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）中具有很好的治疗潜力，目前正在进行首次临床试验。然而，在受影响的肺的确定的小生境内的损伤或病原体相关的有益作用的分子机制大多是未知的。同样，治疗应用的不同来源的MSC，如骨髓或脂肪组织，和肺驻留MSC（rMSC）在病毒性肺感染的感觉损伤的机制还没有得到全面检查。该项目的重点是MSC的功能异质性，并表征病毒感染的肺细胞与具有专用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，我们使用了大量和单细胞（sc）RNA-Seq分析，结合先进的肺类器官建模和体内感染模型，以证明骨髓来源的MSC（BM-MSC），白色脂肪组织来源的MSC（WAT-MSC）和rMSC代表了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（再肺泡化）。不同来源的MSC可以以损伤特异性的方式致敏，以有效地支持肺泡隔室的（再）生成，例如通过IV特异性预处理和通过TLR 3配体离体，以及在体内IV和LPS损伤模型中。关于MSC的有效抗病毒和组织保护作用，我们鉴定了I型IFN依赖性串扰作为BM-MSC和IV感染的AEC之间的潜在信号传导事件，无论是在体外模型中还是在体内将BM-MSC肺内递送到IV感染的WT或IFNAR-/-小鼠中之后。我们将（i）从分子和功能上表征由不同基因表达谱定义的MSC簇，其高度支持肺类器官模型中的（再）肺泡化和体内病毒感染模型中的肺泡修复。我们将确定例如在我们的RNA-seq分析中鉴定为在损伤的肺MSC中上调的EGFR、SERPINF 1、IGF 1和CSF 1/2是否解释了MSC的再生潜力，或者甚至可能替代MSC上清液。为了研究进一步的候选分子，我们将（ii）在肺损伤模型中使用体内功能获得和丧失方法，确定在IV感染和/或LPS攻击期间MSC中诱导的TLR应答的功能相关性;和（iii）在人离体模型中验证我们在人GCP产生的临床级MSC中的发现，应用诸如疾病特异性预处理，基因转移，和基因组编辑，赋予人类MSC定义的遗传程序和有益因子，最终将其用于首次人体试验。

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.