Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells [scMultiome]

Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although the signaling circuits and gene regulatory networks regulating pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations that occur during state transitions remains a fundamental open question. Here, we discover that stem cell fate transitions are gated by two critical signals - nuclear envelope fluctuations and osmotic stress - that emanate from growth factor signaling-controlled changes in nuclear volume and nucleoplasm viscosity/density to subsequently trigger changes in nuclear architecture and transcription. We observe that fate transitions in the early human embryo and in an in vitro pluripotency exit model are guided by rapid changes in nuclear volume and nuclear envelope mechanics. These changes alter nuclear mechanosensitivity and trigger changes in nucleoplasmic viscosity and nuclear condensates that together prime chromatin for a cell fate transition. However, while this mechanical priming accelerates fate transitions, sustained biochemical signals are required for robust induction of differentiation. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics. iPS cells were compressed for 5 or 30 minutes, or pulsed with 30min compression followed by 24hr recovery. Subsequently, single nuclei were harvested. Two independent biological replicates were prepared for each condition, analyzed seperately to ensure reproducibility, and subsequently pooled and analyzed together, treating each cell in the total pool as a biological replicate. Single nuclei were isolated and libraries were prepared for simultaneous ATAC and RNA sequencing on the same single cell.

特定细胞形状与形态的建立是细胞命运转变的核心环节。尽管调控多能干细胞（pluripotent stem cell）分化的信号通路与基因调控网络已得到广泛深入研究，但这些网络如何在时空维度上与细胞状态转变过程中发生的形态学转变及机械形变相整合，仍是一个尚未解决的核心科学问题。本研究发现，干细胞命运转变受两类关键信号调控：核被膜波动与渗透压应激，这类信号源自生长因子信号通路调控的细胞核体积与核质黏度/密度变化，进而触发核结构与转录过程的改变。我们观察到，人类早期胚胎以及体外多能性退出模型中的细胞命运转变，均由细胞核体积与核被膜力学特性的快速变化所介导。这些变化会改变细胞核的机械敏感性，并引发核质黏度与核凝集体的改变，二者共同为细胞命运转变预激活染色质。不过，尽管这类机械预激活可加速细胞命运转变，但要高效诱导分化，仍需要持续的生化信号支持。本研究揭示了一种关键的机械化学反馈机制，该机制将核力学特性、细胞形状与体积同生化信号及染色质状态相整合，从而调控细胞命运转变的动态过程。将诱导多能干细胞（iPS cells）分别施加5分钟或30分钟的压缩处理，或先施加30分钟的压缩脉冲处理，随后恢复培养24小时。随后收集单个细胞核。每种处理条件均设置两组独立生物学重复，先分别进行分析以确保实验可重复性，之后将所有样本混合并统一分析，将混合后总样本中的每个细胞视为一个生物学重复单元。分离单个细胞核并构建文库，以实现对同一单细胞同时开展ATAC测序（Assay for Transposase-Accessible Chromatin sequencing）与RNA测序。