Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment
- Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, United States
- Department of Orthopaedics, The First Hospital of China Medical University, China
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China
- Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, United States
- Renal Electrolyte and Hypertension Division, Department of Medicine and Genetics, University of Pennsylvania, United States
- Department of Orthopaedics, Shandong University Qilu Hospital, Shandong University, China
- Division of Hematology, Children's Hospital of Philadelphia, United States
- Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, United States
- Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, United States
- Translational Musculoskeletal Research Center (TMRC), Corporal Michael J. Crescenz Veterans Affairs Medical Center, United States
- Division of Transnational Medicine and Human Genetics, Perelman School of Medicine, University of Pennsylvania, United States
- Translational Research Program in Pediatric Orthopaedics, The Children's Hospital of Philadelphia, United States
- Department of Oral and Maxillofacial Surgery/Pharmacology, University of Pennsylvania, School of Dental Medicine, United States
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 5 supplements
Clustering of bone marrow mesenchymal lineage cells by single cell transcriptomics reveals the in vivo identity of novel mesenchymal subpopulations.
(A) Fluorescent image of distal femur of 1-month-old Col2:Td mice. (B) The tSNE plot of 7585 Td+ mesenchymal lineage cells isolated from endosteal bone marrow of 1–1.5 month-old Col2:Td mice (n = 5 …
Figure 1—figure supplement 1
Bone marrow Td+ cells from Col2:Td mice contain the entire set of mesenchymal lineage cells.
(A) Fluorescent images of distal femur show that chondrocytes (a), osteoblasts and osteocytes in trabecular bone (b) and cortical bone (c), CD45- stromal cells (d), adipocytes (e), and pericytes …
Figure 1—figure supplement 2
Large scale scRNA-seq analysis of top 1% Td+ cells from endosteal bone marrow of 1–1.5 month-old Col2:Td mice.
(A) Violin plots show numbers of genes, UMIs, and percentages of mitochondrial genes among two batches of samples (1 and 1.5 month). Red box indicates cells within the selection criteria of quality …
Figure 1—figure supplement 3
The expression patterns of Tomato and Col2a1 in tSNE plots.
Mesenchymal lineage cells were circled. Color bar on the right of each panel indicates the expression level.
Figure 1—figure supplement 4
No batch effect was detected in our analysis.
(A) The UMAP plot of 7585 Td+ mesenchymal lineage cells isolated from endosteal bone marrow of 1–1.5 month-old Col2:Td mice (n = 5 mice). (B) The distribution of cells from 1 month and 1.5 month …
Figure 1—figure supplement 5
The expression patterns of previously reported mesenchymal progenitor markers.
(A) The expression patterns are shown in tSNE plots that only contain mesenchymal lineage cells. (B) The expression patterns are shown in tSNE plots that contain all sequenced cells. Green, red, and …
Figure 2
The bifurcated osteo- and adipo-lineage differentiation routes of in vivo bone marrow mesenchymal progenitors.
(A) Pseudotemporal depiction of differentially expressed transcription factors (TFs) starting from branching point (dashed lines) toward osteo- (right) and adipo- (left) lineage differentiation. …
Figure 3 with 2 supplements
Large scale scRNA-seq analyses of bone marrow mesenchymal lineage cells from 3- and 16-month-old Col2:Td mice confirm the same in vivo mesenchymal subpopulations as 1-month-old mice.
(A) CFU-F assays of endosteal bone marrow cells from 1-, 3-, and 16-month-old Col2:Td mice. n = 3 mice/group. Td+ and Td- CFU-F colonies are quantified. (B) The tSNE plots of Td+ mesenchymal lineage …
-
Figure 3—source data 1
Raw data for Figure 3G.
- https://cdn.elifesciences.org/articles/54695/elife-54695-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Large scale scRNA-seq analysis of top 1% Td+ cells from the endosteal bone marrow of 3- and 16-month-old Col2:Td mice.
