成骨分化过程中牙髓干细胞单层和 3D 球体中 DNA 甲基化和基因表达的时间调节：一项比较研究。
Temporal Modulation of DNA Methylation and Gene Expression in Monolayer and 3D Spheroids of Dental Pulp Stem Cells during Osteogenic Differentiation: A Comparative Study.
机构信息
Department of Biophysics, Post Graduate Institution of Medical Education and Research (PGIMER), Chandigarh, 160012, India.
Department of Biochemistry, Post Graduate Institution of Medical Education and Research (PGIMER), Chandigarh, 160012, India.
出版信息
Tissue Eng Regen Med. 2022 Dec;19(6):1267-1282. doi: 10.1007/s13770-022-00485-x. Epub 2022 Oct 11.
BACKGROUND
Human mesenchymal stem cells are being used for various regenerative applications in past decades. This study chronicled a temporal profile of the transcriptional pattern and promoter methylation status of the osteogenic related gene in dental pulp stem cells (DPSCs) derived from 3-dimensional spheroid culture (3D) vis a vis 2-dimensional (2D) monolayer culture upon osteogenic induction.
METHODS
Biomimetic properties of osteogenesis were determined by alkaline phosphatase assay and alizarin red staining. Gene expression and promoter methylation status of osteogenic genes such as runt-related transcription factor-2, collagen1α1, osteocalcin (OCN), and DLX5 (distal-homeobox) were performed by qPCR assay and bisulfite sequencing, respectively. Furthermore, µ-Computed tomography (micro-CT) was performed to examine the new bone formation in critical-sized rat calvarial bone defect model.
RESULTS
Our results indicated a greater inclination of spheroid culture-derived DPSCs toward osteogenic lineage than the monolayer culture. The bisulfite sequencing of the promoter region of osteogenic genes revealed sustenance of low methylation levels in DPSCs during the progression of osteogenic differentiation. However, the significant difference in the methylation pattern between 2D and 3D derived DPSCs were identified only for OCN gene promoter. We observed differences in the mRNA expression pattern of epigenetic writers such as DNA methyltransferases (DNMTs) and methyl-cytosine dioxygenases (TET) between the two culture conditions. Further, the DPSC spheroids showed enhanced new bone formation ability in an animal model of bone defect compared to the cells cultivated in a 2D platform which further substantiated our in-vitro observations.
CONCLUSION
The distinct cellular microenvironment induced changes in DNA methylation pattern and expression of epigenetic regulators such as DNMTs and TETs genes may lead to increase expression of osteogenic markers in 3D spheroid culture of DPSCs which make DPSCs spheroids suitable for osteogenic regeneration compared to monolayers.
背景
在过去的几十年中，人类间充质干细胞被用于各种再生应用。本研究记录了牙髓干细胞（DPSCs）在三维球体培养（3D）和二维（2D）单层培养条件下，成骨诱导后，与成骨相关基因的转录模式和启动子甲基化状态的时间进程。
方法
通过碱性磷酸酶测定和茜素红染色来确定成骨的仿生特性。通过 qPCR 测定和亚硫酸氢盐测序分别对成骨基因如 runt 相关转录因子 2、胶原 1α1、骨钙素（OCN）和 DLX5（远-同源盒）的基因表达和启动子甲基化状态进行检测。此外，通过微计算机断层扫描（micro-CT）检测在大鼠临界颅骨骨缺损模型中的新骨形成。
结果
我们的结果表明，与单层培养相比，球体培养衍生的 DPSCs 更倾向于成骨谱系。成骨基因启动子区的亚硫酸氢盐测序显示，在成骨分化过程中，DPSCs 中的低甲基化水平得以维持。然而，只有 OCN 基因启动子的 2D 和 3D 衍生 DPSCs 之间的甲基化模式存在显著差异。我们观察到两种培养条件之间表观遗传写入器（如 DNA 甲基转移酶（DNMTs）和甲基胞嘧啶双加氧酶（TET））的 mRNA 表达模式存在差异。此外，与在 2D 培养平台上培养的细胞相比，DPSC 球体在骨缺损动物模型中表现出增强的新骨形成能力，进一步证实了我们的体外观察结果。
结论
不同的细胞微环境诱导 DNA 甲基化模式和表观遗传调节因子（如 DNMTs 和 TETs）的表达变化可能导致 DPSCs 三维球体培养中骨生成标志物的表达增加，与单层培养相比，这使得 DPSCs 球体更适合骨生成再生。
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