1. Cancer Biology
  2. Immunology and Inflammation
Download icon

Osterix-Cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics

  1. Biancamaria Ricci
  2. Eric Tycksen
  3. Hamza Celik
  4. Jad I Belle
  5. Francesca Fontana
  6. Roberto Civitelli
  7. Roberta Faccio  Is a corresponding author
  1. Department of Orthopedics, Washington University School of Medicine, United States
  2. Genome Technology Access Center, Department of Genetics, Washington University School of Medicine, United States
  3. Department of Medicine, Division of Oncology, Washington University School of Medicine, United States
  4. Department of Medicine, Division of Bone and Mineral Diseases, Washington University School of Medicine, United States
  5. Shriners Children Hospital, United States
Research Article
Cite this article as: eLife 2020;9:e54659 doi: 10.7554/eLife.54659
8 figures, 3 tables, 1 data set and 1 additional file

Figures

Figure 1 with 1 supplement
Embryonic and adult-derived Osx+ cells are present in primary tumors at extra-skeletal sites.

(A) Doxycycline (doxy)-repressible Sp7-cre/loxP mouse model used to activate Ai9/TdTomato expression for lineage tracing experiments. In no doxy-fed mice, TdT is expressed in embryonic-derived osteolineage cells (left), while in mice fed a doxy diet until weaning, TdT is expressed in adult-derived osteolineage cells. (B–I) Flow cytometry analysis and fluorescence images of primary tumors showing presence of TdTOSX+ cells in no-doxy fed Osx-cre;TdT mice or WT;TdT controls inoculated with B16-F10 melanoma subcutaneously (B–C), or with PyMT breast cancer cells in the mammary fat pad (MFP) (D–E), and in doxy-fed mice injected with B16-F10 subcutaneously (F–G), or with PyMT in the MFP (H–I). Slides for fluorescence images were counterstained with DAPI (blue), magnification 200X. Inserts are further magnified 4.5 folds. Data representative of a single experiment, experiments were repeated between 2 and 5 times. p values represent Student t-test statistical analysis.

Figure 1—figure supplement 1
No difference in tumor growth and body weight between sex and age matched cre positive (Osx-cre;TdT) and negative (WT;TdT) mice.

. (A) Tumor growth in Osx-cre;TdT and WT;TdT male mice injected subcutaneously with B16-F10 tumor cells. (B) Body weight of the mice showed in (A) at the day of tumor injection (day 0) and at the end point (day 14 post-injection). (C) Tumor growth in Osx-cre;TdT and WT;TdT female mice injected in the MFP with PyMT tumor cells. (D) Body weight of the mice showed in (C) on day 0 and day 14 post-injection.

TdTOSX+ cells from primary tumors express mesenchymal markers.

(A) Experimental design for isolating TdTOSX+ cells from primary tumors isolated from doxy-fed Osx-cre;TdT mice inoculated with B16-F10. TdTOSX- cells comprise all TdT negative cells including the tumor cells. Immortalized CAFs (iCAFs) and crushed long bones were used as reference controls. (B–L) Quantitative Real-Time PCR for (B) Sp7(Osterix), (C) Runx2, (D) Bglap(Osteocalcin), (E) Ibsp (Bone sialoprotein), (F) Alpl (Tnap, Tissue non-specific alkaline phosphatase), (G) S100a4 (Fsp1), (H) Acta2 (α-SMA) (I) Collagen1a1 (J) Collagen1a2, (K) Mmp2 (Metalloproteinase2) and (L) Mmp9 (Metalloproteinase9) genes in TdTOSX+, TdTOSX- cells, iCAFs and bone. Each data point includes cells isolated from 3 to 4 mice and from two independent experiments. (M–N) Real-Time PCR of Sp7(Osterix) in B16-F10 or PyMT cell lines, CAFs isolated from primary B16-F10 or PyMT tumors or from the remaining cells comprising the tumor minus the CAFs (susp) (n = 3–4 mice). (O) Sp7(Osx) expression based on the Finak Breast Cancer Stroma gene set in the Oncomine cancer microarray database. Significance was determined by one-way ANOVA with Tukey post-hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3 with 2 supplements
Tumor resident but not bone marrow resident TdTOSX+ cells increase tumor growth.

