概要: Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining tissue integrity. We have previously shown that mouse skin connective tissue, the dermis, is comprised of functionally distinct fibroblast lineages. However, the extent of fibroblast heterogeneity in human skin is unknown. Here, using a combination of spatial transcriptional profiling of human and mouse dermis and single cell transcriptional profiling of human dermal fibroblasts, we show that there are at least four distinct fibroblast populations in adult human skin. We define markers permitting prospective isolation of these cells and show that although marker expression is rapidly lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signalling, T cell communication and the ability to support human epidermal reconstitution in organotypic culture. Furthermore, while some fibroblast subpopulations are spatially segregated, others are not. These findings have profound implications for normal wound healing and diseases characterized by excessive fibrosis, and suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications.
项目整体设计: Spatial RNA sequencing of human papillary versus reticular dermis for 3 individuals, and single cell RNA sequencing of dermal fibroblasts for a single individual
Spatial RNA sequencing was performed using fresh human skin samples (from three separate individuals). Skin samples were incubated with Dispase II (Stemcell Technologies) for 1h at 37℃ permitting separation of the epidermis. The dermis was separated into papillary (upper 100 μm) reticular (200-500 μm) dermis by microdissection under a dissecting microscope. For single cell RNA sequencing, single cells were isolated by flow cytometry and sorted into individual wells of a 96 well plate containing 2μl lysis buffer (0.2% (vol/vol) Triton X-100 and 2 U/ul recombinant RNase inhibitor (Clontech).
提取方案:
Separated papillary and reticular dermis samples were transferred to lysis buffer containing 2-mercaptoethanol (PureLink RNA micro scale kit, Invitrogen) and homogenized for 2 minutes using a mechanical homogenizer (Polytron, Kinematica). Subsequent RNA extraction was performed using the PureLink micro kit according to the manufacturer' instructions.
建库方案:
Library preparation was performed with SmartSeq2 followed by the Nextera XT protocol (Illumina). Sequencing was performed on the Illumina HiSeq 2000 or HiSeq 2500 with TruSeq SBS v3 chemistry.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2000; Illumina HiSeq 2500
链特异性:
Unspecific
样本
基本信息:
样本描述:
生物条件:
实验变量:
方案:
测序信息:
质量评估:
数据来源
GEN样本编号
GEN数据集编号
系列编号
项目编号
样本编号
样本名称
生物样本编号
样本访问号
实验访问号
释放时间
提交时间
最后更新时间
物种
种族
族裔
年龄
年龄单位
性别
来源名称
组织
细胞类型
细胞亚型
细胞系
疾病
疾病状态
发育阶段
突变/变异
表型
Condition Detail
生长方案
处理方案
提取方案
建库方案
分子类型
库的片段类型
链特异性
库的链类型
加标（Spike-In）
测序方法
测序平台
测序仪型号
细胞数
测序片段数
碱基数
平均测序片段长度_1
平均测序片段长度_2
唯一比对率
多重比对率
覆盖度
文章
Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.
The Journal of investigative dermatology . 2018-01-31 [PMID:
29391249]
