UNS: Stem Cell Differentiation and Teratoma-forming Potential in hiPSC-derived Neural Cultures

UNS：hiPSC 来源的神经培养物中的干细胞分化和畸胎瘤形成潜力

• 批准号: 1511914
• 负责人: Brian Kirby
• 金额: $ 33.49万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1511914&HistoricalAwards=false
• 关键词: UNS; Stem; Cell; Differentiation; Teratoma

PI: Kirby, Brian J. Proposal Number: 1511914The investigators will develop techniques that allow human cells from patients to be reprogrammed into neural cells that can be used to repair nerve damage or neurodegenerative disease. Although techniques exist to reprogram cells, these reprogramming techniques at present pose risks to the patient because some reprogrammed cells can form tumors instead of serving a therapeutic purpose. This work will process populations of reprogrammed cells to remove those with the potential to form tumors. This work is broadly significant because, by eliminating the potential for tumor formation, it will enable tissue regeneration to be performed with therapeutic benefit and minimal risk of harming the patient.The long-term goal of this research is to enable efficient, teratoma-free engineering of neuronal cells. The objective of the proposed work is to delineate the relationships between adhesive mechanobiology, teratoma-related markers and differentiation in neurally reprogrammed human induced pluripotent stem cell (hiPSC) populations. The central hypotheses are that (a) neural rosette cells will have an adhesive signature (i.e., integrins, focal adhesions, and adhesive strength), distinct from contaminating cells, that will lead to their selective removal using the micro stem cell high-efficiency adhesion-based recovery (uSHEAR) platform, and (b) teratoma-forming cells will have a unique expression pattern of integrins and glycans, including stage-specific embryonic antigen (SSEA)-5, which will enable microfluidic-based selective capture of teratoma-forming cells. This hypothesis has been formulated based on the previous discovery of differential adhesiveness of hiPSCs and neural cells and label-free isolation of hiPSCs for partially reprogrammed cultures, as well as selective rare cell isolation with geometrically enhanced differential immunocapture (GEDI) devices. The rationale of the proposed work is that the relationships between adhesive mechanobiology, teratoma-related markers, and differentiation are most directly applicable to contaminant-extraction approaches that improve the time, yield, and purity of neural differentiation and facilitate its direct application in regenerative medicine. This work will determine relationships between adhesion signature, surface markers, and teratoma formation through two specific aims, which determine the relationships between teratoma-related markers, adhesive signature, and directed differentiation of hiPSCs along the neural lineage, and determine the relationship between SSEA5-based negative selection in differentiating cells and removal of teratoma-forming risk in progenitor/neuron cultures. The proposed work will determine relationships between surface expression and adhesion during the differentiation process, and evaluate the potential for differentiation and teratoma formation in immune-compromised mice. By exploiting the differences in adhesive signature and molecular fingerprints of cells at different stages of differentiation from hiPSC to neuron, immunocapture of SSEA5+ cells using geometrically enhanced differential immunocapture will be used to eliminate teratoma-forming cells and reduce teratoma formation in vivo. Flow cytometry, immunostaining, and in vivo teratoma formation studies will be used to evaluate the potential for negative selection to limit teratoma-forming risk and link teratoma outcomes to surface markers in flowthrough and captured subpopulations, and these results will allow (a) early purification of radially structured, multipotent neural rosettes for accelerated and enhanced yield and purity of neural differentiation, (b) purification of terminally differentiated neural cell populations, and (c) enhanced characterization of teratoma-specific surface markers. Taken together, the expected outcome of the specific aims is identification of the links between adhesive signature of neural cells in continuously differentiating hiPSC cultures, their surface expression of integrins and SSEA5, and teratoma formation in SCID mice, quantified by measurement of reduction of teratoma formation upon SSEA5-based negative cell selection in a novel microfluidic device. The broad impact of this work is that the knowledge gained will inform devices that use adhesion strength for hiPSC isolation and immunocapture for isolation of rare populations of teratoma-forming cells. The Investigators plan to broaden STEM participation through a series of summer activities designed to use GEDI devices to explain cellular transport to high school women as part of a Cornell leadership academy.

