Implantation of physiologically functional bioengineered innervated IAS construct

描述(申请人提供)：挑战领域(11)再生医学：11-DK-101：促进消化系统、肝脏、胰腺、血液、肾脏和泌尿系统的再生和修复。大便失禁是一种情况，其后果远远超出身体表现。许多人发现自己退出了社交生活，并试图向家人、朋友甚至医生隐瞒这个问题。与这些疾病相关的羞耻、尴尬和耻辱对寻求专业治疗构成了重大障碍，导致许多患有这些疾病的人得不到帮助。随着婴儿潮一代步入60多岁，大小便失禁的发病率和公共卫生负担可能会增加。大便失禁的负担分为经济和非经济两类，每一种都是复杂的。大小便失禁的人可能会经历“意外”、抑郁、社会孤立和社会排斥的焦虑。大小便失禁本身的管理是繁重的。大小便失禁需要更多的非正式和正式护理。这在2007年12月的美国国立卫生研究院科学现状会议上得到了强调[1]。我们需要提供解决方案，以提高大便失禁患者的生活质量。新的假说表明，有效利用关于平滑肌细胞、器官、括约肌的知识，以及可能通过植入生物工程IAS括约肌来替代有缺陷的IAS括约肌是合理的预期。这项拨款提案为解决大便失禁提供了一种有效的方法。我们成功地植入了由IAS平滑肌细胞和神经前体细胞构建的生物工程环。从本质上讲，我们已经在培养中开发出了可以用于植入的内在神经支配的IAS结构。这项资助计划的目标是植入从人或小鼠的间隔膜平滑肌细胞构建的生物工程功能性间隔膜，这些细胞的内在神经连接到外部神经网络。这些生物工程的IAS结构将拥有生物工程的内在神经元电路，并将连接到动物的外部神经网络。我们的初步数据表明：(A)在培养中，与永生化小鼠胚胎肠神经元(IM-Fen)共培养的小鼠IAS平滑肌细胞在中央柱周围形成紧密的环状结构。环周可见神经细胞在紧密的IAS环周围伸长、分支并形成网络。支配的结构：1)对乙酰胆碱和PdBU的收缩和产生持续的力；2)对VIP和电场刺激(EFS)的松弛。(B)使用人IAS平滑肌细胞与IM-Fen细胞共培养构建的生物工程构建物也得到了同样的结果。(C)神经化的生物工程小鼠IAS构建成功地植入了品系匹配的小鼠的皮肤下。环变得血管化，并在动物体内存活了长达27天，没有任何排斥迹象。(D)类似地，神经化的生物工程人IAS结构被成功地植入免疫缺陷小鼠的皮肤下。环被血管化，并在动物体内存活，没有任何排斥的迹象。(E)在收获后，血管化的神经支配环保持了植入前观察到的生理特征。我们的生理学研究证实，这些环保持其功能特性，能够形成基础音调，并对收缩和松弛神经递质以及EFS产生反应。我们的初步结果证实了生物工程环在植入后可以血管化的概念。这些动物可以耐受植入，没有排斥的迹象。这是首次展示具有生理功能的生物工程神经肌肉结构。这些结构在植入后被受体动物耐受，并成为血管化的。这些发现代表了胃肠道组织替代和移植的实质性进展。基于此，该提案的具体目标是：1.制定ENS前体细胞的培养方案，并将其分化为肠神经和神经胶质细胞。2.利用生物工程技术，从人或小鼠和小鼠神经前体细胞的IAS中分离出平滑肌细胞，建立IAS的三维生理功能模型。3.将具有神经功能的3-D IAS组织植入小鼠体内。这是一种创新的方法，可以从自体细胞植入生物工程的IAS。这有可能提高大便失禁患者的生活质量。此外，这将对关注尿失禁患者的研究产生积极影响。 与公共卫生相关：这项拨款提案代表了一种创新的方法，可能会导致从自体细胞植入生物工程内肛门括约肌(IAS)。这种生物工程的IAS将拥有来自胚胎神经前体细胞的内在神经电路。这有可能提高大便失禁患者的生活质量。此外，这对患有尿失禁的人也有积极的意义。

DESCRIPTION (provided by applicant): Challenge Area (11) Regenerative Medicine: 11-DK-101: Promote regeneration and repair in the digestive system, liver, pancreas, hematology, kidneys and urological system. Fecal incontinence is a condition with ramifications that extend well beyond the physical manifestations. Many individuals find themselves withdrawing from their social lives and attempting to hide the problem from their families, friends, and even their doctors. The shame, embarrassment, and stigma associated with these conditions pose significant barriers to seeking professional treatment, resulting in many persons who suffer from these conditions without help. As baby boomers approach their sixties, the incidence and public health burden of incontinence are likely to increase. The burdens of fecal incontinence fall into economic and non- economic categories, and each is complex. Individuals who are incontinent may experience anxiety about "accidents," depression, social isolation, and social exclusion. The management of incontinence itself is burdensome. Incontinence requires greater amounts of informal and formal care giving. This was emphasized in the NIH State-of-the-Science Conference in December 2007 [1]. We need to provide solution to enhance the quality of life for individuals with fecal incontinence. Novel hypothesis suggests the effective utilization of knowledge about smooth muscle cells, organs, sphincters and possible replacement of defective IAS sphincters with implantation of bioengineered IAS sphincters are reasonable expectations. This grant proposal offers an effective approach to tackle fecal incontinence. We were able to successfully implant bioengineered rings that were constructed from IAS smooth muscle cells and neuronal precursor cells. In essence we have developed intrinsically innervated IAS constructs in culture that could be used for implantation. The