treatment. It is highly prevalent affecting 30–40% of all women during their lifetime. Midurethral slings are the standard of gold standard treatment for female patients with SUI. They have excellent short‐term cure rates; however, their efficacy tends to decrease over time and patients often report urinary incontinence recurrence. This paper addresses the applicability of regenerative medicine and tissue engineering for the treatment of SUI in female patients. Cell‐based treatment with periurethral injection of autologous adipose or muscle‐derived stem cells have been used for SUI; however, the cure rates and SUI recurrence at 1 year were 40% and 70%, respectively. Novel minimally invasive approaches, such as low‐intensity extracorporeal shock wave therapies have shown promising results in SUI animal models. In addition, local injection of growth factors, chemokines, and specific antibodies have shown histological evidence of neoangiogenesis, nerve, and sphincter regeneration in rodents and nonhuman primates with SUI. The use of bioactive factors and proteins secreted by cells, which is called secretomes, have been recognized as key regulators of various mechanisms, such as immunomodulation, angiogenesis, inflammation, apoptosis, and tissue repair. Emerging therapies aiming to replace or restore tissues and organ functionality may improve the long‐term efficacy and in the near future may represent the standard of care for the treatment of SUI.     

Autologous bone‐marrow‐derived mesenchymal stem cell transplantation into injured rat urethral sphincter

Objectives: To evaluate the functional and histological recovery by autologous bone‐marrow‐derived mesenchymal stem cell (BMSC) transplantation into injured rat urethral sphincters. Methods: BMSC were harvested from female Sprague–Dawley retired breeder rats for later transplantation. The cells were cultured, and transfected with the green fluorescence protein gene. The urethral sphincters were injured by combined urethrolysis and cardiotoxin injection. One week after injury, the cultured BMSC were injected autologously into the periurethral tissues. Controls included sham‐operated rats and injured rats injected with cell‐free medium (CFM). Abdominal leak point pressures (LPP) were measured before and after surgery during the following 13 weeks. The urethras were then retrieved for histological evaluation. The presence of green‐fluorescence‐protein‐labeled cells and the regeneration of skeletal muscles, smooth muscles, and peripheral nerves were evaluated by immunohistochemical staining. Results: LPP was significantly reduced in the injured rats. It increased gradually after transplantation, but there was no significant difference between the BMSC and CFM groups. In the BMSC group, transplanted cells survived and differentiated into striated muscle cells and peripheral nerve cells. The proportions of skeletal muscle cells and peripheral nerves in the urethra were significantly greater in the BMSC group compared to the CFM group. Conclusions: Despite a clear trend towards recovery of LPP in BMSC‐transplanted urethras, no significant effect was detected. Further study is required for clinical applications for the treatment of stress urinary incontinence.     

Persistence and survival of autologous muscle derived cells versus bovine collagen as potential treatment of stress urinary incontinence

PURPOSE: We explored the use of autologous muscle derived cells as a method of treating stress urinary incontinence. We determined whether urethral muscle derived cell injection is feasible and compared it with bovine collagen injection. MATERIALS AND METHODS: Muscle derived cells isolated from female Sprague-Dawley rats were first transduced with retrovirus carrying the transgene for beta-galactosidase. We injected approximately 1 to 1.5 x 106 cells into the bladder wall and proximal urethra of 6 autologous animals. Tissue was harvested after 3 and 30 days, sectioned, stained for fast myosin heavy chain and assayed for beta-galactosidase. To compare muscle derived cell and bovine collagen injections 100 microl. of commercially available bovine collagen were also injected in Sprague-Dawley female rats. Tissue was harvested in 3 animals each after 3 and 30 days, sectioned and stained for trichrome. Subsequently, 3 adult SCID mice were used to compare the level of transgene expression at each time point after injecting 1.5 x 106 cells per injection, which were transduced with adenovirus carrying the transgene for beta-galactosidase. RESULTS: A large number of cells expressing beta-galactosidase were observed in the bladder and urethral wall 3 and 30 days after autologous cell injection in Sprague-Dawley rats. The persistence of primary muscle derived cells at 3 days was similar to that of collagen. However, at 30 days there was significant cell persistence while only a minimal amount of injected bovine collagen was detectable. Approximately 88% of the beta-galactosidase expression at day 3 remained at day 30 in SCID mice. CONCLUSIONS: We present 2 new findings important for the emerging field of urological tissue engineering, including the feasibility of injecting autologous skeletal muscle derived cells into the lower urinary tract and the greater persistence of such injected cells versus injected bovine collagen. Therefore, autologous muscle derived cell injection may be an attractive alternative treatment option for stress urinary incontinence.     

