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

PI: Kirby, Brian J.提案号：1511914研究人员将开发技术，使患者的人类细胞被重新编程为神经细胞，用于修复神经损伤或神经退行性疾病。虽然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以形成肿瘤而不是用于治疗目的。这项工作将处理重编程细胞群，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在治疗益处和伤害患者的风险最小的情况下进行。这项研究的长期目标是实现高效、无畸胎瘤的神经细胞工程。这项工作的目的是描述粘附力学生物学、畸胎瘤相关标记物和神经重编程人类诱导多能干细胞（hiPSC）群体分化之间的关系。主要假设是：(a)与污染细胞不同，神经花环细胞将具有粘附特征（即整合素、局灶粘附和粘附强度），这将导致它们使用微干细胞高效粘附恢复（uSHEAR）平台选择性去除；(b)畸胎瘤形成细胞将具有独特的整合素和聚糖表达模式，包括阶段特异性胚胎抗原(SSEA)-5。这将使基于微流体的选择性捕获畸胎瘤形成细胞成为可能。这一假设是基于先前发现的hipsc和神经细胞的差异粘附性，部分重编程培养的hipsc的无标记分离，以及使用几何增强的差异免疫捕获（GEDI）设备选择性分离稀有细胞而制定的。这项工作的基本原理是，粘附力学生物学、畸胎瘤相关标记物和分化之间的关系最直接适用于污染物提取方法，这些方法可以提高神经分化的时间、产量和纯度，并促进其在再生医学中的直接应用。本研究将通过两个特定目标确定粘附特征、表面标记物和畸胎瘤形成之间的关系，即确定畸胎瘤相关标记物、粘附特征和hiPSCs沿神经谱系定向分化之间的关系，并确定基于ssea5的分化细胞负选择与祖细胞/神经元培养中畸胎瘤形成风险去除之间的关系。这项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。利用hiPSC向神经元分化不同阶段细胞粘附特征和分子指纹的差异，利用几何增强的差异免疫捕获SSEA5+细胞，在体内消除畸胎瘤形成细胞，减少畸胎瘤形成。流式细胞术、免疫染色和体内畸胎瘤形成研究将用于评估负面选择的潜力，以限制畸胎瘤形成的风险，并将畸胎瘤结果与流动和捕获亚群中的表面标记物联系起来，这些结果将允许(a)早期纯化放射状结构、多能神经花环，以加速和提高神经分化的产量和纯度；(b)纯化终分化神经细胞群，(c)增强畸胎瘤特异性表面标记物的表征。综上所述，具体目标的预期结果是确定持续分化的hiPSC培养物中神经细胞的粘附特征、其表面整合素和SSEA5的表达与SCID小鼠中畸胎瘤形成之间的联系，通过测量基于SSEA5的阴性细胞选择在新型微流控装置中畸胎瘤形成的减少来量化。这项工作的广泛影响是，所获得的知识将为使用粘附强度分离hiPSC和免疫捕获分离罕见畸胎瘤形成细胞的设备提供信息。作为康奈尔大学领导学院的一部分，研究人员计划通过一系列夏季活动来扩大STEM的参与，这些活动旨在使用GEDI设备向高中女性解释细胞运输。

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.