(A) Model showing isolation of TdTOSX+ cells from B16-F10 primary tumors injected into doxy-fed Osx-cre;TdT mice and re-inoculation of TdTOSX+ cells together with B16-F10 tumor cells into WT recipient mice at the ratio 5:1. Mice injected with B16-F10 alone were used as controls. (B) Tumor growth of experimental model described in (A) determined by caliper measurement. n = 3/group, experiment repeated twice. Significance was determined by one-way ANOVA with Tukey post-hoc test. (C) Fluorescence images of bone sections showing presence of TdTOSX+ within the bone and bone marrow of naïve Osx-cre;TdT, Osx-cre;TdT mice inoculated subcutaneously with B16-F10 tumors or WT;TdT mice used as negative control. Sections were counterstained with DAPI (blue), magnification 200X. (D–E) Quantification of TdTOSX+ cells in the bone marrow of tumor-free and B16-F10 tumor bearing (D) Osx-cre;TdT mice (doxy-fed) and (E) Osx-creERT2;TdT (TAM-treated) determined by FACS. Experiment repeated twice. Significance was determined by student t-test statistical analysis. (F) Tumor growth of WT mice inoculated with bone marrow-derived TdTOSX+ cells from B16-F10 bearing doxy-fed Osx-cre;TdT mice together with B16-F10 tumor cells (ratio 5:1). Mice injected with B16-F10 alone were used as controls. n = 3/group. Significance was determined by two-way ANOVA followed by Tukey post-hoc test. ***p<0.001, ****p<0.0001.

Figure 3—figure supplement 1
TdTOSX+ cells increase in the bone marrow of PyMT tumor bearing mice.

(A) Fluorescence images of WT;TdT and Osx-cre;TdT mice inoculated with PyMT tumors in the MFP. Slides for fluorescence images were counterstained with DAPI (blue), magnification 200X. (B) Quantification of TdTOSX+ cells in the bone marrow of tumor-free and PyMT bearing mice. Experiment repeated twice. p value represents Student t-test statistical analysis.

Figure 3—figure supplement 2
Differential expression of mesenchymal markers in TdTOSX+ cells isolated from tumor site or bone marrow.

(A–K) Quantitative Real-Time PCR for (A) Sp7 (Osterix), (B) Runx2, (C) Bglap (Osteocalcin), (D) Ibsp (Bone sialoprotein), (E) Alpl (Tnap, Tissue non-specific alkaline phosphatase), (F) S100a4 (Fsp1), (G) Acta2 (α-SMA), (H) Collagen 1a1, (I) Collagen 1a2, (J) Mmp2 (Metalloproteinase2) and (K) Mmp9 (Metalloproteinase9) genes in TdTOSX+ isolated from the B16-F10 melanoma primary tumors, bone marrow of B16-F10 bearing mice and bone marrow of naïve tumor-free mice.

Figure 4 with 2 supplements
TdTOSX+ cells express the immune surface marker CD45.

(A–B) Representative FACS dot plots and quantification of TdTOSX+ cells in the peripheral blood of tumor-free and B16-F10 or PyMT tumor bearing mice. Significance was determined by student t-test statistical analysis for each tumor model relative to no tumor controls. (C–D) Representative FACS dot plots and quantification of TdTOSX+;CD45+ and TdTOSX+;CD45- populations in the blood of doxy-fed Osx-cre;TdT mice injected with B16-F10 or PyMT cells. (E–H) Representative FACS dot plots and quantification of TdTOSX+;CD45+ and TdTOSX+;CD45- populations in the bone marrow (E–F) or tumor site (G–H) of doxy-fed Osx-cre;TdT mice injected with B16-F10 or PyMT cells, respectively. Experiments were repeated at least twice. (I) Immunohistochemistry staining of paraffin-embedded B16-F10 tumors inoculated into Osx-cre;TdT reporter mice, pseudo colored with red representing TdTOSX+ cells (RFP stained), green representing CD45+ cells (DAB stained) and blue representing nuclei (hematoxylin), magnification 200X.

Figure 4—source data 1

Relates to FACS analysis in panels B, C, D, E, F, G, H.

https://cdn.elifesciences.org/articles/54659/elife-54659-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Presence of TdTOSX+ cells in bone marrow (BM) of TAM-pulsed Osx-creERT2;TdT reporter mice.

(A) Schematic representation of TAM administration and tumor injection in Osx-creERT2;TdT mice. (B) Representative image of flow cytometry dot plots and quantification of TdTOSX+;CD45+ and TdTOSX+;CD45- populations in the bone marrow of B16-F10 bearing mice. A cre negative mouse was used as control to set gates. Experiment was repeated three times.

