单细胞基因组学比较人诱导多能干细胞来源巨噬细胞与外周血来源巨噬细胞的异质性

doi:10.3969/j.issn.1674-8115.2024.12.001

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (12): 1477-1489.doi: 10.3969/j.issn.1674-8115.2024.12.001

• 创新团队成果专栏 •

单细胞基因组学比较人诱导多能干细胞来源巨噬细胞与外周血来源巨噬细胞的异质性

张羽桐(), 侯国俊, 沈南()

上海交通大学医学院附属仁济医院风湿科，上海风湿病学研究所，上海 200127

收稿日期:2024-04-02 接受日期:2024-05-16 出版日期:2024-12-28 发布日期:2024-12-28
通讯作者: 沈南 E-mail:zhangyutong135@126.com;nanshensibs@gmail.com
作者简介:张羽桐（1997—），女，硕士生；电子信箱：zhangyutong135@126.com。
基金资助:
国家自然科学基金(31930037);上海市高水平地方高校创新团队(SSMU-ZDCX20180100)

Comparison of human-induced pluripotent stem cell-derived macrophages with peripheral blood-derived macrophages using single-cell genomics

ZHANG Yutong(), HOU Guojun, SHEN Nan()

Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Rheumatology, Shanghai 200127, China

Received:2024-04-02 Accepted:2024-05-16 Online:2024-12-28 Published:2024-12-28
Contact: SHEN Nan E-mail:zhangyutong135@126.com;nanshensibs@gmail.com
Supported by:
National Natural Science Foundation of China(31930037);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

摘要/Abstract

摘要：

目的·探究人诱导多能干细胞（induced pluripotent stem cell，iPSC）来源的巨噬细胞（iPSC-derived macrophage，IPSDM）与人外周血来源的巨噬细胞（peripheral blood-derived macrophage，PBDM）的单细胞基因组学异质性。方法·利用无饲养层无血清方案体外诱导iPSC分化为IPSDM，通过流式细胞术及反转录实时荧光定量聚合酶链反应（real-time reverse transcription quantitative polymerase chain reaction，RT-qPCR）分别检测IPSDM表面白细胞分化抗原14（cluster of differentiation antigen 14，CD14）与单核-巨噬细胞标志基因的表达情况。随后对IPSDM进行单细胞测序，同时从基因表达综合数据库中下载单细胞测序数据集GSE126085作为PBDM参考数据集。通过R软件seurat包处理分析IPSDM与PBDM测序数据，利用singleR包注释PBDM。以PBDM的高变基因构建参考数据集，利用scmap包将IPSDM的高变基因投影于PBDM，根据高变基因的相似性推断IPSDM的细胞身份。随后参考细胞类型注释工具及相关研究完成对IPSDM的注释，并比较IPSDM和PBDM巨噬细胞标志基因的表达分布。最后，利用seurat包寻找IPSDM与PBDM的差异表达基因（differentially expressed gene，DEG），通过基因本体（Gene Ontology，GO）分析与京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析探究DEG潜在的生物学功能。结果·体外诱导分化29 d后得到悬浮的IPSDM，经流式细胞术及RT-qPCR检测证实约23.1%的IPSDM表达CD14，且与U-937细胞系相比，IPSDM表达更高水平的单核-巨噬细胞标志基因。PBDM数据集中的细胞全部被注释为巨噬细胞，随后构建scmap参考数据集，将IPSDM的高变基因映射于PBDM参考数据集后发现59.8%的IPSDM可以依据PBDM的高变基因索引被注释为巨噬细胞，而余下40.2%的IPSDM细胞无法匹配PBDM的高变基因索引。进一步人工注释IPSDM发现其由97.15%巨噬细胞、2.71%造血前体细胞样细胞和0.14%树突状细胞组成。对IPSDM和PBDM的巨噬细胞标志物表达情况进行对比，发现两者均高表达典型的巨噬细胞标志物CD68基因，IPSDM还高表达组织驻留巨噬细胞相关的标志基因。对2种来源的巨噬细胞的DEG进行GO分析，发现分子功能主要富集于泛素样蛋白连接酶结合，细胞组分主要定位于核斑点和核膜，生物学过程主要富集于翻译调控；而KEGG通路富集分析的结果提示IPSDM与PBDM的DEG可能与多种胞内病原体感染相关。结论·人类IPSDM与PBDM在单细胞转录谱系上存在一定相似性及异质性，在转录组层面IPSDM更具有组织驻留巨噬细胞的特征，IPSDM与PBDM的差异基因可能与胞内感染免疫相关。

