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1 DOI: /nc2014 SM/C-2.6(-) CD31, CD45, SM/C-2.6 PDGFRα CD CD CD CD CXCR4 1.3 c-kit 0.1 Sc Integrin α7 0.1 PDGFRα PDGFRα(-)SM/C-2.6(+) CD31, CD45, PDGFRα SM/C-2.6 CD CD CD CD CXCR4 2.7 c-kit 1.1 Sc Integrin α SM/C-2.6 CD31+ or CD45+ SM/C-2.6(+) SM/C-2.6(-)PDGFRα(-) ±1.7 <0.1 <0.1 DAPI C/EBPα PPARγ DAPI Px7 c Figure S1 (Uezumi et l.) Figure S1 Phenotype of PDGFRα + cells. () Freshly isolted CD31 CD45 SM/C-2.6 PDGFRα + cells were nlysed for the expression of indicted cell surfce ntigens y FACS. Expression of cell surfce ntigens on stellite cells (CD31 CD45 PDGFRα SM/C cells) ws lso exmined. Positive nd negtive gtes were set y nlysing isotype or unstined control smple in ech nlysis (shown on the right). Vlues indicte the percentges of positive cells in ech cell popultion. () The four freshly isolted frctions were sujected to cytospin nd stined with nti-px7 ntiody. The percentges of Px7 + cells re expressed s mens ± s.e. of 24 rndomly selected fields from three independent experiments. (c) C/EBPα nd PPARγ stining of the four freshly isolted frctions. Scle r represents 40 µm. 1

2 4d 10d 100 SM/C-2.6(+) SM/C-2.6(+) DAPI Px7 MyoD Phse MyoD DAPI myogenin myogenin 98.3 Phse DAPI MyoD srcomeric α-ctinin c 4d d Px7 DAPI positive cells (%) DAPI Phse 0 Px7 MyoD 5.5 myogenin Px7 MyoD myogenin Figure S2 Stellite cells differentite into myotues even under dipogenic conditions. (, ) Cultured CD31 CD45 SM/C cells were fixed t the time points indicted. Expression of MyoD, Px7, nd myogenin t dy 4 of culture, nd MyoD nd srcomeric α-ctinin t dy 10 ws exmined y immunofluorescent stining. (c) Cultured CD31 CD45 PDGFRα + cells were stined with nti-myod nd nti-px7, or nti-myogenin ntiodies. (d) Quntittive nlysis of the percentges of cells positive for myogenic trnscription fctors. Dt re represented s mens ± s.d. (see Methods for detils). Scle r represents 40 µm. 2

3 c Adipogenic colony Non-dipogenic colony Numer of colonies 52 (9.0%) PPARγ n 576 Adipogenic Non-dipogenic 41 (78.8%) 11 (21.2%) Myogenic 0 (0%) Phse DAPI Figure S3 (Uezumi et l.) Figure S3 Clonl ssy of PDGFRα + cells. () Typicl imge of single PDGFRα + cell-derived colony. () After 8 dys of culture, colonies were sujected to dipogenic tretment. Adipogenic differentition ws ssessed y PPARγ stining. (c) Numer of colonies nd composition of colonies re shown. Scle rs, 200 µm () nd 50 µm (). 3

4 3T3-L1 1.00E+01 Leptin/β-ctin Reltive Quntity 1.00E E E E-03 WAT 3T3-L1 Figure S4 (Uezumi et l.) Figure S4 Leptin expression in PDGFRα + cells, 3T3-L1 cells, nd WAT. () 3T3-L1 cells nd PDGFRα + cells were induced to differentite into dipocytes. Phse contrst imges of differentited 3T3-L1 cells nd differentited PDGFRα + cells re shown. () RNA ws extrcted from differentited 3T3-L1 cells, differentited PDGFRα + cells, nd WAT. Equl mount of RNA (500 ng) ws sujected to reverse trnscription rection nd rel-time quntittive PCR nlysis ws performed in triplicte to evlute the levels of leptin expression. Rw vlues of leptin were normlized to tht of β-ctin. Dt re represented s the reltive quntity to WAT nd s mens ± s.d. Scle r represents 50 µm. 4