objective of this grant proposal is to implant bioengineered functional IAS constructed from either human or mouse IAS smooth muscle cells with intrinsic innervations connected to extrinsic neural network. These bioengineered IAS constructs will have bioengineered intrinsic neuronal circuitry and will be connected to extrinsic neural network from the animal. Our preliminary data indicates that: (A) in culture, mouse IAS smooth muscle cells co- cultured with Immortomouse Fetal Enteric Neurons (IM-FEN), formed a tight ring around a central post. Peripheral to the ring, the neuronal cells were observed to elongate, branch and form networks around the tight IAS ring. The innervated constructs: 1) contracted and generated sustained force in response to acetylcholine and PdBU; and 2) relaxed in response to VIP and Electrical Filed Stimulation (EFS). (B) Same results were obtained from constructs bioengineered using human IAS smooth muscle cells co-cultured with IM-FEN cells. (C) The innervated bioengineered mouse IAS constructs were successfully implanted under the skin of a strain matched mouse. Rings became vascularized and survived in the animals without any signs of rejection for up to 27 days. (D) Similarly, the innervated bioengineered human IAS constructs were successfully implanted under the skin of an immuno-deficient mouse. Rings became vascularized and survived in the animals without any signs of rejection. (E) Upon harvesting, the vascularized innervated rings maintained their physiological characteristics observed prior to implantation. Our physiological studies confirm that the rings maintain their functional properties are able to develop basal tone and response to contractile and relaxant neurotransmitters as well as EFS. Our preliminary results confirm the proof of concept that bioengineered rings are vascularized upon implantation. The animals tolerate implantation without signs of rejection. This is the first demonstration of physiologically functional bioengineered innervated smooth muscle constructs. These constructs upon implantation were tolerated by recipient animal and became vascularized. These findings represent a substantial advance in GI tissue replacement and transplantation. Based on this, the specific aims of the proposal are: 1. Develop protocols for the culture of ENS progenitor cells and their differentiation into enteric neural and glial cells. 2. Bioengineer a 3-D physiologically functional model of the IAS produced in culture from smooth muscle cells isolated from the IAS of either human or mouse and mouse ENS progenitor cells. 3. Implant an innervated physiologically functional 3-D IAS tissue into a mouse. This is an innovative approach that could result in implantation of bioengineered IAS from autologous cells. This has potential to provide enhanced quality of life to persons with Fecal Incontinence. Furthermore it would have positive implications for research focusing on people suffering from urinary incontinence. PUBLIC HEALTH RELEVANCE: This grant proposal represents an innovative approach that could result in implantation of Bioengineered Internal Anal Sphincter (IAS) from autologous cells. This Bioengineered IAS would have intrinsic neural circuitry derived from embryonic neural progenitor cells. This has potential to provide enhanced quality of life to persons with fecal incontinence. Furthermore it would have positive implication for people suffering from urinary incontinence.