# 2.2.7 研究 | 2021-多个组织的成纤维细胞图谱

## 速览

* 作者：Genentech团队

  > 基因泰克公司（英语：Genentech Inc.），全名基因工程科技公司（英语：Genetic Engineering Technology），是一家生物科技公司，由创投人罗伯特·史旺森（Robert A. Swanson）与生物化学家赫伯特·博耶（Herbert Boyer）博士于1976年成立，这件事也被视为是生物科技产业的起点。罗氏药厂于2009年3月26日以大约468亿美元完成了基因泰克的收购，并完全拥有此公司。 截至2019年2月为止，基因泰克有超过13,697名员工。
* 链接：<https://www.nature.com/articles/s41586-021-03549-5>
* 发表在：Nature
* 日期：12 May 2021
* 还发了一个在线工具：<https://www.fibroxplorer.com/> **FibroXplorer** is an interactive data portal that allows users to examine gene expression at the single-cell level in \~230,000 mouse fibroblasts from 17 tissues, 50 datasets and 12 disease states

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-014620.png)

## 摘要

* 很多研究都表明：成纤维细胞在单个组织中存在异质性，但是在不同健康和患病器官的组织中并没有证明
* 文章整合了2个物种的**17个组织**，涵盖**11种疾病状态**，50个数据集，得到了**230,000**个细胞scRNA数据
* 比较了人和小鼠的成纤维细胞图谱发现，成纤维细胞的转录状态是保守的
* 文章提出假设，成纤维细胞的异质性是由于稳态组织和疾病组织之间的波动差异导致的

## 实验设计

重点还是落在小鼠

* Mouse tissue digestion and stromal cell isolation or identification by FACS
* qPCR, RNA extraction and cDNA synthesis
* Mouse scRNA-seq and cell hashing
* Tissue processing for histology
* RNAscope in situ hybridization
* DSS-induced colitis
* Tumour inoculation
* Mouse bulk RNA-seq analysis
* Mouse bulk ATAC–seq analysis
* Human tissue digestion and stromal cell isolation
* Open chromatin regions (OCRs)  identification
* Motif enrichment analysis
* ATAC–seq and RNA-seq concordance
* Mouse scRNA-seq meta-analysis
* Human scRNA-seq meta-analysis
* Pseudo-bulk analytical strategy
* Projection of human gene sets onto mouse perturbed-state atlas

## 首先看稳态小鼠组织的成纤维细胞

先做了bulk RNA和ATAC-Seq：

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-022931.png)

单细胞图谱构建思路（图a）：

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-032730.png)

得到10个细胞群，marker分别是：Pi16+, Col15a1+, Ccl19+, Coch+, Comp+, Cxcl12+, Fbln1+, Bmp4+, Npnt+ and Hhip+

> Ccl19+ fibroblastic reticular cells (FRCs), Coch+ red pulp fibroblasts, Cxcl12+ mesenchymal stromal cells and osteolineage cells, Fbln1+ and Bmp4+ intestinal fibroblasts, Comp+ fibroblasts, Npnt+ alveolar fibroblasts Hhip+ peribronchial fibroblasts

几乎所有的组织都有Pi16+ and Col15a1+ clusters

> DEGs in the **Pi16**+ cluster (Pi16, Dpp4 and Ly6c1) suggested an identity similar to adventitial stromal cells **Col15a1**+ cluster exhibited an association with the basement membrane, evidenced by expression of Col4a1, Hspg2 and Col15a1

图d是：Slingshot lineage inference identified trajectories that emerged **from the Pi16+** cluster, passed **through the Col15a1+** cluster, and **ended at specialized** clusters

scRNA和bulkRNA的一致性还是很高的，说明scRNA在提高精度的同时，没有引入其他的技术误差：

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-022527.png)

结论是：在稳态小鼠组织中，Pi16+ and Col15a1+的成纤维细胞亚群普遍存在，当然还有一些特异的亚群，它们之间可能存在进化的关联

## 受伤或患病小鼠紊乱组织的成纤维细胞

组织的紊乱包括：感染、损伤、癌症、纤维化、代谢变化和关节炎

探索了**17 publicly** available scRNA-seq datasets across **13 tissues** to generate a perturbed-state fibroblast atlas (*n* = **99,596** cells

也是得到10个cluster：Pi16+, Col15a1+, Ccl19+, Cxcl12+, Comp+, Npnt+, Hhip+, Adamdec1+, Cxcl5+ and Lrrc15+

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-030407.png)

发现： Cxcl12+, Ccl19+, Comp+, Npnt+, and Hhip+ 这几个cluster在基因表达和组织分布和稳态组织的结果很相似； Cxcl5+, Adamdec1+ and Lrrc15+ 这几个是紊乱组织特有的，激活了稳态中没有的细胞状态

## 人的成纤维细胞图谱

假设：小鼠在稳定和紊乱状态的成纤维细胞亚型方面可能与人类表现出一些相似性

使用3个胰腺癌病人的癌和癌旁样本：n = 21,262 cells （图a-c），分成了CAF （c3和c8）和normal fibroblasts

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-033633.png)

在c8的20个高表达基因中，有12个也在稳态中高表达：Dpt+Pi16+ (DPT, IGFBP5, IGFBP6, C3, APOD) or Dpt+Col15a1+ (CXCL12, SMOC2, C7, FBLN5, MFAP4, LUM, FMO2)

接着，拿C3 PDAC CAFs + colon fibroblasts + lung fibroblasts from individuals with non-small cell lung cancer (NSCLC), IPF or COVID-19 (n = 10,355 cells)进行整合，得到6个cluster

> NPNT+ alveolar fibroblasts derived from individuals with lung disease
>
> ADAMDEC1+ and CCL19+31 clusters primarily from colitis samples
>
> PI16+ cluster, in which the human universal signature was most enriched
>
> two myofibroblast clusters defined by LRRC15+ and COL3A1+ expression:
>
> * LRRC15+ myofibroblasts were enriched in cells from individuals with pancreatic and lung cancer,
> * COL3A1+ cluster was enriched in cells from patients with COVID-19

进而验证了小鼠的图谱： LRRC15+ myofibroblasts and ADAMDEC1+ fibroblasts

另外还发现了COVID-19病人中存在一个独特的myofibroblast细胞群

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-13-033733.png)

最后留下疑问：It remains unclear **why two universal Dpt+ fibroblast subtypes exist**, though we speculate that this may represent a necessary division of labour within the lineage.