主要研究者：Kirby，Brian J.提案编号：1511914研究人员将开发技术，使患者的人类细胞重新编程为神经细胞，可用于修复神经损伤或神经退行性疾病。 尽管存在重编程细胞的技术，但这些重编程技术目前对患者构成风险，因为一些重编程细胞可能形成肿瘤而不是用于治疗目的。 这项工作将处理重新编程的细胞群，以去除那些有可能形成肿瘤的细胞。 这项工作具有广泛的意义，因为通过消除肿瘤形成的可能性，它将使组织再生能够以治疗益处和最小的伤害患者的风险进行。这项研究的长期目标是实现有效的，无畸胎瘤的神经元细胞工程。拟议的工作的目的是描绘之间的关系粘附机械生物学，畸胎瘤相关的标志物和分化的神经重编程的人诱导多能干细胞（hiPSC）的人口。中心假设是（a）神经玫瑰花结细胞将具有粘附特征（即，整联蛋白、粘着斑和粘着强度），与污染细胞不同，这将导致使用基于微干细胞高效粘着的恢复（uSHARE）平台选择性去除它们，和（B）畸胎瘤形成细胞将具有独特的整联蛋白和聚糖表达模式，包括阶段特异性胚胎抗原（SSEA）-5，这将使得能够基于微流体选择性捕获畸胎瘤形成细胞。 这一假设是基于先前发现的hiPSC和神经细胞的差异性扩增和用于部分重编程培养物的hiPSC的无标记分离，以及使用几何增强差异免疫捕获（GEDI）装置的选择性稀有细胞分离而制定的。提出的工作的基本原理是，粘附机械生物学，畸胎瘤相关标志物和分化之间的关系是最直接适用于污染物提取方法，提高时间，产量和纯度的神经分化，促进其直接应用于再生医学。 这项工作将通过两个特定目标确定粘附特征、表面标志物和畸胎瘤形成之间的关系，这两个特定目标确定畸胎瘤相关标志物、粘附特征和hiPSC沿着神经谱系定向分化之间的关系，并确定分化细胞中基于SSEA 5的负选择与消除祖细胞/神经元培养物中形成畸胎瘤的风险之间的关系。 拟议的工作将确定分化过程中表面表达和粘附之间的关系，并评估免疫受损小鼠分化和畸胎瘤形成的潜力。 通过利用从hiPSC到神经元的不同分化阶段的细胞的粘附特征和分子指纹的差异，使用几何增强的差异免疫捕获的SSEA 5+细胞的免疫捕获将用于消除畸胎瘤形成细胞并减少体内畸胎瘤形成。流式细胞术、免疫染色和体内畸胎瘤形成研究将用于评估阴性选择的潜力，以限制畸胎瘤形成风险并将畸胎瘤结果与流通和捕获的亚群中的表面标志物联系起来，这些结果将允许（a）早期纯化放射状结构的多能神经细胞，以加速和提高神经分化的产率和纯度，（B）纯化终末分化的神经细胞群，和（c）增强畸胎瘤特异性表面标志物的表征。 总之，特定目标的预期结果是鉴定连续分化的hiPSC培养物中神经细胞的粘附特征、其整合素和SSEA 5的表面表达以及SCID小鼠中的畸胎瘤形成之间的联系，通过测量在新型微流体装置中基于SSEA 5的阴性细胞选择后畸胎瘤形成的减少来定量。这项工作的广泛影响是，所获得的知识将为使用粘附强度进行hiPSC分离和免疫捕获分离罕见畸胎瘤形成细胞群体的设备提供信息。研究人员计划通过一系列旨在使用GEDI设备的夏季活动来扩大STEM参与，以向高中女性解释细胞运输，作为康奈尔大学领导学院的一部分。