Elucidation and improved control of human induced pluripotent stem cell cardiac differentiation by using single-guide RNA-based cellular barcoding to track and manipulate lineages

通过使用基于单向导 RNA 的细胞条形码来跟踪和操纵谱系，阐明并改进对人类诱导多能干细胞心脏分化的控制

• 批准号: 10752369
• 负责人: Sogu Sohn
• 金额: $ 3.92万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752369
• 关键词: Accounting; Address; Adult; Affect; Affinity; Bar Codes; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Categories; Cause of Death; Cell Lineage; Cells; Cues; Data; Disease; Disease model; Frequencies; Gene Expression; Gene Expression Profile; Genes; Genetic; Goals; Guide RNA; Heart; Heart Atrium; Heritability; Heterogeneity; Human; Label; Libraries; Measures; Methods; Modeling; Monitor; Outcome; Pacemakers; Pathway interactions; Patients; Population; Population Heterogeneity; Pre-Clinical Model; Predisposition; Procedures; Process; Production; Public Health; Research; Risk; Somatic Cell; Source; Specific qualifier value; Stem Cell Research; Stem cell pluripotency; Stimulus; Teratoma; Testing; Time; Tissue Model; United States; Ventricular; Work; cell type; clinical translation; clinically relevant; cost; heart function; improved; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; next generation sequencing; pluripotency; preclinical development; response; small molecule; stem cell differentiation; stem cell function; stem cell population; theories; tool; transcriptome; transcriptomics; tumor

PROJECT SUMMARY/ABSTRACT Human induced pluripotent stem cells (hiPSCs) have emerged as useful tools in research due to their capacity to differentiate into any adult somatic cell type, including cardiomyocytes (CMs). These hiPSC-CMs have potential use for developing preclinical models of both normal, disease-state, and even patient-specific heart function. However, hiPSC-CM are currently limited in their clinical translatability. One of the primary challenges in deriving cardiac tissue models from hiPSCs is heterogeneity of cardiac differentiation outcomes. Heterogeneity in hiPSC cardiac differentiation results in low, inconsistent CM yield as well as limited control over CM subtype. Overcoming differentiation heterogeneity will improve scalability and lower the cost of utilizing hiPSC-CM based tissue models, as current differentiation methods achieve increased homogeneity by discarding cells that fail to differentiate into CMs or specific CM subtypes, which is inefficient. This proposal hypothesizes that heterogeneity in terminally differentiated hiPSCs arises from heterogeneity between hiPSC clonal lineages that leads to variable response to differentiation cues. Accordingly, accounting for these variable responses should improve homogeneity and consistency of hiPSC cardiac differentiation. To test this theory, a cell barcoding platform, ClonMapper, will be used to address heterogeneity of hiPSC-CM differentiation. ClonMapper uses unique, heritable single-guide RNA (sgRNA) barcode sequences to label cells and track clonal lineage dynamics in response to experimental conditions, such as differentiation cues. This can be used to resolve the transcriptomic heterogeneity of clonal lineages in hiPSCs and connect it to the transcriptomic heterogeneity of those same lineages at different timepoints in cardiac differentiation. Aim 1 of this proposal will verify if ClonMapper is compatible with hiPSC cardiac differentiation by labelling hiPSC populations with sgRNA barcodes and characterizing their pluripotency. Aim 2 will connect transcriptomic heterogeneity of hiPSC lineages to heterogeneity at different timepoints. This will allow identification of which lineages diverge from an hiPSC-V-CM fate and when, characterization of lineages as having high (HDE) or no/low differentiation efficiency (n/LDE), and creation of gene expression signatures associated with HDE or n/LDE lineages. The gene expression signatures of HDE and n/LDE lineages will be used in Aim 3 to identify gene modulators that can be used as added differentiation stimuli to shift n/LDE lineage gene expression to mimicking HDE lineage gene expression, i.e., shift towards an hiPSC-V-CM state. The results from these studies will identify a potential source of heterogeneity in hiPSC cardiac differentiation outcomes, elucidate the underlying mechanisms that cause this, and establish methods for reducing heterogeneity to improve quantity and consistency of specific hiPSC-CM subtype yield (in this case hiPSC-V-CM), advancing the clinical relevance of hiPSC-CMs.

项目摘要/摘要 人类诱导多能干细胞（hiPSC）由于其能力而成为研究中的有用工具。 分化成任何成体细胞类型，包括心肌细胞（CM）。这些hiPSC-CM具有 开发正常、疾病状态甚至患者特异性心脏的临床前模型的潜在用途 功能然而，hiPSC-CM目前在其临床可翻译性方面受到限制。的主要挑战之一 在从hiPSC衍生心脏组织模型中，心脏分化结果的异质性。异质性 在hiPSC中，心脏分化导致低的、不一致的CM产量以及对CM亚型的有限控制。 克服差异化异质性将提高可扩展性并降低利用基于hiPSC-CM的 组织模型，因为目前的分化方法通过丢弃未能分化的细胞来实现增加的同质性。 区分为CM或特定的CM亚型，这是低效的。该提案假设异质性 在终末分化的hiPSC中，hiPSC克隆谱系之间的异质性导致 对分化线索的不同反应。因此，对这些可变响应的解释应该得到改进。 hiPSC心脏分化的均一性和一致性。为了验证这一理论，一个细胞条形码平台， ClonMapper将用于解决hiPSC-CM分化的异质性。ClonMapper使用独特的， 可遗传的单向导RNA（sgRNA）条形码序列，以标记细胞并跟踪克隆谱系动态， 对实验条件的反应，如分化线索。这可以用来解决转录组学 hiPSC中克隆谱系的异质性，并将其与那些相同克隆谱系的转录组异质性联系起来。 在心脏分化的不同时间点的谱系。本提案的目标1将验证ClonMapper是否 通过用sgRNA条形码标记hiPSC群体与hiPSC心脏分化相容， 表征其多能性。目的2将hiPSC谱系的转录组异质性与 不同时间点的异质性。这将允许鉴定哪些谱系与hiPSC-V-CM不同 命运以及何时，将谱系表征为具有高（HDE）或无/低分化效率（n/LDE）， 以及与HDE或n/LDE谱系相关的基因表达标记的产生。基因表达 HDE和n/LDE谱系的特征将用于目标3，以鉴定可用作 添加分化刺激物以将n/LDE谱系基因表达转变为模拟HDE谱系基因表达， 也就是说，向hiPSC-V-CM状态转移。这些研究的结果将确定一个潜在的来源 hiPSC心脏分化结果的异质性，阐明了导致这种情况的潜在机制， 并建立降低异质性的方法，以提高特异性hiPSC-CM的数量和一致性 亚型产率（在这种情况下为hiPSC-V-CM），提高了hiPSC-CM的临床相关性。