Generation of hPSCs Using Reprogramming Proteins-Encapsulated Nanoparticles

使用重编程蛋白封装的纳米颗粒生成 hPSC

• 批准号: 8176809
• 负责人: HSIAN-RONG TSENG
• 金额: $ 18.59万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8176809
• 关键词: Adult; Apoptosis; Apoptotic; Biological Assay; Categories; Cell Cycle; Cells; Clinical; Consent; Detection; Encapsulated; Fibroblasts; Generations; Histone Deacetylase Inhibitor; Human; Image Cytometry; Institutional Review Boards; Interdisciplinary Study; Joints; Libraries; Ligands; Measures; Medical; Methods; Microfluidics; Mitosis; Monitor; Motivation; Nuclear; Patients; Peptides; Performance; Phenotype; Polymers; Preparation; Process; Production; Proteins; Regenerative Medicine; Reporter; Research; Research Institute; Research Proposals; Safety; Screening procedure; Signal Transduction; Small Interfering RNA; Solutions; Somatic Cell; Staining method; Stains; Stem Cell Research; Stem cells; Synthesis Chemistry; System; Technology; Time; Viral; base; capsule; caspase-3; combinatorial; digital; disorder control; dosage; experience; genetic manipulation; human embryonic stem cell; improved; induced pluripotent stem cell; inhibitor/antagonist; multidisciplinary; nano; nanoparticle; pluripotency; ratiometric; self assembly; stem cell biology; transcription factor; vector

DESCRIPTION (provided by applicant): Stem cells have the potential to revolutionize medical practice by enabling regenerative medicine. This proposal aims to develop highly efficient generation of human induced pluripotent stem cells (hiPSCs) by utilizing a new class of nanoparticle (NP)-based vectors. These NPs are capable of delivering reprogramming transcription factors (TFs), namely OCT4, SOX2, KLF4 and c-MYC. Current reprogramming systems raise safety concerns because they utilize viral expression of the four key TFs, resulting in hiPSCs with multiple viral integrations. Safer strategies for generating hiPSCs have recently been demonstrated by introducing cell- penetrating peptide-fused reprogramming TFs into human somatic cells, averting any potential dangers of genetic manipulation. Still, effective delivery of TFs remains a key obstacle in creating hiPSCs. Three research groups at UCLA with expertise covering synthetic chemistry, nanoparticles, microfluidics (Tseng and Lu) and stem cell biology (Pyle) have collaborated and accomplished preliminary results consisting of the following fundamental proof-of-concept studies: (i) self-assembly production of supramolecular nanoparticles (SNPs) for delivery of TFs, (ii) (ii) a single protein nano-capsule technology, (iii) digital microreactors for large-scale screening, (iv) microfluidic image cytometry (MIC) technology for quantitative phenotyping of single hiPSCs, and (v) extensive experience in generating hiPSCs. The proposed research will leverage the multidisciplinary team to implement the following two specific aims: 1) We will synthesize a variety of polymer building blocks, cross linkers, functional ligands, as well as, nano- capsules containing the four reprogramming TFs for self-assembly of SNP-based delivery vectors. 2) We will generate a combinatorial library of reprogramming TF-encapsulated SNPs (i.e., OCT4/SOX2/ KLF4/c-MYC?SNPs) by performing ratiometric mixing of the four TF-containing nano-capsules, polymer building blocks, cross linkers and functional ligands. Human ESC-derived fibroblast cells with an OCT4-EGFP reporter will be employed as target cells. Subsequently, the MIC technology will be employed to quantify reprogramming performance by measuring pluripotent markers and colony formation in the SNPs-treated cells. If the efficiency is too low, we propose to co-deliver apoptotic inhibitors (e.g., siRNA_p53) with the four reprogramming TFs using siRNA/TFs?SNPs-based vectors. We anticipate that combination of apoptotic inhibitors and reprogramming TFs could dramatically increase the efficiency of reprogramming. PUBLIC HEALTH RELEVANCE: The long-term objective of this research proposal is to explore the use of supramolecular nanoparticles (SNPs) as a new category of artificial vector for simultaneous delivery of the four reprogramming transcription factors (i.e., OCT4, SOX2, KLF4 and c-MYC) into human somatic cells to induce reprogramming to the induced pluripotent cell (iPSC) state.

描述(由申请人提供)：干细胞有可能通过使再生医学产生革命性的医疗实践。该建议旨在利用一类新型的基于纳米颗粒(NP)的载体来高效地产生人诱导多能干细胞(HiPSCs)。这些NPs能够递送重编程转录因子，即OCT4、SOX2、KLF4和c-myc。目前的重编程系统引起了安全问题，因为它们利用四个关键TF的病毒表达，导致具有多个病毒整合的hiPSCs。最近，通过将穿透性多肽融合的TF重新编程到人类体细胞中，避免了任何潜在的遗传操作危险，证明了生成HiPSCs的更安全的策略。尽管如此，有效交付TF仍然是创建HiPSC的关键障碍。加州大学洛杉矶分校拥有合成化学、纳米粒子、微流体(Tseng和Lu)和干细胞生物学(PYLE)专业知识的三个研究小组合作并完成了由以下基本概念验证研究组成的初步成果：(I)自组装生产超分子纳米粒子(SNPs)用于传递转铁蛋白，(Ii)单蛋白质纳米胶囊技术，(Iii)用于大规模筛选的数字微反应器，(Iv)用于单个HIPSCs定量表型的微流控图像细胞仪(MIC)技术，以及(V)在HIPSCs生成方面的丰富经验。这项拟议的研究将利用多学科团队来实现以下两个具体目标：1)我们将合成各种聚合物构建块、交联剂、功能配体以及包含四个重编程因子的纳米胶囊，用于自组装基于SNP的递送载体。2)我们将通过四种含Tf的纳米胶囊、聚合物构建块、交联剂和功能配体的比例混合，生成一个重新编程Tf包裹的SNPs(即OCT4/SOX2/KLF4/c-MYC？SNPs)的组合库。具有OCT4-EGFP报告基因的人胚胎干细胞来源的成纤维细胞将被用作靶细胞。随后，将使用MIC技术通过测量SNPs处理的细胞中的多潜能标记和克隆形成来量化重新编程的性能。如果效率太低，我们建议使用基于siRNA/TFS？SNPs的载体与四个重新编程的TF共传递凋亡抑制因子(例如siRNA_p53)。我们预计，凋亡抑制剂和重新编程TF的组合可以极大地提高重新编程的效率。 与公共健康相关：这项研究建议的长期目标是探索使用超分子纳米颗粒(SNPs)作为一种新的人工载体，同时将四种重编程转录因子(即OCT4、SOX2、KLF4和c-MYC)导入人类体细胞，以诱导重编程到诱导多能细胞(IPSC)状态。