Directed differentiation of embryonic stem cells for urologic applications

描述（由申请人提供）：在此K18干细胞研究职业提升奖申请中，候选人（James Bassuk博士）建议从他的常规专业职责中抽出时间，以通过获得新的研究能力来拓宽他的科学背景。具体的新的研究能力是胚胎干细胞（ESC）在再生医学应用中的应用，如膀胱及其相关管道的组织工程修复。非常需要开发（i）用于患有癌症、间质性膀胱炎或神经性膀胱（脊柱裂或多发性硬化）的患者的替代膀胱和（ii）用于重建由于创伤、狭窄疾病或尿道下裂（男孩中常见的出生缺陷）引起的尿道的上皮管。该项目对他来说是一个真正的职业提升，因为它首次为他的研究项目带来了一个实验设计，该设计专注于如何用基于干细胞的方法研究膀胱形成的发育步骤。该策略涉及找到形成畸胎瘤所需的最小数量的ESC，目的是将这些ESC重新定向为仅形成一种器官类型。胚胎间充质被公认为是一种诱导性组织，它决定了上皮组织的器官特异性编程。在目的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在驱动小鼠ESC进入功能性膀胱组织中的活性。在Carol Ware博士（导师）的指导下，C。Anthony Blau（共同导师）、Simon海沃德（贡献者）和Andrew Farr（贡献者）（候选人）将接受胚胎干细胞正确处理和建立异种移植试验（将重组移植物植入无胸腺裸鼠的肾囊下）的培训。在目标2a中，人ESC将取代小鼠ESC。在目标2b中，来自人婴儿膀胱的间充质将代替大鼠胚胎间充质。婴儿间充质的来源将是西雅图儿童医院的泌尿科服务，由Richard Grady和托马斯Lendvay博士（贡献者）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2年的项目期间，候选人将投入75%的时间在华盛顿大学的干细胞和再生医学研究所接受培训。一旦项目完成，候选人新开发的专业知识将应用于（i）进一步研究诱导型多能干细胞形成膀胱组织发育控制所需的间充质因子，（ii）生成尿路上皮细胞分化的新体外模型和（iii）泌尿外科使用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外科副教授James Bassuk博士提供干细胞技术方面的培训经验。一旦项目结束，Bassuk博士将运用他新获得的专业知识开发新的解决方案，以帮助外科医生修复膀胱及其相关的管道。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结局。

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.