Figure 4—figure supplement 2
Co-expression of Osx-driven TdTomato and the immune marker CD45.

(A) Immunofluorescence on paraffin-embedded B16-F10 tumors isolated from Osx-cre;TdT reporter mice. Staining was done using anti-RFP (red) to detect TdTomato and anti-CD45 (green) to stain immune populations. Slides were counterstained with DAPI (blue), magnification 200X. (B) Staining for CD45 on a single cell suspension from bone marrow of Osx-cre;TdT reporter mice, magnification 200X. Yellow arrows point to TdTOSX+ single cells expressing CD45, while the white arrow points to a TdTOSX+ cell negative for CD45.

Figure 5 with 1 supplement
TdTOSX+;CD45- and TdTOSX+;CD45+ are two functionally distinct populations in the tumor microenvironment.

(A) Multidimensional Principal Component Analysis of RNAseq data obtained from four tumor stroma subsets sorted according to TdTOSX and CD45 expression. (B) Venn diagram depicting uniquely and commonly expressed genes among the four groups. (C–D) First ten GO pathways obtained from the GO analysis of the log two fold-changes for (C) CD45 negative subsets and (D) CD45 positive subsets.

Figure 5—figure supplement 1
Validation of RNAseq data.

Real Time PCR performed on TdTOSX+;CD45+ and TdTOSX+;CD45- populations sorted from B16-F10 tumors in doxy-fed mice. Each sample is a pool of two tumors. Data show three genes (A) Cd163l1, (B) Wdr79 and (C) Spock2 to be upregulated in the TdTOSX+;CD45+ compared to TdTOSX-;CD45+ cells.

Figure 6 with 1 supplement
Subsets of myeloid and lymphoid cells in the tumor microenvironment and the bone marrow are derived from TdTOSX+ cells.

(A–B) Quantification of FACS analysis showing the percentage of TdTOSX+;CD45+ and TdTOSX-;CD45+ populations in the tumor and bone marrow of Osx-cre;TdT mice injected subcutaneously with B16-F10 tumor cells. Data showed as % of total CD45+ cells. (C–D) Quantification of FACS analysis showing the percentage of tumor infiltrating myeloid and lymphoid populations within the TdTOSX+;CD45+ or TdTOSX-;CD45+ subsets, each considered as 100%. (E) Lymphoid over myeloid ratio within the tumor infiltrating TdTOSX+;CD45+ or the TdTOSX-;CD45+ subsets. Statistical analysis was performed by student t-test. (F–G) Quantification of FACS analysis showing the percentage of the bone marrow resident myeloid and lymphoid populations within the TdTOSX+;CD45+ or TdTOSX-;CD45+ subsets, each considered as 100%. n = 3/group. (H) Tumor growth in mice injected with B16-F10 tumor cells alone or together with tumor-derived TdTOSX+;CD45+ or TdTOSX-;CD45+ cells at the ratio 1:5. n = 3–6/group.

Figure 6—figure supplement 1
Gate strategy for immune staining of tumor and bone marrow.

(A) Representative gate strategy analysis of total tumor mass stained for CD45, myeloid immune markers CD11b, F4/80 and Gr1. Analysis performed in both TdTOSX+ and TdTOSX- populations. Identical analysis was performed on BM samples. (B) Representative gate strategy analysis of BM stained for lymphoid immune markers CD3, CD8, CD4 and NK1.1. Analysis performed in both TdTOSX+ and TdTOSX- populations. Identical analysis was performed on tumor samples. Fluorescence minus one (FMO) controls were used to set the gates. WT;TdT mice were used to set the gate for TdT+ cells.

Figure 7 with 1 supplement
TdTOSX+;CD45+ cells derive from TdTOSX+ HSCs.

(A) Schematic of hematopoietic differentiation. (B–E) Flow cytometry quantification of TdTOSX+ cells in the bone marrow of (B–C) doxy-fed Osx-cre;TdT mice and (D–E) TAM-treated Osx-creERT2;TdT mice injected subcutaneously at 7 weeks of age with B16-F10 tumors. In (B and D) LSK and LK subsets are shown, while in (C and E) the LSK population is further divided into HSCs and MPP1-4 subsets. Red numbers represent the average of TdTOSX+ cells in each specific subset. n = 2–6/group.

