Non-genetic differences underlie variability in proliferation among esophageal epithelial clones

Individual cells grown in culture exhibit remarkable differences in their growth, with some cells capable of forming large clusters, while others are limited or fail to grow at all. While these differences have been observed across cell lines and human samples, the growth dynamics and associated cell states remain poorly understood. In this study, we performed clonal tracing through imaging and cellular barcoding of an in vitro model of esophageal epithelial cells (EPC2-hTERT). We found that about 10% of clones grow exponentially, while the remaining have cells that become non-proliferative leading to a halt in the growth rate. Using mathematical models, we demonstrate two distinct growth behaviors: exponential and logistic. Further, we discovered that the propensity to grow exponentially is largely heritable through four doublings and that the less proliferative clones can become highly proliferative through increasing plating density. Combining barcoding with single-cell RNA-sequencing (scRNA-seq), we identified the cellular states associated with the highly proliferative clones, which include genes in the WNT and PI3K pathways. Finally, we identified an enrichment of cells resembling the highly proliferative cell state in the proliferating healthy human esophageal epithelium. Overall design: EPC2-hTERT cells were barcoded and grown in tissue culture, sorted using FACS, and plated sparsely. We then perfomed single-cell RNA sequencing using 10X Genomics sequencing.

体外培养的单个细胞在生长特性上存在显著差异：部分细胞可形成大型细胞簇，而其余细胞的增殖能力受限甚至完全无法增殖。尽管这类差异已在多种细胞系及人类组织样本中被观测到，但其背后的生长动力学与相关细胞状态仍未得到充分阐释。本研究针对食管上皮细胞体外模型（EPC2-hTERT），通过成像技术与细胞条码标记（cellular barcoding）开展克隆追踪实验。研究发现，约10%的克隆呈指数增殖模式，其余克隆的细胞则进入非增殖状态，导致增殖速率停滞。借助数学模型，本研究揭示了两种截然不同的生长行为：指数增殖与逻辑斯蒂增殖。进一步研究表明，呈指数增殖的倾向可通过四次细胞倍增稳定遗传，且低增殖能力的克隆可通过提高接种密度转变为高增殖能力克隆。将细胞条码标记与单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）结合，本研究鉴定出与高增殖克隆相关的细胞状态，其涉及WNT与PI3K信号通路相关基因。最后，本研究在增殖状态的健康人类食管上皮组织中，发现了富集类似高增殖细胞状态的细胞群体。实验设计：对EPC2-hTERT细胞进行条码标记后，开展体外组织培养，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）进行分选，并以低密度接种。随后采用10X Genomics测序平台完成单细胞RNA测序。