干细胞治疗女性压力性尿失禁的研究进展

压力性尿失禁(SUI)是成年女性常见的泌尿系统疾病,给患者生活、工作和社会活动带来极大的影响。目前悬吊带术治疗SUI较成熟,微创简便,疗效确切,但可能出现并发症或者疾病复发。尿道周围注射填充剂,如牛胶原蛋白,远期效果不理想。干细胞是近年来研究的热点,有学者已尝试用其治疗SUI,特别是在大鼠SUI模型中研究较多,经尿道周围注射干细胞,如骨髓来源干细胞,肌肉来源干细胞和脂肪来源干细胞等,其可分化为平滑肌细胞等,提高大鼠尿道括约肌单位的功能,并促进神经细胞再生;注射的干细胞还可旁分泌多种因子,促进尿道组织再生,多种机制共同作用恢复尿道生理性的尿流控制能力。该治疗方法在临床开展较少,但仅有的数据也表明,在超声引导下经尿道周围注射干细胞能明显提高SUI患者的预后,改善排尿状况。     

Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.     

Spiral sling salvage anti-incontinence surgery in female patients with a nonfunctional urethra: technique and initial results

PURPOSE: Female patients with severe urethral incompetence are a unique surgical challenge. Urethral closure and continent diversion are often the next step in the treatment of these patients. We present a technique that provides circumferential coaptation of the urethra as a salvage procedure in this severe subset of patients. MATERIALS AND METHODS: We prospectively evaluated 47 patients who had a spiral sling. A 1 x 15 cm piece of soft polypropylene mesh was prepared with a zero polyglactin suture applied at each end. A clamp was used to pass the mesh between the urethra and pubis. The ends of the mesh were crossed at the ventral aspect of the urethra, creating a complete circle around the urethra. The sutures were transferred to the suprapubic area and tied without tension. The surgical outcome was determined by patient self-assessment, including symptom, bother and quality of life questionnaires. RESULTS: Mean patient age was 59 years. At presentation patients had undergone a mean of 2.6 incontinence procedures and wore a mean of 6 pads daily. Mean daily pad use decreased to 0.9 (p <0.005). Preoperatively mean SUI symptom severity and bother scores were 2.8 and 2.9, respectively, on a scale of 0--none to 3--severe. Postoperatively these values decreased to 0.6 and 0.4, respectively (each p <0.005). There was a mean 87% overall improvement in symptoms. CONCLUSIONS: The spiral sling is an effective salvage transvaginal procedure that may be considered in a small subset of female patients with a nonfunctional urethra as a last resort before urethral closure procedures.     

Location and concentration of estrogen,progesterone, and androgen receptors in the bladder and urethra of the rabbit

