Quasi-spatial single-cell transcriptomics based on physical tissue properties defines early aging-associated niche in liver

Redefining the Early-Aging Microenvironment in the Liver: Quasi-spatial Single-cell Transcriptomics Reveals Fibrotic Niche Formation and Cellular Heterogeneity

Background and Research Motivation

Aging is an inevitable process in life, with one of its most significant features being the accumulation of senescent cells within tissues and organs. These cells are often induced by tissue injury and become increasingly difficult to eliminate as immune surveillance declines with age. The presence of senescent cells and their secretion of inflammation-related molecules (i.e., the senescence-associated secretory phenotype, SASP) have complex effects on the tissue microenvironment, potentially participating in physiological repair as well as inducing chronic inflammation and damaging tissue homeostasis. However, current technologies still face numerous challenges in resolving the exact distribution, phenotypic diversity, and microenvironmental impacts of these cells within tissues. This is especially true in major organs prone to fibrosis such as the liver, where the morphology, dynamic progression, and intricate spatial interactions of the senescent-cell-associated niche remain poorly understood.

In recent years, advances in single-cell omics have allowed us to dissect complex tissues at the cellular level. However, because of technical limitations and differences in cell type abundance, mainstream single-cell and spatial omics methods often overlook the cellular composition of fibrotic niches—these cells are typically ECM (extracellular matrix)-rich and difficult to isolate with standard dissociation protocols. Therefore, developing a method to enrich, localize, and perform multi-omics profiling of aging-associated fibrotic niches has become a crucial challenge for revealing mechanisms of liver aging and for developing anti-aging interventions.

Source of the Paper and Author Background

The article was published in the internationally recognized journal Nature Aging, Volume 5, May 2025, pages 929-949. The leading authors include Kwon Yong Tak, Juyeon Kim, Myungsun Park, Wooseok Kim, Seoyeong Lee, among others, from KAIST, KRIBB, and several other research institutes in South Korea. Correspondence can be addressed to kimchuna@kribb.re.kr and jp24@kaist.ac.kr. These institutions have strong foundations and innovative capacity in the field of biomedical sciences.

Research Design and Innovative Methods (a)

Research Subjects and Grouping

This study focuses on the fibrotic niche in naturally aged mouse liver, using male mice aged 5-6 months (young group) and 22-24 months (old group) as models. To reveal similarities and differences in the cellular microenvironment of liver tissue at different ages, the authors set up conventional control groups as well as fibrotic niche enrichment sequencing (fini-seq) groups, each subdivided into young and old subgroups. In total, 18 samples were used, with 3-⁴⁄₅-6 mice in the young/old groups, respectively.

Procedural Workflow and Technical Innovation

1. Innovative Niche Enrichment Workflow
   The research team first used Sirius-red staining and collagen immunofluorescence to anatomically localize fibrotic regions in mouse liver sections, confirming that with age, ECM accumulates and collagen is deposited notably around large hepatic vessels.
   To selectively enrich niche cells, the team developed a “double-digestion” separation process based on the physical property that ECM-rich regions are resistant to enzymatic digestion. The residual tissue blocks after initial dissociation were subjected to secondary enzymatic digestion and cultured in vitro, resulting in cells with diverse morphologies, strong expansion capabilities, and greater, denser ECM secretion. Atomic force microscopy further showed these ECMs have greater stiffness, and proteomics revealed higher expression of multiple ECM proteins and the inflammatory chemokine CXCL12.
   This technique, named fibrotic niche enrichment sequencing (fini-seq), provides a crucial enrichment strategy for subsequent single-cell RNA sequencing (scRNA-seq) and single-nuclei ATAC sequencing (snATAC-seq).

2. Single-cell/single-nucleus Omics and Data Analysis
   Cells obtained through fini-seq were subjected to scRNA-seq and snATAC-seq, with samples from a standard single-digestion serving as controls to quantify enrichment of rare, niche-related cells. In total, 84,351 cells (RNA) and 22,377 nuclei (ATAC) were analyzed.
   Data analysis was performed using harmony for batch correction, Leiden for clustering, and known lineage markers for cell annotation. To identify and exclude dissociation-induced batch effects and data bias, the team used Non-negative Matrix Factorization (NMF) to identify and filter high dissociation-bias genes. For spatial analysis, the study integrated RNA in situ hybridization (RNAscope), spatial transcriptomics (Visium and Stereo-seq), immunofluorescence/flow cytometry, and other approaches for multi-dimensional, full-space systematic analysis.

3. Novelty and Proprietary Techniques

   • Physical-property-based niche cell enrichment overcomes the bottleneck of isolating ECM-rich niche cells with conventional single-cell omics.
   • fini-seq multi-omics integration, systematically reveals phenotypic, transcriptomic, and epigenetic heterogeneity in the microenvironment.
   • Spatial omics & in situ hybridization accurately map niche cell distribution in specific liver anatomical regions (portal venous area).

Major Findings and Detailed Results (b)

1. Aging Leads to Remodeling of the Cellular Lineage in Liver Fibrotic Niches

The enrichment group better represented age-related niche microenvironments than controls, with obvious enrichment of fibrotic cells, endothelial cells (ECs), immune cells, etc., especially in livers from aged mice. fini-seq samples showed stronger ECM production, forming denser and stiffer matrices. Proteomics revealed a significant increase in collagen XIV (Col14a1), whereas other common collagens like Col1 and Col3 showed no significant change.

