携带CD163报告载体的猪诱导多能干细胞株的建立

Development of porcine induced pluripotent stem cells with a CD163 reporter system

摘要:

猪肺泡巨噬细胞(porcine alveolar macrophage, PAM)是包括猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)在内的多种高致病病毒的受体细胞，是研究病毒与宿主互作机制的重要模型。然而PAM来源有限，难以满足当前需求。利用猪诱导性多能干细胞(induced pluripotent stem cells, iPSCs)向巨噬细胞定向诱导是解决PAM细胞数量不足的有效方法。CD163是PAM细胞的重要标记，也是PRRSV等病毒的主要受体。建立实时报告CD163激活程度的报告系统对于建立并优化猪iPSCs向PAM的诱导分化体系具有指导意义。本研究利用CRISPR/Cas9介导的基因编辑系统，设计靶向CD163终止密码子的sgRNA并构建相应的打靶载体，将其导入到猪PAM中的检测报告系统。进一步将该报告系统导入猪iPSCs中，通过碱性磷酸酶染色、免疫荧光染色和EDU染色等手段来检测其安全性。将猪内源CD163的报告载体系统转染至原代PAM中，检测到了红色荧光的表达，证明了该载体系统的可靠性；将CD163-reporter系统转染至猪iPSCs中，获得CD163 reporter-iPSCs。结果表明，CD163 reporter-iPSCs可以维持正常的多能性基因的表达，并具有与正常猪iPSCs一致的克隆形态和增殖能力。证实成功构建了CD163的报告载体，并将其转染至猪iPSCs，获得含有CD163报告载体的猪iPSCs系。该报告载体既不影响猪iPSCs的多能性，又能够实时指示CD163的表达，为深入解析猪iPSCs分化为PAM的机制以及解析PRRSV等重大病原与宿主的互作研究奠定了基础。

Abstract:

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.