Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells [ATAC-seq]. Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells [ATAC-seq]

Mesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB. Integrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes. In conclusion, the observed changes in AP1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication. Overall design: MSCs: PoieticsTM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (hMSC) are isolated from normal (non-diabetic) adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of normal volunteers. Three PoieticsTM hMSC cells, namely 36015, 36461, and 36550, sourced from three distinct donors, were acquired from Lonza. These cells were designated as follows: 1 (batch 18TL 169252 from donor 36015), 2 (batch 18TL 241909 from donor 36461), and 3 (batch 18TL 262066 from donor 36550). The MSC cells were grown in a humidified chamber maintained at 37°C and 5% CO2, utilizing MSCBMTM Basal Media (PT-3238) and MSCGMTM SingleQuots Supplement Kit (PT-4105) obtained from Lonza. Media was changed every three days. MSCs were cultured in media supplemented with 10 ng/ml PDGF-BB (Invitrogen) for 48 hours as the treated sample, while the control sample was cultivated without PDGF-BB. Control and PDGF-BB treated MSCs were concurrently collected from distinct plates for ATACseq analyses.

间充质干细胞（Mesenchymal stem cells, MSCs）是一类多能干细胞，因其具备自我更新能力且可在特定条件下分化为多种细胞类型，目前正被纳入临床试验研究范畴。然而，MSCs的治疗效果仍受限于低植入率、细胞融合及细胞存活能力不足等问题。目前已有多种策略被探索用于提升MSCs的治疗效能，其中血小板衍生生长因子（platelet-derived growth factor, PDGF）-BB展现出极具潜力的应用前景。
为深入解析PDGF-BB对MSCs基因表达谱及染色质可及性的影响，本研究对三组经PDGF-BB刺激与未刺激的体外培养人原代MSCs开展了RNA测序及染色质可及性谱分析。对基因表达与染色质可及性的整合分析显示，PDGF-BB处理可重塑染色质可及性景观，通过AP-1家族转录因子TEAD、CEBP及RUNX2标记激活或抑制的染色质区域。上述AP1转录因子的表达变化，进而介导细胞增殖，并促使细胞向成骨细胞、脂肪细胞或软骨细胞方向分化。
不同供体分离得到的MSCs，其分化程度存在显著差异。在所有差异表达基因中，亦富集到外泌体相关基因。
综上，本研究观测到的AP1转录因子表达变化不仅诱导了细胞增殖与分化，还揭示了基于供体特异性差异的MSCs分化程度差异。此外，差异表达基因中外泌体相关基因的富集现象，提示PDGF-BB在促进细胞间通讯方面可能发挥重要作用。

### 总体实验设计
本研究使用的间充质干细胞为Poietics™ 正常人骨髓来源间充质干细胞（human bone marrow-derived Mesenchymal Stem Cells, hMSC），其分离自正常志愿者双侧髂后嵴穿刺抽取的非糖尿病成人骨髓。本研究从Lonza公司获取了来自3名不同供体的3株Poietics™ hMSC，分别为36015、36461与36550，对应批次信息如下：样本1（供体36015，批次18TL 169252）、样本2（供体36461，批次18TL 241909）、样本3（供体36550，批次18TL 262066）。
MSCs的培养条件为：在37℃、5% CO₂的湿润培养箱中培养，使用Lonza公司提供的MSCBMTM基础培养基（PT-3238）及MSCGMTM SingleQuots添加剂套装（PT-4105），每3天更换一次培养基。处理组细胞在添加10 ng/ml PDGF-BB（Invitrogen）的培养基中培养48小时，对照组则在不含PDGF-BB的培养基中常规培养。随后分别从对应培养板中收集对照组与PDGF-BB处理组的MSCs，用于ATAC-seq分析。