Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner. Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner

Fibrotic scar tissue formation is conserved throughout the central nervous system in humans and mice, and impairs tissue regeneration and functional recovery. However, the origin of scar-forming stromal fibroblasts is controversial. Here, we show that stromal fibroblasts found after spinal cord injury derive from two populations of perivascular cells that are anatomically and transcriptionally defined as pericytes and perivascular fibroblasts. We identify two distinct perivascular cell populations, which activate and transcriptionally converge on the generation of stromal myofibroblasts after injury. Our results suggest potential targets to improve tissue regeneration and functional recovery after central nervous system injuries. Overall design: We crossed GLAST-CreERT2;R26R-tdTomato mice to the Pdgfrb-eGFP reporter line, which drives EGFP expression in all PDGFRb+ perivascular cells (pericytes, vSMCs and fibroblasts). We isolated GLAST-expressing perivascular cells from the uninjured spinal cord and 5 days after spinal cord injury by fluorescence activated cell sorting (FACS) based on tdTomato and EGFP expression. To define the molecular identity and compare GLAST-expressing perivascular cells (tdTomato+EGFP+) to other perivascular cells, we also sorted dTomato-EGFP+ cells. Sorted cells were subjected single cell sequencing using the SmartSeq 2 protocol.

纤维化瘢痕组织形成在人类与小鼠的中枢神经系统中均保守存在，可损害组织再生与功能恢复。然而，瘢痕形成相关基质成纤维细胞（stromal fibroblasts）的来源仍存在争议。本研究证实，脊髓损伤后出现的基质成纤维细胞源自两类经解剖学与转录组学特征定义的血管周细胞：分别为周细胞（pericytes）与血管周成纤维细胞（perivascular fibroblasts）。我们鉴定出两类截然不同的血管周细胞群体，它们在损伤后被激活，并在转录层面共同导向基质肌成纤维细胞的生成。本研究结果为改善中枢神经系统损伤后的组织再生与功能恢复提供了潜在干预靶点。

整体实验设计：我们将GLAST-CreERT2;R26R-tdTomato小鼠与Pdgfrb-eGFP报告基因品系进行杂交，该报告品系可在所有PDGFRβ+血管周细胞（包括周细胞、血管平滑肌细胞（vascular smooth muscle cells, vSMCs）与成纤维细胞）中驱动EGFP的表达。我们基于tdTomato与EGFP的表达特征，通过荧光激活细胞分选（fluorescence activated cell sorting, FACS）从未损伤脊髓以及脊髓损伤后5天的脊髓组织中分离出表达GLAST的血管周细胞。为明确分子特征并将表达GLAST的血管周细胞（tdTomato+EGFP+）与其他血管周细胞进行比较，我们同时分选了dTomato-EGFP+细胞群。分选获得的细胞采用SmartSeq 2技术开展单细胞测序。