Mechanisms of stretch-mediated skin expansion at single cell resolution [ATAC-seq]

The ability of the skin to expand in response to stretching has, for decades, been exploited in reconstructive surgery. Several studies have investigated the response of stretching epidermal cells in vitro. However, it remains unclear how mechanical forces affect epidermal stem cell behaviour in vivo. Here, we develop a mouse model in which the temporal consequences of the stretching the skin epidermis can be studied. Using a multidisciplinary approach that combines clonal analysis and mathematical modelling, we show that mechanical force induces skin expansion by promoting the renewal of epidermal stem cells. This occurs through a structured response in which cell fates are coordinated locally by stem cells that switch between states primed for renewal or differentiation. Transcriptional and chromatin profiling identifies the gene regulatory networks modulated by mechanical force. Using a combination of pharmacological inhibition and several conditional gene loss-of-function mouse mutants, we dissect the signalling pathways that control force-mediated tissue expansion. We used ATACseq to profile the chromatin landscape of basal cells isolated from the interfolliular epidermis during force-mediated tissue expansion. Interfollicolar basal epideramal cells form the back skin were FACS sorted for the positive expression of ITGA6 and negative expression of CD34. One CD1 mouse was subjected to force-mediated tissue expansion and an untreted animal was used as control. For each sample, 10000 basal cells were FACS sorted and used for ATAC preparation.

数十年来，皮肤响应牵拉而扩张的能力已被应用于重建外科手术中。已有多项研究对体外培养的表皮细胞牵拉响应展开了探究，但目前仍不明确机械力如何在体内影响表皮干细胞的行为。本研究构建了一种可用于研究皮肤表皮牵拉后随时间变化效应的小鼠模型。本研究结合克隆分析与数学建模的多学科方法，证实机械力通过促进表皮干细胞的自我更新来诱导皮肤扩张。该过程依托于一种程序化应答：表皮干细胞可在预定向自我更新或分化的状态间切换，并以此在局部协调细胞命运。转录组学与染色质谱分析（chromatin profiling）鉴定出了受机械力调控的基因调控网络。本研究结合药物抑制实验与多种条件性基因功能缺失小鼠突变体，解析了调控力介导组织扩张的信号通路。本研究利用ATAC测序（ATAC-seq）对力介导组织扩张过程中，从毛囊间表皮分离的基底细胞的染色质景观进行了谱分析。研究人员从背部皮肤分离毛囊间表皮基底细胞，并通过荧光激活细胞分选（FACS）以ITGA6阳性、CD34阴性的表型进行分选。选取1只CD1小鼠进行力介导组织扩张处理，并以未处理的动物作为对照。每个样本均分选得到10000个基底细胞，用于ATAC文库的制备。