Farhang Niloofar, Brunger Jonathan M, Stover Joshua D, Thakore Pratiksha I, Lawrence Brandon, Guilak Farshid, Gersbach Charles A, Setton Lori A, Bowles Robby D
1 Department of Bioengineering, University of Utah , Salt Lake City, Utah.
2 Department of Biomedical Engineering, Duke University , Durham, North Carolina.
Tissue Eng Part A. 2017 Aug;23(15-16):738-749. doi: 10.1089/ten.TEA.2016.0441. Epub 2017 Feb 28.
Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1β. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1β, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1β, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.
肌肉骨骼疾病与炎性细胞因子的作用有关，尤其是肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的作用。这些炎性细胞因子可促进患病组织中细胞的凋亡和衰老以及细胞外基质的分解。基于干细胞的疗法正在被考虑用于治疗肌肉骨骼疾病，但这些炎性细胞因子的存在将对输送到这些环境中的治疗性细胞产生类似的有害作用。需要以细胞和途径特异性的方式来预防炎症诱导的凋亡和促炎信号传导的方法。在本研究中，我们展示了使用基于成簇规律间隔短回文重复序列（CRISPR）的表观基因组编辑，通过抑制炎性细胞因子细胞受体，特别是肿瘤坏死因子受体1（TNFR1）和白细胞介素1受体1（IL1R1），来改变细胞对炎性环境的反应。我们将基于CRISPR/Cas9的阻遏物靶向人脂肪来源干细胞（hADSCs）中的TNFR1和IL1R1基因调控元件，并研究了这些基因抑制的功能结果。在工程化的hADSCs中证明了有效的信号调节，因为在存在TNF-α或IL-1β的情况下，下游转录因子核因子-κB（NF-κB）的活性显著降低或维持在基线水平。未分化hADSCs的微球培养表明，在用TNF-α或IL-1β处理的工程化hADSCs中存活率提高，而对其免疫调节特性影响很小。此外，如在这种炎性环境中糖胺聚糖的产生增加所示，工程化hADSCs在存在TNF-α或IL-1β的情况下表现出改善的软骨形成分化能力。总体而言，这项工作展示了一种调节细胞对炎性信号反应的新方法，该方法可应用于工程化输送到炎性环境中的细胞，以及作为一种直接基因疗法来保护暴露于慢性炎症的内源性细胞，这在广泛的退行性肌肉骨骼病理学中都有观察到。