The objective of this study was to determine location and concentration of estrogen, androgen and progesterone receptors in the bladder and urethra of the rabbit. Two urethral and two bladder specimens were obtained from four 12-week-old female New Zealand white rabbits. Rat monoclonal antibody (AN 1–15) to human androgcn receptor and (H222) to human estrogen receptor and mouse monoclonal antibody (PR6) to chicken progesterone receptor were used. Immunocytochemical staining was performed and specimens were evaluated lor presence and location of steroid receptors. Androgen receptors were found in the highest concentrations in urethral and bladder epithelium. Low to low/moderate concentration were found in smooth muscle. Estrogen receptors were found in moderate to moderate/high concentrations in urethral epithelium and bladder and urethral smooth muscle. Progesterone receptors were not found in appreciable concentrations from any location, though the animals were not pretreated with estrogen. The rabbit model suggests a mechanism by which estrogen therapy can be effective in treating postmenopausal lower urinary tract symptoms. Progesterone receptors were not found in appreciable concentrations, suggesting progesterone therapy may not diminish the effectiveness of estrogen therapy by acting on urethral progesterone receptors. The effect of androgcns on the lower urinary tract needs further investigation to determine if androgen therapy can alleviate lower urinary tract symptoms. © 1995 Wiley-Liss, Inc.     

Periurethral injection of autologous adipose-derived stem cells for the treatment of stress urinary incontinence in patients undergoing radical prostatectomy: Report of two initial cases

Objectives:   To report a novel cell therapy using autologous adipose tissue-derived stem cells (ADSC) for stress urinary incontinence caused by urethral sphincteric deficiency and the outcomes in two initial cases undergoing periurethral injection of stem cells for the treatment of urinary incontinence after radical prostatectomy.
Methods:   Two patients with moderate stress incontinence after radical prostatectomy were enrolled. After liposuction of 250 mL of adipose tissue from the abdomen, we isolated ADSC from this tissue by using the Celution system. Subsequently, the isolated ADSC and a mixture of stem cells and adipose tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. Short-term outcomes during a 12-week follow-up were assessed by a 24-h pad test, a validated patient questionnaire, urethral pressure profile, transrectal ultrasonography, and magnetic resonance imaging.
Results:   Urinary incontinence progressively improved after 2 weeks of injection up to 12 weeks in terms of decreased leakage volume in a 24-h pad test, decreased frequency and amount of incontinence, and improved quality of life as per the questionnaire. In urethral pressure profile, both maximum urethral closing pressure and functional profile length increased. Ultrasonography and magnetic resonance imaging showed sustained presence of the injected adipose tissue. Enhanced ultrasonography showed a progressive increase in the blood flow to the injected area. No significant adverse events were observed peri- and postoperatively.
Conclusion:   This preliminary study showed that periurethral injection of the autologous ADSC is a safe and feasible treatment modality for stress urinary incontinence.     

骨骼肌肌源细胞自体移植在压力性尿失禁治疗中的价值

目的探讨自体骨骼肌肌源细胞(MDC)移植在压力性尿失禁治疗中的价值。方法采用差速贴壁法分离、纯化培养6只雌性SD大鼠的MDC,用携带Lac-Z基因的腺病毒载体转染MDC,转染成功后将MDC 5×106注射于自体膀胱颈周围,5、15 d处死大鼠切取膀胱及后尿道,组织学检查及X-gal染色观察注射组织形态学改善及MDC存活情况。结果5、15 d注射部位组织未见明显炎症改变及炎性细胞浸润,局部细胞层数增多。X-gal染色显示各时间点大量细胞被蓝染,提示移植细胞在体内成活。结论MDC自体膀胱尿道周围注射可长期存活,自体MDC移植有可能成为压力性尿失禁的一种有效的治疗方法。     

肌源性干细胞注射对压力性尿失禁的治疗作用

目的观察尿道周围注射肌源性干细胞是否可以提高压力性尿失禁大鼠的漏尿点压力。方法运用Preplate技术从健康成年雌性SD大鼠的腓肠肌内培养获取肌源性干细胞（MDSC），切断大鼠双侧的坐骨神经建立压力性尿失禁的动物模型，将MDSC注射到压力性尿失禁大鼠的尿道周围，4周后检测尿失禁大鼠的漏尿点压力，并与注射生理盐水的大鼠比较。结果切断双侧的坐骨神经可导致大鼠漏尿点压力进行性下降，大鼠尿道周围注射MDSC后4周可显著提高其漏尿点压力（P〈0．05）。结论MDSC注射治疗大鼠压力性尿失禁简便、有效，作用持久，其作用机制值得进一步探讨，该实验为今后临床开展此项工作提供了依据。     

Purification of human muscle‐derived cells using an immunoselective method for potential use in urological regeneration

OBJECTIVE

To purify human muscle‐derived cells (hMDCs) that could be used for managing urinary incontinence, erectile dysfunction and for bladder reconstitution.