5 Cell sorting Adipo IM Adipo MM dy PDGFRα PPARγ SM/C-2.6(+) c C/EBPα C/EBPβ CD31( )CD45( ) CD31( )CD45( )SM/C-2.6(+) PPARγ leptin C/EBPα DAPI diponectin GAPDH Oil Red O Phse Figure S5 Direct dipogenic induction of freshly isolted muscle-derived cells. () Protocol for direct dipogenic induction. Freshly isolted cells were directly cultured in Adipo IM for 10 dys followed y tretment with Adipo MM for 3 dys. () CD31 CD45 SM/C cells or CD31 CD45 PDGFRα + cells were stined with nti-pdgfrα, nti-c/ebpα, nd nti-pparγ ntiodies or were stined with Oil Red O. (c) Expression of dipogenic genes in CD31 CD45 SM/C cells nd CD31 CD45 PDGFRα + cells fter direct dipogenic induction. Scle r represents 40 µm. 5

6 H2O PC H2O PC Runx2 αsma Osteoclcin SM22 Osteopontin smmhc GAPDH Clponin1 GAPDH Figure S6 (Uezumi et l.) Figure S6 Expression of osteogenic nd smooth muscle genes in PDGFRα + cells. (, ) Expression of osteogenic genes in BMP-7-treted PDGFRα + cells () nd smooth muscle genes in TGF-β1-treted PDGFRα + cells () ws exmined y RT- PCR. RNA extrcted from whole emryos ws used s positive control (PC). 6

7 GFP C57BL/6 CTX Cell sorting 1 CD31+ or CD45+ 2 PDGFRα(-) 3 trnsplnttion CD31, CD45 1 Counts (dy) nlysis GFP PDGFRα CD31+ or CD45+ PDGFRα(-) c Numer of GFP+ myofiers / section p<0.001 GFP lminin α2 DAPI 0 CD31+, CD45+ PDGFRα(-) Figure S7 (Uezumi et l.) Figure S7 Muscle regenertive potentil of muscle-derived cells. () Trnsplnttion protocol. () Trnsplnted muscles were nlyzed for the expression of GFP nd lminin α2. (c) Quntittive nlysis of trnsplnttion experiments. n = 5 in ech group; dt re represented s mens ± s.d. Scle r indictes 20 µm. 7

8 M-cd PDGFRα lminin α2 M-cd PDGFRα lminin α2 DAPI PDGFRα Dlk1 Dlk1 PDGFRα pn-lminin DAPI Figure S8 (Uezumi et l.) Figure S8 Locliztion of PDGFRα + cells in muscle. (, ) Muscle sections were stined with ntiodies ginst M-cdherin (M-cd), PDGFRα, nd lminin α2 (); Dlk1, PDGFRα, nd pn-lminin (). Arrows indicte PDGFRα + cells locted in the interstitil spce of muscle tissue. Arrowheds in indicte stellite cells. Scle rs represent 10 µm. 8

9 0d Glycerol 4d GFP PDGFRα DAPI GFP CD45 DAPI GFP PDGFRα DAPI GFP CD45 DAPI c Glycerol 14d GFP C/EBPα GFP CD45 C/EBPα DAPI Oil Red O DAPI HE Figure S9 Bone mrrow cells do not give rise either to PDGFRα + cells or to dipocytes in glycerol-injured muscle. C57BL/6 mice were lethlly irrdited nd trnsplnted with whole BM cells from GFP Tg mice. Five months fter BM trnsplnttion, mice showing BM chimerism greter thn 90% were sujected to nlysis. () Uninjured muscles of BM-trnsplnted mice were nlysed for the expression of GFP nd PDGFRα or GFP nd CD45. () Four dys fter glycerol injection, muscles of BM-trnsplnted mice were nlysed for the expression of GFP nd PDGFRα or GFP nd CD45. (c) Two weeks fter glycerol injection, muscle sections of BM-trnsplnted mice were sujected to immunofluorescent stining for GFP, CD45, nd C/EBPα, followed y Oil Red O stining nd then HE stining. Arrows indicte GFP CD45 C/EBPα + Oil Red O + uniloculr dipocytes. We identified more thn 1000 dipocytes in glycerol-injected muscles from four BM-trnsplnted mice, ut could not find GFP + BM-derived dipocytes. Almost ll of the GFP + cells were positive for hemtopoietic mrker CD45 during glycerolinduced ftty degenertion. Scle rs indicte 20 µm. 9

