Single Cell RNA Sequencing of Adult Mouse Intervertebral Disc Cells

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-beta and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich extracellular matrix. Cells were isolated from intervertebral discs of P30 shh-cre:R26R-tdTomato mice. TdTomato+ cells were enriched using fluorescence activated cell sorting and analyzed using single cell RNA-sequencing. Three replicate sequencing batches were performed, each comprising pooled cells from litter matched animals. Libraries were generated using the 10x Genomics Chromium Single Cell 3' Reagent kit v3 per manufacturer's instructions. Libraries were uniquely indexed using the Chromium dual Index Kit, and paired-end sequencing was performed at a targeted depth for each library of 20,000 mean reads per cell (NovaSeq 6000; Illumina, San Diego, USA). Nucleus pulposus cells were identified within the total sequenced cell population by expression of established markers Krt8, Krt18, Krt19 and T.椎间盘退变（Intervertebral disc degeneration）是慢性下腰痛的首要诱因。旨在通过再生中央髓核（nucleus pulposus）治疗椎间盘退变的细胞治疗策略具备显著应用前景，但仍存在诸多关键挑战。其中一大难题在于治疗性细胞无法有效模拟天然髓核细胞的功能：这类细胞在骨骼细胞类群中独具特性，其起源为胚胎脊索（notochord）。 本研究采用单细胞RNA测序（single cell RNA sequencing）技术，解析了出生后小鼠椎间盘内脊索来源的髓核细胞的新兴异质性。具体而言，我们证实了早、晚两个发育阶段的髓核细胞的存在，分别对应脊索祖细胞与成熟髓核细胞。晚期髓核细胞的细胞外基质相关基因（包括聚集蛋白聚糖（aggrecan）、Ⅱ型胶原及Ⅵ型胶原）的表达水平显著升高，同时转化生长因子-β（TGF-β）与磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-Akt）信号通路的活性显著增强。 此外，我们鉴定出Cd9可作为晚期髓核细胞的新型表面标志物，并证实这类细胞定位于髓核外周区域，其数量随出生后日龄增加而增多，且与新生的糖胺聚糖富集型细胞外基质共定位。 实验材料取自P30龄shh-cre:R26R-tdTomato小鼠的椎间盘。通过荧光激活细胞分选（fluorescence activated cell sorting）富集tdTomato阳性细胞，随后采用单细胞RNA测序开展分析。本研究共设置3次重复测序批次，每批次均包含同窝配对动物的混合细胞。文库构建严格按照厂商操作指南，采用10x Genomics Chromium单细胞3'端试剂试剂盒v3（10x Genomics Chromium Single Cell 3' Reagent kit v3）完成。采用Chromium双索引试剂盒为文库添加唯一索引标记，随后在NovaSeq 6000测序平台（Illumina，美国加利福尼亚州圣地亚哥）上进行双端测序，每例文库的目标测序深度为平均每个细胞20000条读段。通过已确立的标志物Krt8、Krt18、Krt19及T的表达特征，在总测序细胞群中鉴定出髓核细胞。