关键词: 诱导多能干细胞, 细胞分化, 巨噬细胞, 单细胞测序

Abstract:

Objective ·To explore the heterogeneity in single-cell genomics between human-induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM) and human peripheral blood-derived macrophages (PBDM). Methods ·iPSCs were differentiated into IPSDMs in vitro using a feeder-free and serum-free protocol. The expression of cluster of differentiation antigen 14 (CD14) and monocyte-macrophage marker genes in IPSDMs was analyzed using flow cytometry and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Single-cell sequencing was then performed on IPSDMs. Simultaneously, the single-cell sequencing dataset GSE126085 was downloaded from the Gene Expression Omnibus database as a reference dataset for PBDMs. Sequencing data for both IPSDMs and PBDMs were processed and analyzed using the seurat package in R software, with PBDMs annotated using the singleR package. A reference dataset was constructed with highly variable genes from PBDMs, and the highly variable genes of IPSDMs were projected onto the PBDM dataset using the scmap package to infer IPSDMs cell identities based on variable gene similarity. IPSDMs were annotated using cell-type annotation tools and referenced against relevant studies. The expression distribution of macrophage marker genes was compared between IPSDMs and PBDMs. Differentially expressed genes (DEGs) between IPSDMs and PBDMs were identified using the seurat package, and their potential biological functions were explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Results ·Suspended IPSDMs were obtained after 29 d of in vitro differentiation. Flow cytometry and RT-qPCR confirmed that approximately 23.1% of IPSDMs expressed CD14, and IPSDMs exhibited higher expression of monocyte-macrophage marker genes compared to the U-937 cell line. All cells in the PBDM dataset were annotated as macrophages. After constructing a scmap reference dataset using PBDMs, 59.8% of IPSDMs were annotated as macrophages through mapping their highly variable genes to the PBDM dataset. The remaining 40.2% of IPSDM cells could not be matched to the variable genes of PBDMs. Further manual annotation of IPSDMs revealed a composition of 97.15% macrophages, 2.71% hematopoietic precursor cell-like cells, and 0.14% dendritic cells. When comparing the expression of macrophage markers, both IPSDMs and PBDMs highly expressed the classical macrophage marker CD68 gene, while IPSDMs exhibited higher expression of markers associated with tissue-resident macrophages. GO analysis of DEGs showed enrichment in the molecular functions such as ubiquitin-like protein ligase binding, cellular components such as the nuclear speck and nuclear envelope, and biological processes such as the regulation of translation. KEGG pathway enrichment indicated that the DEGs between IPSDMs and PBDMs might be related to various intracellular pathogen infections. Conclusion ·Human IPSDMs and PBDMs exhibit certain similarities and heterogeneity at the single-cell transcriptional level. Transcriptomic analysis indicates that IPSDMs display more characteristics of tissue-resident macrophages. The DEGs between IPSDMs and PBDMs are potentially associated with intracellular infection immunity.

Key words: induced pluripotent stem cell (iPSC), cell differentiation, macrophage, single-cell gene expression analysis

中图分类号:

R392.12

张羽桐, 侯国俊, 沈南. 单细胞基因组学比较人诱导多能干细胞来源巨噬细胞与外周血来源巨噬细胞的异质性[J]. 上海交通大学学报（医学版）, 2024, 44(12): 1477-1489.

ZHANG Yutong, HOU Guojun, SHEN Nan. Comparison of human-induced pluripotent stem cell-derived macrophages with peripheral blood-derived macrophages using single-cell genomics[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1477-1489.

图/表 7

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR.

Gene	Forward primer (5´→3´)	Reverse primer (5´→3´)
HLA-DRA	AGCTGTGGACAAAGCCAACCTG	CTCTCAGTTCCACAGGGCTGTT
CD68	CGAGCATCATTCTTTCACCAGCT	ATGAGAGGCAGCAAGATGGACC
CD86	CCATCAGCTTGTCTGTTTCATTCC	GCTGTAATCCAAGGAATGTGGTC
MRC1	AGCCAACACCAGCTCCTCAAGA	CAAAACGCTCGCGCATTGTCCA
RPL13A	CTCAAGGTGTTTGACGGCATCC	TACTTCCAGCCAACCTCGTGAG

图1 iPSC分化为IPSDM的过程及鉴定Note： A. Flowchart of iPSCs differentiation into IPSDMs. B. Morphology of iPSCs used for differentiation (×100). C. Morphology of iPSCs on the 3rd day of differentiation into EBs (×40). D. Morphology of EBs on the 7th day of differentiation into monocytes (×100). E. Morphology of cells in the medium on the 21st day of EB differentiation into monocytes (×100). F. Flow cytometry analysis results of CD14 marker expression in IPSDMs. G. RT-qPCR results of HLA-DRA, CD68, CD86 and MRC1 gene expression levels in IPSDMs and U-937 cells.

