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
基本信息
- 批准号:10752369
- 负责人:
- 金额:$ 3.92万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AccountingAddressAdultAffectAffinityBar CodesCardiacCardiac MyocytesCardiovascular DiseasesCategoriesCause of DeathCell LineageCellsCuesDataDiseaseDisease modelFrequenciesGene ExpressionGene Expression ProfileGenesGeneticGoalsGuide RNAHeartHeart AtriumHeritabilityHeterogeneityHumanLabelLibrariesMeasuresMethodsModelingMonitorOutcomePacemakersPathway interactionsPatientsPopulationPopulation HeterogeneityPre-Clinical ModelPredispositionProceduresProcessProductionPublic HealthResearchRiskSomatic CellSourceSpecific qualifier valueStem Cell ResearchStem cell pluripotencyStimulusTeratomaTestingTimeTissue ModelUnited StatesVentricularWorkcell typeclinical translationclinically relevantcostheart functionimprovedinduced pluripotent stem cellinduced pluripotent stem cell derived cardiomyocytesnext generation sequencingpluripotencypreclinical developmentresponsesmall moleculestem cell differentiationstem cell functionstem cell populationtheoriestooltranscriptometranscriptomicstumor
项目摘要
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.
项目概要/摘要
人类诱导多能干细胞 (hiPSC) 因其能力而成为研究中有用的工具
分化成任何成体细胞类型,包括心肌细胞(CM)。这些 hiPSC-CM 具有
潜在用于开发正常、疾病状态甚至患者特异性心脏的临床前模型
功能。然而,hiPSC-CM 目前的临床可转化性有限。主要挑战之一
从 hiPSC 衍生心脏组织模型的一个重要因素是心脏分化结果的异质性。异质性
hiPSC 心脏分化导致 CM 产量低且不一致,并且对 CM 亚型的控制有限。
克服分化异质性将提高可扩展性并降低利用基于 hiPSC-CM 的成本
组织模型,因为当前的分化方法通过丢弃未能分化的细胞来提高同质性
分化为 CM 或特定的 CM 亚型,效率低下。该提议假设异质性
在终末分化的 hiPSC 中,hiPSC 克隆谱系之间的异质性导致了
对分化线索的可变反应。因此,对这些可变响应的解释应该得到改进
hiPSC 心脏分化的同质性和一致性。为了测试这个理论,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、
可遗传的单引导 RNA (sgRNA) 条形码序列可标记细胞并跟踪克隆谱系动态
对实验条件的反应,例如分化线索。这可用于解决转录组学问题
hiPSC 中克隆谱系的异质性,并将其与相同细胞的转录组异质性联系起来
心脏分化中不同时间点的谱系。该提案的目标 1 将验证 ClonMapper 是否
通过用 sgRNA 条形码标记 hiPSC 群体,与 hiPSC 心脏分化兼容
表征它们的多能性。目标 2 将 hiPSC 谱系的转录组异质性与
不同时间点的异质性。这将允许识别哪些谱系与 hiPSC-V-CM 不同
命运和时间,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 谱系相关的基因表达特征。基因表达
HDE 和 n/LDE 谱系的特征将用于目标 3 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仿 HDE 谱系基因表达,
即,转向 hiPSC-V-CM 状态。这些研究的结果将确定潜在的来源
hiPSC 心脏分化结果的异质性,阐明导致这种情况的潜在机制,
建立减少异质性的方法,以提高特定 hiPSC-CM 的数量和一致性
亚型产量(在本例中为 hiPSC-V-CM),提高了 hiPSC-CM 的临床相关性。
项目成果
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