Garibyan Mher, Hoffman Tyler, Makaske Thijs, Do Stephanie, March Alexander R, Cho Nathan, Pedroncelli Nico, Lima Ricardo Espinosa, Soto Jennifer, Jackson Brooke, Khademhosseini Ali, Li Song, McCain Megan, Morsut Leonardo
bioRxiv. 2023 May 20:2023.05.19.541497. doi: 10.1101/2023.05.19.541497.
Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.
合成Notch（synNotch）受体是模块化的合成组件，通过基因工程改造进入哺乳动物细胞，以检测相邻细胞呈现的信号，并通过激活规定的转录程序做出反应。迄今为止，synNotch已被用于对治疗性细胞进行编程以及在多细胞系统中塑造形态发生模式。然而，对于需要空间精度的应用，如组织工程，细胞呈现的配体通用性有限。为了解决这个问题，我们开发了一套材料来激活synNotch受体，并作为生成用户定义的材料与细胞信号通路的通用平台。首先，我们证明了synNotch配体，如绿色荧光蛋白（GFP），可以通过对成纤维细胞产生的纤连蛋白进行基因工程，与细胞生成的细胞外基质（ECM）蛋白偶联。然后，我们使用酶促或点击化学方法将synNotch配体共价连接到明胶聚合物上，以激活在水凝胶上或水凝胶内生长的细胞中的synNotch受体。为了在细胞单层中实现对synNotch激活的微观控制，我们将synNotch配体微接触印刷到表面上。我们还通过设计具有两种不同合成途径的细胞，并将它们培养在微流控图案化有两种synNotch配体的表面上，对包含多达三种不同表型细胞的组织进行图案化。我们通过将成纤维细胞共转分化为骨骼肌或内皮细胞前体，以用户定义的空间模式构建具有规定血管网络的肌肉组织，展示了这项技术。总的来说，这一套方法扩展了synNotch工具包，并为在哺乳动物多细胞系统中空间控制细胞表型提供了新途径，在发育生物学、合成形态发生、人体组织建模和再生医学等方面有许多广泛应用。
