Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering.

中文翻译：

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无外源生长因子的协同 CRISPRa 调节的软骨细胞外基质沉积。

干细胞疗法已显示出对肌肉骨骼疾病进行再生治疗的前景，但它们的成功情况参差不齐。为了增强再生效果，利用生长因子诱导分化为天然细胞类型，但无法控制的体内条件会抑制分化，并且难以实现对表达的基质蛋白的精确控制。为了解决这些问题，我们研究了一种通过直接上调主要软骨组织蛋白、蛋白聚糖 ( ACAN ) 和胶原蛋白 II ( COL2A1 )来增强再生表型的新方法。) 使用 dCas9-VPR CRISPR 基因激活系统。我们证明了在颗粒培养中独立于外源生长因子的靶蛋白的表达和沉积增加。的奇异上调COL2A1 / ACAN有趣表示COL2A1上调介导的最高硫酸化糖胺聚糖聚糖（sGAG）沉积，除了II型胶原沉积。通过 RNA-seq 分析，这被证明是通过COL2A1上调介导更广泛的软骨基因表达变化而发生的。COL2A1和ACAN 的多重上调一起导致最高的 sGAG 和胶原蛋白 II 沉积，其水平与软骨生长因子分化颗粒中的水平相当。总的来说，这项工作表明 dCas9-VPR 系统可以在没有生长因子的情况下稳健地上调COL2A1和ACAN沉积，为软骨和椎间盘细胞治疗和组织工程提供一种新的、精确的控制干细胞表型的方法。