PDGFR Marks Distinct Mesenchymal and Pericyte Populations Within the Periosteum With Overlapping Cellular Features

Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine Orthopedics
periosteum PDGFRβ mesenchymal progenitor cells pericytes bone regeneration single-cell RNA sequencing

New Developments in Bone Regeneration: The Discovery of PDGFRβ-marked Distinct Mesenchymal and Pericyte Subpopulations in the Periosteum — Interpretation of “pdgfr marks distinct mesenchymal and pericyte populations within the periosteum with overlapping cellular features”

1. Research Background and Scientific Question

The periosteum, a thin vascularized outer layer of bone tissue, has long been regarded as a crucial cellular reservoir for bone growth and repair. In recent years, the diversity of mesenchymal stem/progenitor cells (MSCs) within the periosteum and their roles in bone regeneration have attracted widespread attention. Multiple studies have identified a series of surface markers for periosteal progenitor cells, including CD73, CD164, PDPN, PDGFRα (platelet-derived growth factor receptor alpha), leptin receptor, and nestin. However, the functional heterogeneity, cellular attribution, and regenerative efficacy associated with these markers remain unsettled.

Vascular-associated cells, especially microvascular pericytes, have also been demonstrated to possess multilineage differentiation potential and play important roles in bone repair. Related studies have shown that CXCR4+ pericytes derived from the periosteum exhibit stronger osteogenic capabilities than pericytes from other tissues, highlighting the critical importance of vascularization in bone regeneration. Existing literature also suggests that the proliferation and differentiation of periosteal pericytes contribute additional sources of osteoprogenitors. However, the phenotypic overlap, functional redundancy, marker differentiation, and respective regenerative roles of periosteal pericytes and MSCs have not been clearly elucidated for a long time.

PDGFRβ (platelet-derived growth factor receptor β) is a common surface marker for mesenchymal progenitor cells and pericytes, involved in regulating cell proliferation, migration, and survival. Previous studies have indicated that PDGFRβ-positive cells possess significant osteogenic potential crucial for fracture healing and bone homeostasis. Yet, within the periosteal tissue, how many cell types express PDGFRβ? Do these cells possess shared or unique molecular and functional characteristics? These are the scientific core questions this current study addresses.

2. Article Information and Author Team

This paper, titled “pdgfr marks distinct mesenchymal and pericyte populations within the periosteum with overlapping cellular features,” is an original basic research article. The main authors include Ziyi Wang, Qizhi Qin, Neelima Thottappillil, Mario Gomez Salazar, Masnsen Cherief, Mary Archer, Deva Balaji, and Aaron W. James, all from the Department of Pathology, Johns Hopkins University. The paper was published on April 16, 2025, by Oxford University Press in “Stem Cells” (2025, Vol. 43, sxaf020).

3. Detailed Research Design and Technical Approach

1. Research Subjects and Overall Design

This study focuses on the mouse long bone periosteum, systematically analyzing the diversity and functional characteristics of PDGFRβ-expressing periosteal cell subpopulations by integrating single-cell RNA sequencing (scRNA-seq), transgenic mouse models (Pdgfrβ-CreERT2; mT/mG reporter mice), immunohistochemistry, flow cytometry sorting, and in vitro functional assays.

Below is a detailed account of the main research procedures and experimental verification:

2. Single-Cell Transcriptomics Analysis

(1) Sampling and Preparation

  • The study selected four 16-week-old male C57BL/6J mice and dissected periosteal cells from the left femur and tibia, followed by enzymatic digestion and single-cell transcriptome library preparation using the 10X Genomics platform, targeting 10,000 sequenced cells per sample.
  • Data were processed using Cell Ranger and Seurat, with stringent filtering of low-quality cells (number of genes >500 and <8000, mitochondrial transcripts <20%), SCTransform normalization, dimension reduction (UMAP), and pathway enrichment using KEGG databases.

(2) Main Analyses and Discoveries

  • A total of 4,042 single-cell transcriptomes were obtained and eight major cell clusters identified, including mesenchymal cells, pericytes, vascular endothelium, hematopoietic, and immune subpopulations. PDGFRβ was found not only to be widely distributed among pericyte clusters but also expressed in 30%-76% of mesenchymal cell subgroups.
  • Detailed analysis of mesenchymal cell subclusters M1-M5 revealed PDGFRβ is highly co-expressed with bone formation-related genes (such as BGLAP, Spp1, Alpl, Ctsk).
  • GO and KEGG pathway analysis showed that PDGFRβ+ cells were enriched in biological processes such as proliferation, adhesion, osteogenesis, and chondrogenesis, mainly activating PI3K-AKT, JAK-STAT, and MAPK signaling, while PDGFRβ- cells were more involved in adipogenic differentiation and angiogenesis.

3. Spatial Localization Analysis Using Transgenic Fluorescent Reporter Mice

Pdgfrβ-CreERT2;mT/mG mice were used, in which tamoxifen-induced Cre recombination enables PDGFRβ-expressing cells to express EGFP (green fluorescence), while other cells express tdTomato (red). Periosteal tissues were analyzed via immunofluorescence staining and histological sectioning. Co-localization was assessed with markers for pericytes (CD146), endothelial cells (Endomucin), MSCs (PDGFRα), and the osteoblastic transcription factor (Runx2).

The main findings are as follows:

  • Both perivascular and non-perivascular regions of the periosteum and bone marrow showed PDGFRβ+ cells, with EGFP signal highly concentrated in the primary spongiosa.
  • Among CD146+ periosteal cells, 33% were also PDGFRβ+, and among Endomucin+ endothelial cells, 47% expressed PDGFRβ.
  • A high proportion of PDGFRα+ and Runx2+ cells also expressed PDGFRβ, suggesting that PDGFRβ spans functionally overlapping but spatially distinct progenitor populations.

