Regeneration of the Lower Urinary Tract in Nonhuman Primates

描述(申请人提供)：使用选择性细胞移植再生受损组织的概念已被临床应用于各种肌肉疾病，包括压力性尿失禁(SUI)，这是几种损伤和导致括约肌缺陷(SMD)的先天性异常的表现。最近使用自体肌源性祖细胞(MPC)治疗女性SMD的临床试验结果令人振奋。然而，目前尚不清楚注射的MPC在括约肌再生中是被动(膨胀)还是主动(细胞整合)作用，最近人们对这些临床试验的短期和长期成功率提出了担忧。重复的临床试验将需要时间，而且仍然缺乏对其结果的机械解释。我们建议在一项研究中解决这两个问题，该研究旨在使用损伤性SMD的食蟹猴(Cyno)模型来评估MPC作为积极参与者在恢复排尿反射控制中的作用，在该模型中，成功的衡量标准可以在相对较短的几年内实现。我们实验室的两个发现(一个在老鼠身上，一个在狗身上)支持这样的工作假设，即注射的MPC整合并在雌性括约肌再生中发挥积极作用。基于目前与这种治疗方法相关的不确定性以及对更直接可用于人类的结果的需求，我们建议将我们的研究扩展到成年女性Cyno‘s-相当于45-50岁的女性-她们的遗传、解剖、生理、姿势、盆腔内膀胱位置、年龄和生殖泌尿系统中激素相关的变化，使尿括约肌暴露于中年女性常见的结构性和药物应激源。具体目的为：1.建立损伤后SMD的CyNO模型。这将通过将我们的犬括约肌切除模型应用于成年雌性Cyno的犬，然后在3mo、6mo、12mo和24mo测量尿括约肌结构的纵向变化(对植入特征的组织学和分子分析，例如：标记细胞的括约肌含量、注入的细胞活力、功能性运动终板的形成和基因表达数据)和功能(静息和反射尿动力学、对药物和电场刺激的反应)。损伤后(这相当于人类6年的随访研究)；以及2.在损伤诱导的SMD模型中测量MPC细胞治疗的效果。首先，我们将优化培养系统，以产生足够数量的Lenti-LacZ转基因MPC用于治疗SMD，然后将标记的MPC注射到受损的括约肌中，然后测量这些细胞对括约肌功能和结构的纵向(与SA1相同的时间点)影响。预计从这个灵长类动物模型获得的结果将直接翻译给这个年龄段的女性，其他SMD患者，以及其他肌肉组织和基于肌肉细胞的生物工程器官的再生。公共卫生相关性：对于括约肌缺乏症(SMD)相关的压力性尿失禁的细胞疗法，一个悬而未决的问题是，注射的细胞在临床改善中起到了被动还是积极的作用。这项拟议的研究将利用成年雌性非人灵长类动物来测量肌肉前体细胞在SMD损伤模型中结构和功能变化的纵向影响。我们预计，对结构(MPC活性、功能和对运动终板形成的贡献)和功能(静息和反射尿动力学测量)的纵向和平行评估将有助于确定MPC细胞疗法在恢复功能性括约肌中的作用(主动或被动)。

DESCRIPTION (provided by applicant): The concept of regenerating damaged tissue using selective cell transplantation has been applied clinically for a variety of muscle disorders, including stress urinary incontinence (SUI), which is a manifestation of several kinds of injuries and congenital abnormalities leading to sphincteric muscle deficiency (SMD). Results of recent clinical trials using autologous muscle-derived progenitor cells (MPCs) to treat SMD in women are promising. However, it remains unclear whether injected MPCs play a passive (bulking), or active (cell integration) role in sphincter regeneration and recent concern has been raised about the short- and long-term success rates of these clinical trials. Repeated clinical trials will take time and still lack a mechanistic explanation for their results. We propose to address these two issues in a study designed to evaluate the role of MPCs as active participants in restoration of reflex control over micturition using a cynomolgus monkey (Cyno) model of injury- induced SMD, where measures of success can be achieved in relatively few years. Two findings from our lab (one in rats and one in dogs) support the working hypothesis that injected MPCs integrate and play an active role in sphincter regeneration of females. Based on the current uncertainties associated with this therapeutic approach and the need for results that are more directly translatable to human beings, we propose to extend our studies to adult female Cyno's - at the age equivalent of 45-50 year-old women - whose genetic, anatomic, physiologic, postural, intra-pelvic bladder location, age and hormone-related changes in the genitourinary system, expose the urinary sphincter to structural and pharmacologic stressors common to middle-aged women. The specific aims are: 1. To establish a Cyno model of injury-induced SMD. This will be achieved by applying our canine model of sphincter muscle removal to adult female Cyno's and then measuring longitudinal changes in urinary sphincter structure (histological and molecular analysis of engraftment characteristics such as: sphincter content of labeled cells, injected cell viability, formation of functional motor endplates and gene expression data) and function (resting and reflex urodynamics, responses to pharmacologic and electric field stimulation) at 3mo., 6mo., 12mo., and 24 mo. post injury ( this will be equivalent to a 6 year follow-up study in human beings); and 2. To measure the effects of MPC cell therapy in the Cyno model of injury-induced SMD. First, we will optimize the culture system to produce sufficient numbers of functional lenti-LacZ transfected Cyno-derived MPCs for use in the treatment of SMD and then inject the labeled MPCs into the damaged sphincter muscle and then measure the longitudinal (same time- points as SA1) effects of these cells on sphincter function and structure. It is anticipated that the results obtained from this primate model will be directly translatable to women of this age-group, to other patients with SMD and to regeneration of other muscle tissues and muscle cell-based bioengineered organs. PUBLIC HEALTH RELEVANCE: An unanswered question about cell therapy for sphincter muscle deficiency (SMD) -associated stress urinary incontinence is whether the injected cells play a passive, or an active role in clinical improvement. The proposed studies will utilize adult female nonhuman primates to measure the longitudinal effects of muscle progenitor cells on both structural and functional changes in an injury model of SMD. We anticipate that longitudinal and parallel assessments of structure (MPC viability, functionality, and contribution to motor endplate formation) and function (resting and reflex urodynamic measures) will help determine the role (active or passive) of MPC cell therapy in restoration of a functional sphincter.