(A) Endosteal bone marrow cells from 3- and 16 month old mice were FACS sorted into top 1% (<1%) and >1% Td expressing cells. (B) Unsorted and sorted cells were cultured for CFU-F assay. Cell number …
Figure 3—figure supplement 2
Pseudotime trajectory analysis of bone marrow mesenchymal lineage cells from all age groups.
(A) Monocle (left) and Slingshot (right) trajectory plots of bone marrow mesenchymal lineage cells of integrated database from mice at 1, 3 and 16 months of ages. Cells are labeled according to …
Figure 4 with 4 supplements
Mouse bone marrow contains abundant non-proliferative adipocyte precursors.
(A) Representative fluorescent images of 1-month-old Adipoq:Td:Col1a1-GFP (a–d) or Adipoq:Td (f–g) femur reveal many bone marrow Td+ cells. (a) A low magnification image of a distal femur. (b–g) At …
Figure 4—figure supplement 1
Large scale scRNA-seq data predict a large cluster of adipocytes in the bone marrow of adolescent mice.
(A) The expression patterns of known adipocyte markers in tSNE plots. The number in parenthesis below each gene indicates its rank among all genes based on its expression level in the adipocyte …
Figure 4—figure supplement 2
The adipocyte markers predicted by sequencing data are validated by in vitro adipogenic differentiation assay.
Bone marrow mesenchymal progenitors were cultured to confluence and switched to adipogenic differentiation medium. Cells were harvested at indicated time points for qRT-PCR analysis of newly …
Figure 4—figure supplement 3
New born Adipoq:Td mice (P5) have abundant Td+ cells in the bone marrow.
(A) Fluorescent image of the proximal tibia at a low magnification. Arrow points to patellar fat pad. (B–D) At a high magnification, it is obvious that Td does not label osteoblasts and osteocytes …
Figure 4—figure supplement 4
Young AdipoqER:Td mice have abundant Td+ cells in bone marrow.
(A) Low magnification image of a femur from a P23 AdipoqER:Td mouse (Tamoxifen at P14-16) reveals abundant Td+ cells in the bone marrow. Td does not label chondrocytes in articular cartilage and …
Figure 5 with 7 supplements
Non-lipid-laden Td+ cells in Adipoq:Td mice form a vast 3D network through their cell processes ubiquitously distributed inside the bone marrow.
(A) The expression patterns of Pdgfrb and Lamb1 are shown in tSNE plots. (B) Immunofluorescence staining reveals that all PDGFRβ+ and Laminin+ cells with a pericyte morphology are Td+ (pointed by …
-
Figure 5—source data 1
Raw data for Figure 5E.
- https://cdn.elifesciences.org/articles/54695/elife-54695-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
The expression patterns of pericyte markers and angiogenesis factors in UMAP plots.
Figure 5—figure supplement 2
Td+ pericytes are unique to the bone marrow of Adipoq:Td mice.
Td+ pericytes are not detected in muscle, liver, lung, brain, heart, and kidney of 1-month-old Adipoq/Td mice. Emcn: Endomucin. n = 3 mice.
Figure 5—video 1
Confocal fluorescence image of Td+ cells in bone marrow of Adipoq:Td mice to show its 3D network structure made of cell processes protruding from cell bodies.
Scan depth: 50 μm.
Figure 5—video 2
Confocal fluorescence image of Td+ cells with PDGFRβ staining (green) in bone marrow of Adipoq:Td mice.
Scan depth: 50 μm.
Figure 5—video 3
Confocal fluorescence image of Td+ cells with Connexin 43 staining (green, shown as dots on cell processes) in bone marrow of Adipoq:Td mice.
Scan depth: 50 μm.
Figure 5—video 4
Confocal fluorescence image of Td+ cells with Emcn staining (green, vessel) in bone marrow of Adipoq:Td mice.
Scan depth: 50 μm.
Figure 5—video 5
Confocal fluorescence image of Td+ cells with Perilipin staining (green) in bone marrow of Adipoq:Td mice.