4. Flow Sorting and In Vitro Functional Characterization

(1) Adenylamide Sorting and Grouping

  • After excluding CD31+ (endothelial), CD45+ (leucocyte), and ter119+ (erythroid progenitor) cells, periosteal cells were divided by GFP and tdTomato fluorescence into PDGFRβ+ and PDGFRβ- groups, accounting for about 7% and 67% respectively.

(2) Functional Experiments

  • Proliferation ability: PDGFRβ+ cells showed 60.8% higher proliferation at 24h and 41.3% at 48h compared to PDGFRβ- cells in MTS assays.
  • Migration ability: In scratch assays, PDGFRβ+ cells had a 1.41-fold higher migration rate.
  • Osteogenic differentiation: After 21 days of induction, Alizarin Red S staining revealed 2.14-fold greater osteogenic capacity in PDGFRβ+ cells, with upregulation of related osteogenic genes (Bglap, Sp7, Col1a1, Runx2).
  • Stemness genes (Nes, Ly6a) and pericyte markers (Mcam, Acta2) were significantly higher in PDGFRβ+ cells.

5. Dual-Marker Flow Sorting and Subpopulation Functional Heterogeneity Analysis

Cells were further divided into four subpopulations based on CD146 and PDGFRβ fluorescence intensity: PDGFRβ- CD146-, PDGFRβ- CD146+, PDGFRβ+ CD146-, PDGFRβ+ CD146+. Each was tested for gene expression, proliferation, colony formation, and osteogenic differentiation.

  • The PDGFRβ+ CD146+ group showed the strongest proliferation and clonogenicity, with colony-forming unit numbers about four times higher than PDGFRβ- CD146+, and 57.4% higher than PDGFRβ+ CD146-. Colony area was similar between PDGFRβ+ CD146- and PDGFRβ+ CD146+.
  • In osteogenic differentiation, PDGFRβ+ CD146- and PDGFRβ+ CD146+ showed no significant difference, but both had significantly higher osteogenic potential than CD146- groups.
  • Typical pericyte markers (cspg4, rgs5) were much lower expressed in the PDGFRβ+ CD146- group than in PDGFRβ+ CD146+.
  • These results suggest that PDGFRβ+ CD146+ represents a pericyte subtype with the highest proliferative and clonogenic capacity, and highly enriched osteogenic ability.

4. Major Research Conclusions and Their Significance

In summary, this study found that:

  1. There are multiple subpopulations of PDGFRβ+ cells in the mouse periosteum, including both vascular-associated pericyte groups (mainly PDGFRβ+ CD146+) and non-vascular mesenchymal groups (PDGFRβ+ CD146-), with overlapping but distinct molecular and functional characteristics.
  2. PDGFRβ+ cells demonstrate strong stem/progenitor cell traits and high osteogenic capacity, regardless of concurrent CD146 expression.
  3. PDGFRβ+ CD146+ pericytes are the most clonogenic and proliferative subtypes, while PDGFRβ+ CD146- mesenchymal cells are also outstanding in osteogenic differentiation.
  4. The PDGFRβ+ population is an excellent cell source for bone regeneration and tissue engineering, with broad prospects for clinical applications requiring vascularized bone repair.

5. Research Highlights and Innovations

  • By systematically integrating single-cell omics, spatial localization, and functional analysis, this study for the first time reveals the multidimensional spatial, molecular, functional, and phenotypic heterogeneity of PDGFRβ+ cells in the periosteum.
  • The use of Pdgfrβ-CreERT2;mT/mG reporter mice, combined with flow cytometry sorting, enabled high-purity selection and functional comparison among osteoprogenitor subpopulations.
  • The study clearly distinguishes the differences in stemness, proliferation, and osteogenic differentiation between PDGFRβ+ pericyte and mesenchymal subtypes, providing a precise theoretical foundation for using PDGFRβ as a marker for bone regenerative cells.

6. Scientific and Clinical Application Value

This study lays a critical foundation for understanding periosteal cell heterogeneity, stem cell biology, and bone tissue engineering. Clinically, PDGFRβ+ cell-based vascularized bone regeneration strategies will have a direct impact on fracture healing, bone defects, transplantation, and tissue engineering. Moreover, elucidating the functional properties of different progenitor cell subtypes will help refine the theoretical basis and practical strategies for cell therapy in bone regeneration.

7. Author Contributions and Acknowledgments

This study lists Ziyi Wang, Qizhi Qin, and Neelima Thottappillil as co-first authors, with Aaron W. James as corresponding author. The project was funded by NIH/NIAMS, NIH/NIDCR, Maryland Stem Cell Research Foundation, and others. The authors thank the technical support from the transcriptomics, flow cytometry, and microscopy cores at Johns Hopkins University.

8. Further Reading and Data Availability

  • The raw single-cell sequencing data have been uploaded to GEO (Accession Number: GSE272612).
  • Interested readers may refer to the supplementary materials of the paper or contact the corresponding author for more experimental details.

9. Summary

By applying cutting-edge omics and cellular techniques, this paper clarifies the heterogeneity and functional features of PDGFRβ+ cells in the periosteum, providing an important theoretical basis and experimental support for fundamental and translational studies in bone regeneration. Its scientific value and application prospects warrant ongoing attention from the fields of orthopedics, regenerative medicine, and stem cell research.