Figure 7—figure supplement 1
Gate strategy for HSC immunostaining of bone marrow.

(A) Representative gate strategy analysis of bone marrow stained for HSC subsets. TdTOSX+ cells were quantified in each subset. Fluorescence minus one (FMO) controls were used to set the gates. WT;TdT mice were used to set the gate for TdT+ cells.

Sp7 transcripts and Osx protein are expressed in a subset of HSCs isolated from bone marrow of naïve mice.

(A) Real Time PCR analysis comparing sorted hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and whole bone marrow (WBM) from 7 to 9 week old mice. FACS sorted TdTOSX- cells from the tumor of Osx-cre;TdT mice were used as negative control (n = 3–8/group). Statistical analysis was determined by two-way ANOVA followed by Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B) Real Time PCR analysis of isolated mature CD3+ T cells, conventional dendritic cells (cDC) and bone marrow-derived macrophages (mφ) from WT naïve mice. (C–E) Immunofluorescence for Osx in (C) primary osteoblasts differentiated from BMSC cultured for 4 days in osteogenic media, (D) TdTOSX+ HSCs sorted from Osx-cre;TdT reporter mice and (E) whole bone marrow cells from WT mice. DAPI is used for nuclear staining. Magnification 200X.

Tables

Table 1
Top 50 uniquely up-regulated genes in tumor-derived TdTOSX+;CD45- cells.

List of the 50 most highly expressed genes in the TdTOSX+;CD45- cells compared to the TdTOSX-;CD45- cells isolated from the TME.

external_gene_namedescriptionTdTOSX+CD45-
logFC
TdTOSX+CD45-
linearFC
Reg1regenerating islet-derived 17.675801204.477899
Fbln7fibulin 77.184048145.416614
Adcyap1r1adenylate cyclase activating polypeptide 1 receptor 16.864422116.51905
Col24a1collagen, type XXIV, alpha 16.788179110.521192
Moxd1monooxygenase, DBH-like 16.74623107.35382
Fmodfibromodulin6.739645106.864989
4930562D21RikRIKEN cDNA 4930562D21 gene6.14992471.008705
Angpt4angiopoietin 46.13162770.113799
Tmem132ctransmembrane protein 132C5.93300261.095844
Frem1Fras1 related extracellular matrix protein 15.90616659.969876
Cxcl5chemokine (C-X-C motif) ligand 55.8888159.252728
Ccl19chemokine (C-C motif) ligand 195.74835553.756054
Rab15RAB15, member RAS oncogene family5.69605751.842269
Prlrprolactin receptor5.67365151.04334
Msx1msh homeobox 15.65766150.480738
Pabpc4lpoly(A) binding protein, cytoplasmic 4-like5.65730150.46814
Reg3gregenerating islet-derived 3 gamma5.65184150.2775
Rflnarefilin A5.65176550.274843
Adamts3a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 35.5264646.092506
Lrp3low density lipoprotein receptor-related protein 35.46586544.196651
Clstn2calsyntenin 25.4162842.703438
Adam5a disintegrin and metallopeptidase domain 55.3649541.210775
H2-M11histocompatibility 2, M region locus 115.33553140.378937
Ptx3pentraxin related gene5.32056539.962237
Rtl3retrotransposon Gag like 35.25291638.131634
Gm26682predicted gene, 266825.25280338.128648
Morc1microrchidia 15.25159438.096689
Hoxc6homeobox C65.17632936.160165
Mcpt8mast cell protease 85.14657635.422062
Gabrb3gamma-aminobutyric acid (GABA) A receptor, subunit beta 35.11087334.556218
Sbsponsomatomedin B and thrombospondin, type 1 domain containing5.08542633.952042
Akap6A kinase (PRKA) anchor protein 65.07310233.663235
Hmcn1hemicentin 15.06034633.366905
Aqp2aquaporin 25.03126432.701036
Gria3glutamate receptor, ionotropic, AMPA3 (alpha 3)5.01192432.265589
AI464131expressed sequence AI4641315.00141432.03138
Muc13mucin 13, epithelial transmembrane4.99713231.936452
Syt4synaptotagmin IV4.99466631.881898
Spon1spondin 1, (f-spondin) extracellular matrix protein4.91603330.190712
Caln1calneuron 14.8599629.039801
Hoxc4homeobox C44.84415528.723415
Tbx2T-box 24.84204228.681376
Jph2junctophilin 24.81683328.18456
Frzbfrizzled-related protein4.7921427.706253
Galnt5polypeptide N-acetylgalactosaminyltransferase 54.7778227.432606
Gm29100predicted gene 291004.7725527.332593
Tspyl5testis-specific protein, Y-encoded-like 54.77150927.312867
Spag17sperm associated antigen 174.77065327.296671
Ldoc1regulator of NFKB signaling4.747926.86954
4833422C13RikRIKEN cDNA 4833422C13 gene4.73067826.550693
Table 2
Top 50 uniquely up-regulated genes in tumor-derived TdTOSX+;CD45+ cells.