MATERIALS AND METHODS

hMDCs isolated by a modified preplate technique (MPT) from skeletal muscles of limbs were purified by CD34 antibody and magnetic Dynabeads cell selection system (MDCSS). The growth‐doubling time of the hMDCs and purified hMDCs was measured. The purified hMDCs were characterized by flow cytometry, immunofluorescence and confocal laser scanning microscopy.

RESULTS

The growth‐doubling time of hMDCs was ≈24 h, which increased to 35 h after purifying using the MDCSS. There were scanty fibroblasts after purification and the MDCSS‐purified hMDCs were identified by high expression of stem cell markers and myoblast markers. The expression proportion of the stem cell marker CD34 and myoblast marker CD56 in the hMDCs was higher after purification (MDCSS) than before (MPT), at 32.13% vs 4.12% and 21.56% vs 8.60%, respectively.

CONCLUSIONS

The purification of hMDCs showing high expression of stem cell and myoblast markers is feasible. These purified hMDCs could potentially be used for urological regeneration.     

静脉移植骨髓间充质干细胞促进脊髓损伤后GDNF基因的表达

[目的]探讨经静脉移植骨髓间充质干细胞(MSCs)对大鼠脊髓损伤(SCI)后胶质细胞源性神经营养因子(GDNF)表达的影响。[方法]MSCs提取自成年Wistar大鼠的股骨干骨髓,经原代培养、鉴定及5-溴脱氧尿嘧啶核苷(Brdu)核标记。以改良Allen′s打击装置制作大鼠T10节段脊髓损伤(SCl)模型,于伤后立即缝合切口并经尾静脉注射移植MSCs。实验共分为3组MSCs尾静脉移植组(A组)、生理盐水注射组(B组)、正常对照组(C组)。术后不同时间点应用免疫组化法和逆转录聚合酶链反应法(RT-PCR)观察MSCs移植后的存活状态以及不同时间点GDNF基因的表达变化。[结果]免疫组化结果MSCs经尾静脉移植后在损伤脊髓平面可以检测到迁移及存活,标记细胞呈棕黄色的核标记,以损伤区域为多并向周围迁移,移植术后第14d,A组Brdu阳性细胞较多,最远于距离损伤区2.5cm处可检测到。B及C组则均未检测到阳性细胞。RT-PCR结果移植术后第1、3、5d,A组表达量呈逐渐升高趋势,B组呈一过性表达升高,C组无变化。各时间点A组GDNFmRNA的表达量明显高于B组,P<0.05。免疫组化结果移植术后第7、14、28dGDNF的表达量明显高于B组,P<0.05。[结论]骨髓间充质干细胞经尾静脉移植后可迁移至脊髓损伤区域,并上调胶质细胞源性神经营养因子基因的表达,是该移植方式修复大鼠脊髓损伤的机制之一。     

大鼠肌源性干细胞的培养与鉴定

目的探讨大鼠肌源性干细胞体外原代培养的方法，为临床应用自体肌源性干细胞注射治疗压力性尿失禁提供实验依据。方法采用胶原酶和胰蛋白酶等分离大鼠骨骼肌细胞，应用差速贴壁法进行纯化获取肌源性干细胞。在倒置显微镜下观察干细胞的形态，通过免疫组织化学法进行鉴定。结果通过差速贴壁法可获取高纯度的肌源性干细胞，免疫组织化学技术可鉴定其肌源性和干细胞特性。结论肌原性干细胞可通过体外原代培养获取，适用于组织工程和基因治疗，临床应用有待进一步研究。