Novel Imaging Approach to Monitor Chondrogenic Differentiation of iPS Cells

描述（由申请人提供）：关节软骨缺损是关节炎患者疼痛和功能损害的主要来源。目前的治疗方法，虽然减轻了一些临床症状，但不足以治愈潜在的不可逆软骨损失。目前，美国约有4300万老年人患有关节炎引起的残疾，这给我们的社会造成了每年950亿美元的医疗费用和工资损失。干细胞移植提供了一种潜在的治疗选择。人诱导多能干细胞（hiPS）是一种集人胚胎干细胞（hESCs）和人间充质干细胞（hMSCs）优点于一体的新型细胞类型。hiPS细胞是基于非多能性细胞，如成人成纤维细胞，通过引入与多能性相关的转录因子进行重编程。培养几周后，hiPS细胞在表面标记物、形态、增殖、饲养体依赖性、基因表达和体外分化方面与hESCs几乎没有区别。hiPS细胞具有独特的特性，即它们是自体的，因此能够转化为患者特异性干细胞。这一特点有助于避免免疫反应，克服与hESC移植相关的伦理问题。另一方面，hiPS细胞也克服了与骨髓来源的hMSCs或软骨来源的软骨细胞相关的限制，例如侵入性采集程序、产量可变以及老年患者细胞中软骨再生潜力有限。hiPS细胞可以以一致和可复制的方式作为均匀的细胞群收获，易于扩增，并且可以在体内环境中更好地定向形成功能3D组织。因此，髋关节细胞是目前最有希望用于软骨修复的细胞类型。为了使关节炎关节的透明软骨再生，移植的干细胞不仅要存活和移植，而且要分化成软骨细胞。我们的项目致力于研究决定软骨干细胞分化的分子途径。我们试图使用一种新颖的、创新的成像方法来解决一个具有挑战性的技术和概念问题，即我们目前无法诊断体内的细胞分化过程。本研究的目标是开发一种新颖的“智能”成像技术，基于细胞MR成像和基因表达介导的2-半乳糖苷酶敏感MR造影剂EgadMe的激活，用于无创的髋关节细胞向软骨细胞分化的体内可视化。该方法依赖于载体转染的hiPS细胞，这些细胞在软骨分化后具有2-半乳糖苷酶的细胞谱系特异性表达，以及半乳糖苷包被的MR造影剂EgadMe，该造影剂在2-半乳糖苷酶切割后仅产生MR信号。egadme标记的hiPS细胞的软骨分化将通过MR成像检测，当软骨细胞中的2-半乳糖苷酶表达将半乳糖糖外壳劈开，导致造影剂激活，MR图像上显示阳性信号。我们将采用三步法，首先生成软骨分化后表达2-半乳糖苷酶的hiPS细胞，然后在体外研究EgadMe标记的髋关节细胞在软骨分化前后的MR信号特征，最后在已建立的关节炎动物模型中比较EgadMe标记的髋关节细胞在体内软骨分化前后MR信号特征的纵向变化。egadme增强MR成像技术可能为干细胞分化提供一种新的结果测量方法，可以显著改善对MASI的监测，并最终帮助优化我们恢复关节炎患者关节功能的努力。据我们所知，利用“可激活”造影剂进行磁共振成像的hiPS细胞分化的非侵入性体内可视化的方法是新颖的，以前从未被描述过。由于认识到新的细胞成像技术能够改善移植干细胞的体内生理变化表征，我们预测，我们基于核磁共振成像的hiPS细胞分化可视化分析将有助于检测和定量体内hiPS细胞分化结果。一旦建立，这项技术可以扩展到任何干细胞来源的再生组织的体内分化过程的表征。潜在的应用包括不同干细胞类型（hESC, hMSC, hiPS）的体内比较研究，自体和异体干细胞分化特性的比较，基因工程干细胞的研究，不同支架和生长因子的比较，以及对干细胞分化结果的人口统计学影响的评估。这种新的成像技术可以帮助识别和监测干细胞介导的关节修复最有前途的方法，这可能导致更好的基质相关干细胞植入技术的发展，为关节炎患者提供期待已久的功能恢复和疼痛缓解，并消除与长期残疾相关的直接和间接成本。所提出的分子成像技术可作为干细胞疗法临床前评估、相关临床试验设计以及最终在临床实践中评估和优化这些干细胞疗法的关键工具。

DESCRIPTION (provided by applicant): Articular cartilage defects are the major source of pain and functional impairment in patients with arthritis. Current treatments, whilst alleviating some of the clinical symptoms, prove insufficient to cure the underlying irreversible cartilage loss. About 43 million elderly individuals in the US currently suffer from arthritis-induced disabilities, which cause annual costs to our society in medical care and lost wages in the order of $95 billion. Stem cell transplants provide a potentially curative therapeutic option. Human induced pluripotent stem cells (hiPS cells) represent a novel cell type, which integrates advantages of human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs). hiPS cells are based on non-pluripotent cells, such as adult fibroblasts, which are reprogrammed via introduction of transcription factors that are linked to pluripotency. After a few weeks in culture, hiPS cells are virtually indistinguishable from hESCs with regards to their surface markers, morphology, proliferation, feeder dependence, gene expression and in vitro differentiation. hiPS cells have the exceptional characteristic that they are autologous and thus, able to transform into patient specific stem cells. This feature helps to avoid immune reactions and overcomes ethical concerns that are associated with hESC transplants. On the other hand, hiPS cells also overcome limitations associated with bone marrow derived hMSCs or cartilage derived chondrocytes, such as invasive harvesting procedures, variable yields, and limited cartilage regeneration potential in cells obtained from older patients. hiPS cells can be harvested as a homogeneous cell population in a consistent and reproducible manner, are easily expanded, and may be better directed to form functional 3D tissue in an in vivo environment. Thus, hiPS cells currently represent the most promising cell type for cartilage restoration. In order to regenerate hyaline cartilage in arthritic joints, the transplanted stem cells must not only survive and engraft, but also differentiate into chondrocytes. Our project endeavors to investigate molecular pathways that determine chondrogenic stem cell differentiation. We attempt to use a novel, innovative imaging approach to solve a challenging technical and conceptual problem that being our current inability to diagnose cellular differentiation processes in vivo. The goal of this study