List of the 50 most highly expressed genes in the TdTOSX+;CD45+ cells compared to the TdTOSX-;CD45+ cells isolated from the TME.

external_gene_
name
descriptionTdTOSX+CD45+
logFC
TdTOSX+CD45+
linearFC
Cd163l1CD163 molecule-like 16.48211289.394369
Ltblymphotoxin B6.21703774.389998
Klrg1killer cell lectin-like receptor subfamily G, member 15.91569760.367363
Foxp3forkhead box P35.88242358.991007
Trbv19T cell receptor beta, variable 195.8390157.242322
Cd5CD5 antigen5.82296556.609196
Gm4759predicted gene 47595.71223852.426999
Tcrg-C1T cell receptor gamma, constant 15.70992152.342868
Icosinducible T cell co-stimulator5.58885648.129715
Gm19585predicted gene, 195855.57758247.755056
FaslFas ligand (TNF superfamily, member 6)5.57131447.548033
Rln3relaxin 35.51806245.824955
Ubash3aubiquitin associated and SH3 domain containing, A5.43230843.180511
Pcsk1proprotein convertase subtilisin/kexin type 15.37632941.537117
Ccr8chemokine (C-C motif) receptor 85.3242340.06386
Gimap3GTPase, IMAP family member 35.25750838.253185
Slamf1signaling lymphocytic activation molecule family member 15.22064137.288037
Klrb1fkiller cell lectin-like receptor subfamily B member 1F5.20406736.86212
Ikzf3IKAROS family zinc finger 35.18533236.386518
Trdv4T cell receptor delta variable5.17928836.234394
Trat1T cell receptor associated transmembrane adaptor 15.14464835.374755
Pdcd1programmed cell death 15.09201734.107504
Izumo1rIZUMO1 receptor, JUNO5.05363533.212045
Cd3eCD3 antigen, epsilon polypeptide5.04217432.949246
ItkIL2 inducible T cell kinase5.02718232.608632
Ctla4cytotoxic T-lymphocyte-associated protein 44.97804331.516668
Actn2actinin alpha 24.91996730.27316
Trbv1T cell receptor beta, variable 14.89116429.674756
Dkkl1dickkopf-like 14.82062828.258804
Cd300eCD300E molecule4.80135527.883791
Pglyrp2peptidoglycan recognition protein 24.8003227.863797
Cd226CD226 antigen4.76644127.21709
Themisthymocyte selection associated4.76008227.097397
Dpep3dipeptidase 34.69447325.892693
Cd209aCD209a antigen4.65026125.111233
Cd27CD27 antigen4.64872525.084512
Gpr55G protein-coupled receptor 554.62111324.608975
Ccr6chemokine (C-C motif) receptor 64.59600424.18439
Gpr174G protein-coupled receptor 1744.59468324.16225
A630023P12RikRIKEN cDNA A630023P12 gene4.58649924.025577
Cd28CD28 antigen4.56425523.657981
Cd3gCD3 antigen, gamma polypeptide4.54760923.386577
Ltalymphotoxin A4.53429323.171712
Ccr3chemokine (C-C motif) receptor 34.52652723.047318
Gzmbgranzyme B4.51865822.921957
Lrrc66leucine rich repeat containing 664.4714722.184347
Xirp2xin actin-binding repeat containing 24.46293722.053513
St8sia1ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 14.46092422.022769
Sit1suppression inducing transmembrane adaptor 14.41736321.367754
Tnfrsf4tumor necrosis factor receptor superfamily, member 44.38186820.84844
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Mus musculus)Sp7(Osx)NCBI GeneRRID:MGI:2153568NCBI ID:170574
