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

项目摘要/摘要 人类诱导多能干细胞(Hipscs)因其能力而成为研究中有用的工具。 分化为任何成体细胞类型，包括心肌细胞(CMS)。这些HiPSC-CMS具有 开发正常、疾病状态甚至患者特定心脏的临床前模型的潜在用途 功能。然而，目前HiPSC-CM在临床上的可译性有限。主要挑战之一是 从HiPSCs获得心脏组织模型的关键是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。 克服差异化异构性将提高可扩展性并降低使用基于HiPSC-CM的成本 组织模型，因为目前的分化方法通过丢弃未能 区分为CMS或特定的CM亚型，这是低效的。这一提议假设异质性 在终末分化的HiPSC中，源于HiPSC克隆谱系之间的异质性，导致 对分化提示的不同反应。因此，应改进对这些可变反应的核算 HiPSC心脏分化的均质性和一致性。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使用唯一的、 可遗传的单引导RNA(SgRNA)条形码序列，用于标记细胞和跟踪克隆谱系动力学 对实验条件的反应，如分化线索。这可以用来解决转录本问题 HPSCs克隆谱系的异质性及其与其转录异质性的关系 心脏分化过程中不同时间点的谱系。此建议书的目标1将验证ClonMapper是否 通过用sgRNA条形码标记HiPSC群体与HiPSC心脏分化兼容 描述了它们的多能性。AIM 2将把HiPSC谱系的转录异质性与 不同时间点的异质性。这将允许识别哪些谱系与HiPSC-V-CM不同 命运和何时，谱系的特征为具有高(HDE)或无/低分化效率(n/LDE)， 以及创建与HDE或n/LDE谱系相关联的基因表达签名。基因表达 在目标3中将使用HDE和n/LDE谱系的签名来识别可用作 添加分化刺激以将n/LDE谱系基因表达转变为模拟HDE谱系基因表达， 即向HiPSC-V-CM状态转变。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，阐明导致这一结果的潜在机制， 并建立了降低异质性的方法，以提高特异性HiPSC-CM的质量和一致性 亚型产量(在本例中为HiPSC-V-CM)，提高了HiPSC-CMS的临床相关性。

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.