Fig 1 Process and identification of iPSCs differentiation into IPSDMs

图2 GSE126085中PBDM样本细胞聚类过程及注释后的细胞分布特征图Note： A. The cluster tree of the PBDM sample. B. Distribution of the PBDM sample after annotation. C. Feature distribution plots of marker gene expression in the PBDM sample.

Fig 2 Cell clustering process and distribution feature plot of the PBDM samples after annotation in GSE126085

图3 IPSDM样本的单细胞基因组学图景Note：A. Distribution of PBDM index genes. B. The cell flow for projecting the IPSDM dataset onto the PBDM dataset. C. The cluster tree of IPSDM and PBDM samples. D. Distribution of IPSDM and PBDM samples after annotation. E. Bubble plots showing the expression levels of cell marker genes in different cell types. F. Expression distribution of tissue-resident macrophage-specific marker genes of IPSDM and PBDM samples.

Fig 3 Single-cell RNA genomics landscape of IPSDM samples

表2 IPSDM与PBDM中细胞类型、对应的标志基因和细胞数

Tab 2 Cell types, the corresponding marker genes and cell numbers in IPSDM and PBDM

Cell type	Marker gene	IPSDM/n(%)	PBDM/n(%)
Total	‒	9 745	1 796
Macrophage	CD14, CD68, S100A11, and CD163	9 467 (97.15)	1 794 (99.89)
HPC-like cell	HES1, SOX4, CDK6, RUNX1T1, and MECOM	264 (2.71)	2 (0.11)
Dendritic cell	ITGAM and ITGAX	14 (0.14)	0 (0)

表3 PBDM与IPSDM中的巨噬细胞之间上调与下调变化倍数排名前10的DEG

Tab 3 Top 10 DEGs with up- and down-regulated fold changes between the macrophages from PBDM and IPSDM

Gene	Average log₂FC	pct.1	pct.2	P value	Adjusted P value
MAP4K3-DT	10.399	0.497	0	8.45×10^-305	1.66×10^-301
PNRC2	10.246	0.498	0	2.28×10^-305	4.50×10^-302
TENT2	10.154	0.488	0	8.07×10^-297	1.59×10^-293
SELENON	9.884	0.402	0	2.94×10^-226	5.79×10^-223
RAB7B	9.847	0.341	0	2.27×10^-181	4.47×10^-178
FAAP20	9.780	0.396	0	2.85×10^-221	5.61×10^-218
RAB29	9.757	0.405	0	1.58×10^-228	3.11×10^-225
UTP11	9.677	0.370	0	6.19×10^-202	1.22×10^-198
TMEM35B	9.662	0.377	0	2.08×10^-207	4.11×10^-204
ELOA	9.568	0.376	0	7.27×10^-207	1.43×10^-203
EMILIN1	-3.939	0.049	0.278	7.48×10^-244	1.47×10^-240
CCR5	-2.685	0.147	0.319	3.16×10^-102	6.22×10^-99
PHLDA3	-2.645	0.160	0.430	7.26×10^-208	1.43×10^-204
PCM1	-2.429	0.578	0.719	5.15×10^-262	1.02×10^-258
VAMP3	-2.383	0.424	0.679	7.93×10^-295	1.56×10^-291
RRM2	-2.362	0.086	0.307	9.29×10^-164	1.83×10^-160
AGO1	-2.284	0.279	0.432	7.54×10^-97	1.49×10^-93
ITPKB	-2.238	0.166	0.279	1.49×10^-51	2.94×10^-48
SMC4	-2.161	0.235	0.337	6.41×10^-46	1.26×10^-42
MAPK14	-2.077	0.372	0.499	1.82×10^-102	3.58×10^-99

图4 IPSDM和PBDM中的巨噬细胞DEG的功能分析Note： A. GO enrichment analysis results of DEGs in the macrophages from the IPSDM sample vs the PBDM sample. B. KEGG pathway enrichment analysis results of DEGs in the macrophages from the IPSDM sample vs the PBDM sample.

Fig 4 Functional analysis of DEGs between the macrophages from PBDM and IPSDM

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单细胞基因组学比较人诱导多能干细胞来源巨噬细胞与外周血来源巨噬细胞的异质性

Comparison of human-induced pluripotent stem cell-derived macrophages with peripheral blood-derived macrophages using single-cell genomics

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