Novel strategies for therapeutic programming and delivery of mesenchymal stem cells to improve outcome of influenza virus-induced lung injury

间充质干细胞(MSC)在不同形式的急性肺损伤(ALI)中具有良好的治疗潜力，目前处于初步临床试验阶段。然而，在受影响的肺的特定的小生境中，潜在的与损伤或病原体相关的有益影响的分子机制大多是未知的。同样，不同来源的骨髓或脂肪组织等不同来源的骨髓间充质干细胞和肺驻留间充质干细胞(RMSC)在病毒性肺感染期间的治疗作用机制也没有得到全面的研究。该项目关注于MSC的功能异质性，并表征了病毒感染的肺细胞与具有抗病毒和器官修复能力的MSC不同亚群之间的分子相互作用。在第一个资助期，我们使用批量和单细胞(Sc)RNA-Seq分析结合先进的肺器官模型和体内感染模型来证明骨髓来源的MSCs(BM-MSC)、白色脂肪组织来源的MSCs(Wat-MSC)和RMSC代表着单细胞转录谱不同的异质群体，在抗病毒宿主防御、组织保护和肺组织再生(重新形成肺泡)方面发挥着不同的作用。不同来源的MSC可以以损伤特异性的方式启动，以有效地支持(重新)肺泡室的生成，例如通过IV特异性的预适应和体外TLR3配体，以及在IV和LPS损伤的体内模型中。关于MSC强大的抗病毒和组织保护作用，我们在体外模型和将BM-MSC注入IV型感染的WT或IFNAR-/-小鼠体内后，确定I型干扰素依赖的串扰是BM-MSC和IV感染的AEC之间潜在的信号事件。在即将到来的资金阶段，我们将(I)从分子和功能上表征由不同的基因表达谱定义的MSC簇，这对肺器官模型中的肺泡化和体内病毒感染模型中的肺泡修复都是高度支持的。我们将确定例如SPARC、SERPINF1、IGF1和CSF1/2在我们的RNA-SEQ分析中是否在受损的肺MSC中上调，或者甚至可能替代MSC上清液。为了进一步研究候选分子，我们将：(Ii)通过体内肺损伤模型的功能获得和丧失方法来确定MSC在IV感染和/或内毒素攻击期间诱导的TLR反应的功能相关性；(Iii)在人类体外模型中，应用GCP产生的临床级MSC验证我们的发现，应用疾病特异性预适应、基因转移和基因组编辑等策略，为人类MSC赋予明确的遗传程序和有益因素，最终将其用于首例人体试验。

Mesenchymal stem cells (MSC) have promising therapeutic potential in different forms of acute lung injury (ALI), and are now in first clinical trials. However, the molecular mechanisms underlying injury- or pathogen-related beneficial effects within defined niches of the affected lung are mostly unknown. Likewise, the mechanisms by which therapeutically applied MSC of different sources, such as bone marrow or adipose tissue, and lung-resident MSC (rMSC) sense injury during viral lung infections have not been comprehensively examined. This project focuses on the functional heterogeneity of MSC and characterizes the molecular interactions between virus-infected lung cells and different subsets of MSC with dedicated anti-viral and organ repair capacity. In the first funding period, we used bulk- and single cell (sc)RNA-Seq analyses combined with advanced lung organoid modelling and in vivo infection models to demonstrate that bone marrow-derived MSC (BM-MSC), white adipose tissue-derived MSCs (WAT-MSC) and rMSC represent heterogeneous populations that differ in single cell transcriptional profiles, exerting distinct roles in anti-viral host defense, tissue protection, and lung tissue regeneration (re-alveolarization). MSC of different origin could be primed in an injury-specific way to effectively support (re)generation of the alveolar compartment, e.g. by IV-specific preconditioning and by TLR3 ligands ex vivo, and in IV- and LPS-injury models in vivo. Regarding the potent antiviral and tissue-protective effects of MSC, we identified a type I IFN-dependent cross-talk as the underlying signalling event between BM-MSC and the IV-infected AEC, both in in vitro models and after intrapulmonary delivery of BM-MSC into IV-infected WT or IFNAR-/- mice in vivo.In the upcoming funding period, we will (i) molecularly and functionally characterize MSC clusters defined by distinct gene expression profiles, which are highly supportive for (re)alveolarization in lung organoid models and for alveolar repair in in vivo viral infection models. We will determine whether e.g. SPARC, SERPINF1, IGF1 and CSF1/2 identified to be upregulated in injured lung MSC in our RNA-seq analyses account - among others - for the regenerative potential of MSC, or might even substitute for MSC supernatants. To investigate further candidate molecules; we will (ii) define the functional relevance of TLR-responses induced in MSC during IV infection and/or LPS challenge using gain- and loss-of-function approaches in vivo in lung injury models; and (iii) validate our findings in human GCP-produced clinical-grade MSC in human ex vivo models, applying strategies such as disease-specific pre-conditioning, gene transfer, and genome editing to endow human MSC with defined genetic programs and beneficial factors to ultimately use them in first-in-human trials.