Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors [scRNA-seq]

The early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors. scRNA-seq for non limb fibroblasts undergoing reprogramming into limb progenitors, with controls

早期肢芽（early limb bud）由源自侧板中胚层（lateral plate mesoderm, LPM）的间充质祖细胞（mesenchymal progenitors，即肢芽祖细胞limb progenitors）构成，此类祖细胞可产生成熟肢芽的绝大多数组织。侧板中胚层还可发育为躯干、体侧与颈部的中胚层组分。然而，在其他轴位生成的间充质细胞，即便处于调控肢芽发育的信号微环境中，既无法分化为肢芽中所见的多种细胞类型，也不能被诱导形成具有模式化的附肢样结构。本研究借助直接重编程技术，发现一组通常在早期肢芽中表达的因子（Prdm16、Zbtb16与Lin28），可赋予非肢源成纤维细胞肢芽祖细胞样特性。经这些因子重编程的细胞，其基因表达谱与内源性肢芽祖细胞相似，且可分化为相同的细胞类型。额外补充Lin41可增强重编程细胞的增殖能力，同时抑制其分化。上述结果表明，这四种关键因子可能在内源性肢芽祖细胞的特化过程中发挥核心作用。本研究针对重编程为肢芽祖细胞过程中的非肢源成纤维细胞开展了单细胞RNA测序（scRNA-seq）分析，并设置了对照样本。