OPTICAL, FLUIDIC, AND MOLECULAR TECHNOLOGIES FOR SINGLE-CELL OMICS

总结 干细胞分化发生在一个由中间细胞状态连续体定义的复杂景观上， 分支轨迹和高度协调的监管动态。这种复杂性往往隐藏在 总体测量，其通常限于测量群体中标记物的平均值， 细胞高通量单细胞RNA测序的最近和快速出现提供了强有力的 通过测量细胞中数千个基因的表达， 大量的细胞。辅助统计工具可以处理这些大型数据集， 不同的细胞群基于它们的转录谱。然而，RNA转录本丰度并不 总是与蛋白质组成相关，有许多重要的细胞内分子，如脂质和 代谢物，其不是在基因组中特异性编码的。细胞的正交分子测量 国家可以为研究调控动力学提供转录组分析的有力补充 在干细胞分化期间。这项研究计划的重点是技术的发展，以促进 单细胞中的“多组学”测量。我们利用三大核心技术来测量细胞状态 表型我们使用DamID来探测基因组组织和蛋白质-DNA相互作用，单细胞RNAseq 提供全转录组基因表达谱分析和相干拉曼成像来表征 活细胞的化学成分。微流体技术促进了这些技术的整合， 实现单细胞的多模式测量。该平台将为解剖提供一种新的方法， 在分化干细胞中的协调调节网络。我们的最终目标是开发一种工具， 这些测量在原位，以保留单细胞的空间信息和细胞的背景下， 发育中的组织或整个有机体。

SUMMARY Stem cell differentiation takes place on a complex landscape defined by a continuum of intermediate cell states, branching trajectories, and highly coordinated regulatory dynamics. This complexity is often hidden to ensemble measurements, which are typically limited to measuring average values of markers in populations of cells. The recent and rapid emergence of high-throughput single-cell RNA sequencing has provided a powerful solution for dissecting transitions between cell states by measuring the expression of thousands of genes in large populations of cells. Complementary statistical tools can process these large data sets and identify distinct groups of cells base on their transcriptional profile. However, RNA transcript abundance does not always correlate with protein composition and there are many important intracellular molecules, like lipids and metabolites, which are not specifically encoded in the genome. Orthogonal molecular measurements of cell state could provide a powerful complement to transcriptome analysis for investigating regulatory dynamics during stem cell differentiation. This research program focuses on the development of technology to facilitate “multi-omic” measurements in single cells. We take advantage of three core technologies to measure cell state phenotypes. We use DamID to probe genome organization and protein-DNA interactions, single-cell RNAseq to provide whole-transcriptome gene expression profiling, and coherent Raman imaging to characterize the chemical composition of live cells. Microfluidic technology facilitates the integration of these techniques and enables multimodal measurement of single cells. This platform will provide a new approach for dissecting coordinated regulatory networks in differentiating stem cells. Our ultimate goal is to develop a tool to make all of these measurements in situ, in order to retain single-cell spatial information and cellular context in a developing tissue or whole organism.