Genetic reagent (Mus musculus)Tg(Sp7-tTA,tetO-EGFP/cre)1Amc/J
(Osx-cre)
The Jackson LaboratoriesCat#006361
RRID:MGI:3689350
C57Bl/6
Genetic reagent (Mus musculus)B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J
(TdT)
The Jackson LaboratoriesRRID:MGI3813511C57Bl/6
Genetic reagent (Mus musculus)Sp7-creERT2/Ai9tdTomato
(Osx-creERT2;TdT)
PMID:31768488Cat#007909
RRID:MGI:4829803
Prof. Silva MJ (Washington University in St Louis)
C57Bl/6
Cell line (Mus musculus, C57Bl/6)B16-F10 melanoma cell lineATCCRRID:CRL-6475-LUC2C57Bl/6
Cell line (Mus musculus)PyMT breast cancer cell linePMID:27216180Prof. Weilbaecher KN (Washington University in St Louis)
C57Bl/6
Cell line (Mus musculus)Immortalized Cancer Associated Fibroblast cell linePMID:27264173Prof Longmore GD (Washington University in St Louis)
AntibodyRat anti-mouse CD45-APCeFluor780(clone 30-F11)eBioscienceCat #47-0451-82
RRID:AB_1548781
FACS (1:400)
AntibodyRat anti-mouse CD45-PE Cy7 (clone 30-F11)eBioscienceCat #25-0451-82
RRID:AB_2734986
FACS (1:400)
AntibodyRat anti-mouse CD11b-AlexaFluor700 (clone M1/70)eBioscienceCat#56-0112-82
RRID:AB_657585
FACS (1:400)
AntibodyRat anti-mouse Gr1(Ly6G)-FITC (clone RB6-8C5)Miltenyi BiotecCat#130-102-837
RRID:AB_2659858
FACS (1:100)
AntibodyRat anti-mouse F4/80 PerCP Cy5.5 (clone BM8)BiolegendCat#123126
RRID:AB_10802654
FACS (1:400)
AntibodyHamster anti-mouse CD3e-PE (clone 145–2 C11)eBioscienceCat#A14714
RRID:AB_2534230
FACS (1:200)
AntibodyRat anti-mouse CD4-APC (clone RM4-5)BD BiosciencesCat#553051
RRID:AB_398528
FACS (1:200)
AntibodyRat anti-mouse CD8a-FITC (clone 53–6.7)BD BiosciencesCat#561966
RRID:AB_10896291
FACS (1:200)
AntibodyMouse anti-mouse NK1.1 (clone PK136)BiolegendCat#108722
RRID:AB_2132712
FACS (1:200)
AntibodyRat anti-mouse CD45R (B220)-Biotin (clone RA3-6B2)eBioscienceCat#13-0452-82
RRID:AB_466449
FACS (0.4 ul/sample)
AntibodyHamster anti-mouse CD3e-Biotin (clone 145–2 C11)eBioscienceCat#13-0031-82
RRID:AB_466319
FACS (0.4 ul/sample)
AntibodyRat anti-mouse Ter119 (clone TER119)eBioscienceCat# 13-5921-82
RRID:AB_466797
FACS (0.4 ul/sample)
AntibodyRat anti-mouse Ly-6G/Ly-6C Biotin (clone RB6-8C5),eBioscienceCat#13-5931-82
RRID:AB_466800
FACS (0.2 ul/sample)
AntibodyRat anti-mouse CD41Biotin (clone MWReg30)eBioscienceCat#13-0411-82
RRID:AB_763484
FACS (1 ul/sample)
AntibodyStreptavidin-BV510BD BiosciencesCat#563261FACS (0.5 ul/sample)
AntibodyRat anti-mouse Sca-1 BV711 (clone D7)BD BiosciencesCat#563992
RRID:AB_2738529
FACS (0.25 ul/sample)
AntibodyRat anti-mouse c-kit(CD117)-APCeFluor780 (clone ACK2)eBioscienceCat#47-1172-82
RRID:AB_1582226
FACS (1 ul/sample)
AntibodyRat anti-mouse CD16/32-BUV395 (clone 2.4G2)BD BiosciencesCat#740217
RRID:AB_2739965
FACS (0.5 ul/sample)
AntibodyRat anti-mouse CD34-FITC (clone RAM34)eBioscienceCat#11-0341-82
RRID:AB_465021
FACS (4 ul/sample)
AntibodyRat anti-mouse CD150(SLAM)-BV421 (clone TC15-12F12.2)BiolegendCat#115943
RRID:AB_2650881
FACS (1 ul/sample)
