概要: Differentiation of human pluripotent stem cells toward definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA-sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. We further show that differentiation-arrested phenotype is inversely correlated with zinc concentration in the differentiation media. This study improves our understanding of in-vitro DE differentiation and provides actionable options to improve DE differentiation efficiency.
项目整体设计: RNA-sequencing of 329 single cells collected at four time points during a 4-day DE differentiation to identify mechanisms leading to cellular heterogeneity during differentiation
hESCs are routinely maintained in E8 medium (Stem Cell Technologies). For differentiation, Advanced RPMI 1640 (Gibco) was supplemented with l-glutamine and antibiotics, with Activin A (R & D Systems) and CHIR99021 (Tocris) added to specified concentrations and time.
处理方案:
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提取方案:
Single-cell whole transcriptome amplification (WTA) was performed using the Fluidigm C1 Single-Cell Auto Prep System (C1 System) as per the manufacturer recommendations (full details available at Fluidigm.com). Briefly, cells from each time point were single cell collected with Accutase and were resuspended at a concentration of 250 cell per L of complete media, mixed 7:3 with C1 suspension reagent (Fluidigm), and loaded onto C1 Single-Cell Auto Prep chips (C1 chips; Fluidigm). After loading, each of the cell isolation chambers on the C1 chip was optically inspected for the presence of a cell. Subsequently, these cells were lysed and SMART-Seq (Ramskold et al., 2012) whole transcriptome amplified (WTA) products were prepared with the C1 System using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) and protocols provided by Fluidigm.
建库方案:
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测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2500
链特异性:
Unspecific
样本
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数据来源
GEN样本编号
GEN数据集编号
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物种
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组织
细胞类型
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疾病
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突变/变异
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Condition Detail
生长方案
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分子类型
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加标（Spike-In）
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平均测序片段长度_1
平均测序片段长度_2
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多重比对率
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文章
Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.
