Directed differentiation of embryonic stem cells for urologic applications

描述(由申请者提供)：在这份K18干细胞研究职业提升奖的申请中，候选人(James Bassuk博士)建议从他常规的专业职责中抽出时间，通过获得新的研究能力来拓宽他的科学背景。有待学习的特殊新研究能力是将胚胎干细胞(ESCs)用于再生医学应用，如组织工程修复膀胱及其相关管道。有必要开发(I)用于癌症、间质性膀胱炎或神经性膀胱(脊柱裂或多发性硬化症)患者的替换膀胱，以及(Ii)用于因创伤、狭窄疾病或尿道下裂(男孩常见的出生缺陷)而重建尿路的上皮管。这个项目对他来说是一个真正的职业提升，因为它第一次为他的研究项目带来了一个实验设计，重点是如何用基于干细胞的方法来研究膀胱形成的发育步骤。该策略包括寻找形成畸胎瘤所需的最小胚胎干细胞数量，目标是将这些胚胎干细胞重新定向为只形成一种器官类型。胚胎间充质是一种诱导性组织，它决定了上皮组织的器官特异性编程。在目标1中，将通过尿路上皮细胞终末分化的体视学定量来测量大鼠胚胎膀胱间充质细胞驱动小鼠胚胎干细胞向功能性膀胱组织分化的活性。在Carol Ware博士(导师)、C.Anthony Blau博士(共同导师)、Simon Hayward博士(贡献者)和Andrew Farr博士(贡献者)的指导下，候选人将接受正确处理胚胎干细胞的培训，并建立异种移植试验，将重组移植物植入无菌裸小鼠的肾被膜下。在目标2a中，人类胚胎干细胞将取代小鼠胚胎干细胞。在目标2b中，人婴儿膀胱间充质细胞将替代大鼠胚胎间充质细胞。婴儿间充质的来源将是西雅图儿童医院的泌尿科服务，由理查德·格雷迪博士和托马斯·伦德维博士(撰稿人)采购。因此，问题是人类胚胎干细胞是否会对膀胱间充质发育控制做出适当的反应？在为期两年的项目期间，候选人将把75%的时间用于在华盛顿大学干细胞和再生医学研究所进行培训。一旦项目完成，候选人新开发的专业知识将被应用于(I)进一步研究由可诱导多能干细胞形成膀胱组织的发育控制所需的间充质因子，(Ii)产生新的尿路上皮细胞分化的体外模型，以及(Iii)用于泌尿外科的组织工程解决方案。 公共卫生相关性：该项目将为华盛顿大学泌尿学副教授詹姆斯·巴苏克博士提供干细胞技术方面的培训经验。一旦该项目结束，巴苏克博士将运用他新获得的专业知识来开发新的解决方案，以帮助外科医生修复膀胱及其相关的尿路。这些努力有望改善因癌症、间质性膀胱炎、多发性硬化症或脊柱裂而患有无功能膀胱的患者的预后。

DESCRIPTION (provided by applicant): In this K18 Career Enhancement Award in Stem Cell Reseach application, the Candidate (Dr. James Bassuk) proposes to take time from his regular professional responsibilities in order to broaden his scientific background by acquiring new research capabilities. The specifiic new research capability to be learned is the use of embryonic stem cells (ESCs) in regenerative medicine applications such as tissue engineered repair of the urinary bladder and its associated tract. There is a significant need to develop (i) replacement bladders for patients suffering from cancer, interstitial cystitis or neurogenic bladder (spina bifida or multiple sclerosis) and (ii) epithelial tubes for reconstruction of the urethra due to trauma, stricture disease or hypospadias (a common birth defect in boys). The project represents a true career enhancement for him because it brings to his research program, for the first time, an experimental design that focuses on how developmental steps of bladder formation can be studied with a stem cell-based approach. The strategy involves finding the minimum number of ESCs needed to form a teratoma with the goal of redirecting these ESCs to form only one organ type. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. In Aim 1, the activity of rat embryonic bladder mesenchyme in driving mouse ESCs into functional bladder tissue will be measured by a stereological quantification of urothelial cell terminal differentiation. Under the guidance of Drs. Carol Ware (Mentor), C. Anthony Blau (Co-Mentor), Simon Hayward (Contributor) and Andrew Farr (Contributor), the Candidate will be trained in the proper handling of embryonic stem cells and in setting up xenograft assays where recombinant grafts are implanted under the renal capsule of athymic nude mice. In Aim 2a, human ESCs will be substituted for mouse ESCs. In Aim 2b, mesenchyme from human infant bladder will be substituted for rat embryonic mesenchyme. The source of infant mesenchyme will be the urology service at Seattle Children's Hospital, as procured by Drs. Richard Grady and Thomas Lendvay (Contributors). Thus, the question asked is will human ESCs respond appropriately to bladder mesenchymal developmental control? Over a 2-year project period, the Candidate will devote 75% of his time to train at the Institute for Stem Cell and Regenerative Medicine at the University of Washington. Once the project has been completed, then the Candidate's newly developed expertise will be applied to (i) the further study of mesenchymal factors necessary for developmental control of bladder tissue formation from inducible pluripotent stem cells, (ii) generating new in vitro models of urothelial cell differentiation and (iii) tissue engineered solutions for urologic use. PUBLIC HEALTH RELEVANCE: This project will provide a training experience in stem cell technology for Dr. James Bassuk, an Associate Professor of Urology at the University of Washington. Once the project is over, Dr. Bassuk will apply his newly acquired expertise to developing new solutions to assist the surgeon in repairing the urinary bladder and its associated tract. These efforts are expected to lead to improved outcomes for patients suffering with a non-functional bladder due to cancer, interstitial cystitis, multiple sclerosis or spina bifida.