Neuroscience Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA.
Regenerative Sciences Training Program, Center for Regenerative Medicine, Mayo Clinic, Jacksonville, FL, USA.
Stem Cell Res Ther. 2023 Oct 5;14(1):289. doi: 10.1186/s13287-023-03515-0.
Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRS) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.
We used CRISPR/Cas9 homology-directed repair to stably integrate MetRS into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRS was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRS-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.
Our results showed successful integration of MetRS into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRS-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.
The MetRS-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.
间充质基质细胞 (MSCs) 具有动态的分泌组，在组织修复和再生中起着关键作用。然而，在混合培养疾病模型中研究 MSC 分泌组仍然具有挑战性。本研究旨在开发一种基于突变甲硫氨酰-tRNA 合成酶的工具包 (MetRS)，以选择性分析混合培养系统中 MSC 的分泌蛋白，并展示其用于研究 MSC 对病理刺激的反应的潜力。
我们使用 CRISPR/Cas9 同源定向修复将 MetRS 稳定整合到细胞中，使非典型氨基酸叠氮亮氨酸 (ANL) 的掺入成为可能，并利用点击化学方便地进行选择性蛋白质分离。MetRS 被整合到 H4 细胞和诱导多能干细胞 (iPSCs) 中，进行了一系列概念验证研究。在 iPSC 分化为诱导-MSCs 后，我们验证了它们的身份，并将表达 MetRS 的 iMSCs 与未处理或脂多糖 (LPS) 处理的 THP-1 细胞共培养。然后，我们使用抗体阵列对 iMSC 分泌组进行了分析。
我们的结果表明 MetRS 成功整合到靶细胞中，允许从混合培养环境中特异性分离蛋白质。我们还证明，表达 MetRS 的 iMSCs 的分泌组可以与共培养中的 THP-1 细胞区分开来，并且与未经处理的 THP-1 细胞相比，与 LPS 处理的 THP-1 细胞共培养时会发生改变。
我们生成的基于 MetRS 的工具包可用于在混合培养疾病模型中选择性分析 MSC 分泌组。这种方法广泛适用于研究不仅是 MSC 对病理条件模型的反应，还适用于从 iPSCs 分化而来的任何其他细胞类型。这可能揭示新的 MSC 介导的修复机制，并推进我们对组织再生过程的理解。