Large yellow circles are lipid-laden adipocytes. Scan depth: 50 μm.
Figure 6
Bone marrow Adipoq+ cells are critical for supporting marrow vasculature.
(A) Ablation of Adipoq-Cre labeled cells changes the color of long bones from red to pink. One-month-old Adipoq:Td:DTR mice received either veh or DT injections (50 μg/kg) every other day for 2 …
-
Figure 6—source data 1
Raw data for Figure 6F.
- https://cdn.elifesciences.org/articles/54695/elife-54695-fig6-data1-v3.xlsx
Figure 7 with 3 supplements
Bone marrow Adipoq+ cells inhibit bone formation.
(A) 3D μCT images of Adipoq:Td:DTR femurs reveal drastic de novo bone formation in the midshaft region after 2 weeks of DT injections. (B) H and E staining of femoral sections. (C) 3D reconstructed …
-
Figure 7—source data 1
Raw data for Figure 7D.
- https://cdn.elifesciences.org/articles/54695/elife-54695-fig7-data1-v3.xlsx
-
Figure 7—source data 2
Raw data for Figure 7H.
- https://cdn.elifesciences.org/articles/54695/elife-54695-fig7-data2-v3.xlsx
Figure 7—figure supplement 1
Ablation of Adipoq+ cells reduces overall bone marrow (BM) cellularity but has little effect on hematopoietic cells.
(A) 2 weeks of DT injections significantly reduce femoral BM cellularity in 1-month-old Adipoq:Td:DTR mice. (B) Peripheral blood (PB) and BM frequency of hematopoietic lineage cells were assessed by …
Figure 7—figure supplement 2
Ablation of Adipoq+ cells increases trabecular bone mass in vertebrae.
(A) 2 weeks of DT injections significantly reduce the number of bone marrow Td+ cells in vertebrae of 1-month-old Adipoq:Td:DTR mice. (B) Vessel staining reveals that DT injections alter vessel …
Figure 7—figure supplement 3
Fat transplantation does not rescue bone phenotypes induced by ablation of Adipoq+ cells.
(A) Blood glucose level was measured in Adipoq:Td:DTR mice received vehicle injections (veh group), DT injections (DT group), and DT injections after fat transplantation (DT+Fat group). n = 3 …
Figure 8 with 2 supplements
A schematic diagram depicts a model of bone marrow mesenchymal lineage cells and the role of MALPs.
OB: osteoblast; Ocy: osteocyte; EMP: early mesenchymal progenitor; LMP: late mesenchymal progenitor; LCP: lineage committed progenitors; MALPs: marrow adipogenic lineage precursors; LiLA: …
Figure 8—figure supplement 1
A comparison of large scale scRNA-seq data of bone marrow mesenchymal lineage cells between our group (Zhong et al.) and Tikhonova et al. that was recently published (Tikhonova et al., 2019).
An alternative annotation of cell clusters based on our data is indicated by red in parentheses below Tikhonova’s annotation.
Figure 8—figure supplement 2
The comparison of large scale scRNA-seq data of bone marrow mesenchymal lineage cells between our group (Zhong et al.) and Baryawno et al. that was recently published (Baryawno et al., 2019) confirms the same distribution of mesenchymal subpopulations but with different annotation of cell clusters.
(A) Violin plots of cluster 7 markers, including known adipocyte markers and novel markers in Zhong’s 1–1.5 month-old dataset and Baryawno’s dataset. Red boxes are cluster 7 adipocyte in Zhong …
Author response image 1
Large scale scRNA-seq identifies subpopulations of bone marrow mesenchymal lineage cells from MSCs to mature cells.
Author response image 2
Detection of bone marrow MSCs in vivo.