is to develop a novel, "smart" imaging technique for non-invasive in vivo visualization of the differentiation of hiPS cells into chondrocytes, based on cellular MR imaging and gene expression mediated activation of the 2-galactosidase-sensitive MR contrast agent EgadMe. The approach relies on vector-transfected hiPS cells which are marked by a cell lineage-specific expression of 2-galactosidase after chondrogenic differentiation and the galactopyranose- coated MR contrast agent EgadMe, which generates only an MR signal after cleavage by 2-galactosidase. The chondrogenic differentiation of EgadMe-labeled hiPS cells will be detected with MR imaging when the galactopyranose coat is cleaved by 2-galactosidase expression in chondrocytes, resulting in activation of the contrast agent and a positive signal on MR images. In a three step approach, we will first generate hiPS cells that express 2-galactosidase upon chondrogenic differentiation, then investigate the MR signal characteristics of EgadMe labeled hiPS cells before and after chondrogenic differentiation in vitro, and finally compare longitudinal changes in MR signal characteristics of EgadMe labeled hiPS cells before and after chondrogenic differentiation in vivo, in an established animal model of arthritis. The EgadMe-enhanced MR imaging technique may provide a novel outcome measure for stem cell differentiation, which could significantly improve monitoring of MASI and ultimately help to optimize our efforts to restore joint functions of patients with arthritis. To the best of our knowledge, the proposed approach for a non-invasive in vivo visualization of hiPS cell differentiation with an "activatable" contrast agent for MR imaging is novel and has not been described before. With the realization that novel cellular imaging techniques enable an improved in vivo characterization of physiological changes of transplanted stem cells, we predict that our MR-based imaging assay for visualizing hiPS cell differentiation will facilitate the detection and quantification of hiPS cell differentiation outcomes in vivo. Once established, this technique could be extended to characterizations of in vivo differentiation processes of any stem cell derived regenerative tissue. Potential applications comprise comparative in vivo investigations of different stem cell types (hESC, hMSC, hiPS), comparisons of the differentiation properties of autologous and allogeneic stem cells, investigations of genetically engineered stem cells, comparisons of different scaffolds and growth factors, and assessments of demographic effects on stem cell differentiation outcomes. This novel imaging technique could help to identify and monitor the most promising approaches for stem cell mediated joint restoration which could lead to the development of better matrix-associated stem cell implantation techniques, provide the long awaited functional restitution and pain relief for patients with arthritis, and eliminate direct and indirect costs associated with long term disabilities. The proposed molecular imaging technique could be used as a critical tool for preclinical assessments of stem cell-based therapies, for the design of related clinical trials, and ultimately, for the assessment and optimization of those stem cell-based therapies in clinical practice. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a magnetic resonance (MR) imaging technique that can detect the critical differentiation of human induced pluripotent stem cells (hiPS cells) into chondrocytes via activation of the novel MR contrast agent EgadMe. EgadMe provides only a signal when cleaved by the enzyme 2-galactosidase, which is produced by chondrocytes, but not undifferentiated hiPS cells. Realization of this new imaging technique could provide direct measures of in vivo stem cell differentiation outcomes which would significantly enhance our ability to identify factors that lead to cartilage regeneration and ultimately direct us to the development of successful techniques for joint restoration and functional improvement.