AntibodyHamster anti-mouse CD48-PE Cy7 (clone HM48-1)eBioscienceCat#25-0481-80
RRID:AB_1724087
FACS (0.15 ul/sample)
AntibodyRat anti-mouse CD135(Flt3)-APC (clone A2F10)eBioscienceCat#17-1351-82
RRID:AB_10717261
FACS (2 ul/sample)
AntibodyRat anti-mouse CD45 (clone 30-F11)InvitrogenCat#14-0451-82
RRID:AB_467251
IF, IHC (1:200)
AntibodyRabbit anti-mouse RFP/TdTRocklandCat#600-401-379
RRID:AB_2209751
IF,IHC (1:500)
AntibodyGoat-anti-Rat IgG-BiotinThermo FisherCat#31830
RRID:AB_228355
IF, IHC (1:500)
AntibodyStreptavidin-HRPBio-RadCat#STAR5BIF, IHC (1:500)
AntibodyGoat-anti-rabbit Alexa-fluor647Thermo FisherCat#A21244
RRID:AB_2535812
IF (4 ug/ml)
AntibodyRabbit anti-mouse Sp7/OsxAbcamCat#ab227820IF (1:500)
AntibodyGoat anti-rabbit AlexaFluor488AbcamCat#ab150077
RRID:AB_2630356
IF (1:1000)
Sequence-based reagentCyclophilinThis paperPCR primers5’-AGC ATA CAG GTC CTG GCA TC-3’ and 5’-TTC ACC TTC CCA AAG ACC AC-3’
Sequence-based reagentSp7(Osx)This paperPCR primers5’-AAG GGT GGG TAG TCA TTT GCA-3’ and 5’-CCC TTC TCA AGC ACC AAT GG-3’
Sequence-based reagentS100A4(Fsp-1)This paperPCR primers5’-TGA GCA ACT TGG ACA GCA ACA-3’ and 5’-TTC CGG GGT TCC TTA TCT GGG-3’
Sequence-based reagentActa2(α-SMA)This paperPCR primers5’-GTC CCA GAC ATC AGG GAG TAA-3’ and 5’-TCG GAT ACT TCA GCG TCA GGA-3’
Sequence-based reagentCol1a1This paperPCR primers5’-GGC CTT GGA GGA AAC TTT GC-3’ and 5’-GGG ACC CAT TGG ACC TGA AC-3’
Sequence-based reagentBglap(Ocn)This paperPCR primers5’-GGA CTG AGG CTC TGT GAG GT-3’ and 5’-CAG ACA CCA TGA GGA CCA TC-3’
Sequence-based reagentRunx2This paperPCR primers5’-GTT ATG AAA AAC CAA GTA GCC AGG-3’ and 5’-GTA ATC TGA CTC TGT CCT TGT GGA-3’
Sequence-based reagentBSPThis paperPCR primers5’-AGG ACT AGG GGT CAA ACA C-3’ and 5’-AGT AGC GTG GCC GGT ACT TA-3’
Sequence-based reagentTNAPThis paperPCR primers5’-GGG GAC ATG CAG TAT GAG TT-3’ and 5’-GGC CTG GTA GTT GTT GTG AG-3’
Commercial assay or kitTSA FITC SystemPerkin ElmerCat#NEL701A001KT
Commercial assay or kitBOND Intense R detection kitLeica BiosystemCat#DS9263
Commercial assay or kitBOND Polymer Refine Red detection kitLeica BiosystemCat#DS9390
Commercial assay or kitAvidin/Biotin Blocking kitVector LabsCat#SP2001
RRID:AB_2336231
Software, algorithmSTARSTARRRID:SCR_015899Version 2.0.4b
Software, algorithmSubreadSubreadRRID:SCR_009803Version 1.4.5
Software, algorithmRRRRID:SCR_001905Version 3.4.1
Software, algorithmEdgeREdgeRRRID:SCR_012802Version 3.20.2
Software, algorithmLIMMALIMMARRID:SCR_010943Version 3.34.4
Software, algorithmGAGEGAGERRID:SCR_017067Version 2.28.0
OtherVECTASHIELD Mounting Medium with DAPIVector LabsCat#H1200
RRID:AB_2336790
one drop
OtherProLong Gold with DAPIInvitrogenCat#P10144one drop

Data availability

Sequencing data are deposited in GEO under accession code GSE143586 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE143586.

The following data sets were generated
  1. 1

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)