2. Discovery of Highly Heterogeneous, Aging-associated Cellular Subpopulations

• Endothelial Cells (ECs):
  In the aged niche regions enriched by fini-seq, canonical liver-specific LSEC (liver sinusoidal endothelial cells) were greatly reduced, while “fibrotic ECs” subgroups emerged in large numbers. These ECs lost their liver zonation transcriptomic features and instead resembled ECs from intestine/heart. Functionally, these ECs highly expressed chemotactic, inflammatory, and senescence-related genes (e.g., CXCL10, IL6, CDKN1A, CDKN2A) and showed loss of endocytic and clearance function, likely exacerbating chronic inflammation and pathological remodeling in the liver.
  Among the four fibrotic EC subtypes, “ec_sema3g”—marked by high Sema3g expression—accounted for more than half, exhibiting active Notch, P53, MAPK signaling, and typical “senescence” molecular features. Spatial in situ hybridization confirmed their unique aggregation around portal veins, with the proportion co-marked by CDKN1A increasing with age.

• Vascular Smooth Muscle Cells (vSMCs):
  Enrichment revealed vSMC_il6high cells characterized by high IL6 expression, increasing 2.2-fold in aged niches. These cells exhibited upregulation of inflammation, p53, immune regulation, and angiogenesis pathways, as well as prominent activity of the senescence gene set (SenMayo+).

• Fibroblasts and Hepatic Stellate Cells (HSCs):
  Detailed subtyping uncovered multiple fibroblast subpopulations. Particularly, fb_wif1 and fb_smoc1 immune-interacting fibroblasts were highly enriched in niche areas. fb_smoc1 participated in both ECM synthesis, PI3K/AKT activity, and also strongly associated with senescence. Spatial staining demonstrated that they co-localize with fibrotic ECs in the portal vein region.

3. Immune Environment Remodeling—T Cell Recruitment and Exhaustion

Eighty percent of the niche area comprised immune cells, with T cells constituting more than half. Further subtyping showed that, with aging, the exhausted PD-1^high subgroup of CD8+ tissue resident memory T cells (CD8+ TRM) markedly increased. Cell interaction analyses and flow cytometry confirmed the niche’s high affinity for, and tendency to exhaust, T cells—hypothesized to be linked to high PD-L1 expression in fibrotic ECs, leading directly to immune tolerance and escape of senescent cells from clearance.

4. Epigenetic Mechanisms and Spatial Heterogeneity

fini-ATAC-seq found that fibrotic ECs’ open chromatin regions were strongly enriched for key regulatory sites such as NF-κB and HIF1a, indicating that hypoxic stress and chronic inflammation signaling may be crucial drivers for niche formation and cell reprogramming. Fibrotic ECs also acquired reprogramming features related to OSKM (Oct4, Sox2, Klf4, c-Myc). Spatial omics revealed that niche cells are patchily and mosaicly distributed around the portal vein, with clear spatial heterogeneity—fb_wif1 dominating early/acute response, fb_smoc1 dominating chronic remodeling.

Study Conclusions and Academic Value (c-d)

This study, for the first time, proposes and applies a fini-seq technological route combining physical property-based niche cell enrichment with multi-omics analysis, systematically revealing the dynamic evolution, multi-lineage remodeling, and molecular drives of the early-aging microenvironment in the liver. The main conclusions and significance are as follows:

1. For the first time, maps the full panorama and spatial distribution of hepatic fibrotic niches in natural aging, providing a theoretical basis for understanding overall liver aging and precise intervention.
2. Identifies new cellular subtypes such as fibrotic EC and fb_smoc1, filling the gap missed by conventional approaches for these important, “hard-to-isolate” rare cells, enabling in-depth functional mining.
3. Elucidates new mechanisms by which hypoxia, chronic inflammation, and cell-cell interaction (e.g., PD-L1/PD-1 axis) drive niche formation, thus providing scientific rationale for anti-aging and anti-fibrosis therapeutic targets.
4. Proposes and validates a multistage functional differentiation model for immune-interacting fibroblasts, refining the temporal window and contextual scenarios for immune recruitment and matrix remodeling in niche progression and disease development.
5. Spatial omics innovatively reveals that niche structures are distributed as heterogeneous patches, providing new perspectives for understanding the origin, spread, and progression routes of tissue abnormalities.
6. fini-seq, as a technological platform, holds generalizable potential for application to other organs and diseases (e.g., early-stage fibrosis, cirrhosis, solid tumors, etc.).

Study Limitations and Future Prospects

The authors noted that fini-seq’s sensitivity and quantitative accuracy for certain cell types (e.g., hepatocytes) is still limited, especially as large, fragile cells (like hepatocytes) may be lost during double digestion. Future approaches could be supplemented by single-nucleus RNA-seq and other strategies. Furthermore, the “resistant-to-digestion” property targeted by physical enrichment may also be influenced by anatomical structure and tissue rigidity, requiring more nuanced biophysical characterization.

Summary

Guided by fini-seq technology and integrating multi-omics, multi-modal, and spatial approaches, this study establishes a paradigm for full-cellular, full-spatial investigation of liver aging niches. Not only does it break through the bottleneck of conventional single-cell omics, but it also unravels the complex interplay between early pathological microenvironments and immune regulation. In the future, related techniques may provide systematic solutions for aging mechanisms, early warning, and targeted intervention in liver and other organs, ushering in new advances for aging research and translational medicine.