Representative images of stromal MSCs in 1-month-old Col2/Td mice. Arrows point to CD34+Td+ cells in A and Sca1+Td+ cells in B.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Col2-Cre | PMID:10686612 | ||
| Genetic reagent (M. musculus) | Dmp1-Cre | PMID:22623172 | ||
| Genetic reagent (M. musculus) | Adipoq-Cre | PMID:21356515 | ||
| Genetic reagent (M. musculus) | Acta2-CreER | PMID:18571490 | ||
| Genetic reagent (M. musculus) | Col1a1-GFP | PMID:11771662 | ||
| Genetic reagent (M. musculus) | Rosa26 iDTR | Jackson Laboratory | ||
| Genetic reagent (M. musculus) | Rosa26 | Jackson Laboratory | ||
| Antibody | goat anti-mouse LepR | R and D system | Cat.#: AF497 | IF(1:200) |
| Antibody | rabbit anti-mouse Laminin | Sigma | Cat.#: L9393 | IF(1:200) |
| Antibody | rat anti-mouse CD45 | Biolegend | Cat.#: 103101 | IF(1:200) |
| Antibody | rat anti-mouse Endomucin | Santa cruz | Cat.#: sc 65495 | IF(1:200) |
| Antibody | rabbit anti-Osterix | Abcam | Cat.#: ab22552 | IF(1:200) |
| Antibody | rabbit anti-Perilipin | Cell signaling | Cat.#: 9349 | IF(1:200) |
| Antibody | rat anti-mouse PDGFRβ | Biolegend | Cat.#: 136002 | IF(1:100) |
| Antibody | rabbit anti-mouse connexin 43 | Cell signaling | Cat.#: 3512 | IF(1:100) |
| Antibody | FITC rat anti-mouse Endomucin | Santa cruz biotechology | Cat.#: sc-65495 | Flow analysis (1:100) |
| Antibody | rat anti-Gr-1 APC-Cy7, | BD | Cat.#: 557661 | Flow analysis (1:100) |
| Antibody | rat anti-Mac-1 APC | eBioscience | Cat.#: 17-0112-83 | Flow analysis (1:100) |
| Antibody | rat anti-B220 FITC | eBioscience | Cat.#: 11-0452-82 | Flow analysis (1:100) |
| Antibody | hamster anti-CD3 PE-Cy7 | eBioscience | Cat.#: 25-0031-82 | Flow analysis (1:100) |
| Antibody | APC rat anti-mouse CD31 | Biolegend | Cat.#: 561814 | Flow analysis (1:100) |
| Antibody | Click-iT EdU Alexa Fluor 647 Imaging Kit | Thermo Fisher | Cat.#: D3822 | |
| Peptide, recombinant protein | Calcein | Sigma | Cat.#: C0875 | 15 mg/kg forin vivo injection |
| Chemical compound, drug | Diphtheria toxin | Sigma-Aldrich | Cat.#: D0564-1MG | 50 µg/kg forin vivo injection |
| Chemical compound, drug | ACK lysing buffer | ThermoFisher Scientific | Cat.#: A1049201 | 500 ul/ 50 million cells |
| Software, algorithm | Cellranger 3.02 | https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installation | ||
| Software, algorithm | Seurat V2, V3 | PMID:25867923 PMID:31178118 | https://satijalab.org/seurat/ | |
| Software, algorithm | Monocle V2 | PMID:24658644 | http://cole-trapnell-lab.github.io/monocle-release/docs | |
| Software, algorithm | Slingshot 1.5.1 | PMID:29914354 | https://bioconductor.org/packages/devel/bioc/vignettes/slingshot/inst/doc/vignette.html | |
| Other | BODIPY dye | ThermoFisher scientific | D3822 | IF (1:500) |
Additional files
-
Source code 1
R script for reading the matrix file of the dataset.
- https://cdn.elifesciences.org/articles/54695/elife-54695-code1-v3.zip
-
Supplementary file 1
Mouse real-time PCR primer sequences used in this study.
- https://cdn.elifesciences.org/articles/54695/elife-54695-supp1-v3.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/54695/elife-54695-transrepform-v3.docx