10 c p<0.001 Glycerol 3d CTX 3d Numer of Px7(+)EdU(+) cells / section CTX 3d Glycerol 3d EdU Px7 EdU Px7 lminin α2 lminin α2 DAPI Figure S10 (Uezumi et l.) Figure S10 Prolifertion of myogenic progenitors is severely impired in glycerol-injured muscle. (, ) EdU ws dministered 2 nd 3 dys fter CTX () or glycerol () injection. Two hours fter lst EdU dministrtion, mice were scrificed nd muscle sections were sujected to immunofluorescent stining for Px7 nd lminin α2 followed y EdU detection. (c) Quntittive nlysis of Px7 + EdU + cells. n = 6 in CTX group nd n = 8 in glycerol group; dt re represented s mens ± s.d. Scle rs indicte 20 µm. 10

11 Tle S1 Primry ntiodies used Antiody Clone Conjugte(s) Dilution or finl concentrtion Source rt nti-cd13 123H1 FITC 1:100 MBL rt nti-cd29 HMβ1-1 Alex 488 1:200 BioLegend rt nti-cd Alex 488, PE, iotin rt nti-cd34 RAM34 FITC 1:100 rt nti-cd45 30-F11 Alex 488, PE, iotin 1:200 BioLegend BD PhrMingen 1:200 BioLegend rt nti-cd90 (Thy-1) CT-TH1 FITC 1:200 Cltg rt nti-cd117 (c-kit) 2B8 FITC 1:100 BioLegend rt nti-cd184 (CXCR4) 2B11 Alex 488 1:100 ebioscience rt nti-sc-1 D7 FITC 1:200 rt nti-integrin α7 CA5.5 DyLight 649 1:1000 rt nti-stellite cells SM/C-2.6 iotin 1:200 got nti-pdgfrα polyclonl unconjugted, iotin rit nti-c/ebpα polyclonl unconjugted 1:400 mouse nti-pparγ E-8 TRITC 1: µg ml -1 R&D BD PhrMingen Gift from F. Rossi S1 Gift from S. Fukd S2 Snt Cruz Biotechnology Snt Cruz Biotechnology rit nti- PPARγ 81B8 unconjugted 1:100 Cell Signling rt nti-dlk1 iotin 1:200 mouse nti-α smooth muscle ctin Gift from A. Miyjim S3 1A4 Cy3 1:200 Sigm rit nti-ng2 polyclonl unconjugted 1:200 Chemicon rit nti-lminin polyclonl unconjugted 1:200 Sigm rit nti-vimentin polyclonl unconjugted 1:50 BioVision rt nti-lminin α2 4H8-2 unconjugted 1:200 Alexis rit nti-ki67 polyclonl unconjugted 1:5 Ylem

12 rit nti-gfp polyclonl unconjugted 1:100 Chemicon chick nti-gfp polyclonl unconjugted 1:800 Chemicon rit nti-collgen IV polyclonl unconjugted 1:200 Chemicon mouse nti-px7 PAX7 unconjugted 1:2 DSHB rit nti-m-cdherin polyclonl unconjugted 1:1000 rit nti-myod polyclonl unconjugted 1:400 mouse nti-myogenin F5D unconjugted 1:100 DSHB rit nti-myogenin polyclonl unconjugted 1:200 mouse nti-srcomeric-α-ctinin Gift from S. Tked S4 Snt Cruz Biotechnology Gift from N. Hshimoto S5 EA-53 unconjugted 1:800 Sigm mouse nti-myhc MF-20 unconjugted 1:100 DSHB mouse nti-emyhc F1.652 unconjugted 1:2 DSHB rit nti-perilipin A/B polyclonl unconjugted 1:250 Sigm Secondry regents used Secondry Regent Conjugte(s) Used Source streptvidin PE-Cy5 BD PhrMingen streptvidin PerCP-Cy5.5 BD PhrMingen donkey polyclonl nti-got IgG PE Jckson ImmunoReserch donkey polyclonl nti-mouse IgG Cy3 Jckson ImmunoReserch donkey polyclonl nti-got IgG Alex 488, Alex 568 Moleculr Proes donkey polyclonl nti-mouse IgG Alex 568 Moleculr Proes donkey polyclonl nti-rit IgG Alex 488, Alex 647 Moleculr Proes donkey polyclonl nti-rt IgG Alex 488 Moleculr Proes chicken polyclonl nti-rt IgG Alex 647 Moleculr Proes got polyclonl nti-mouse IgG Alex 488, Alex 568 Moleculr Proes got polyclonl nti-rt IgG Alex 594 Moleculr Proes got polyclonl nti-rit IgG Alex 488 Moleculr Proes got polyclonl nti-chicken IgG Alex 488 Moleculr Proes

13 Tle S2 Primer sequences used Gene Forwrd Reverse Size Px3 5 CAAACCCAAGCAGGTGACAA 3 5 CAGGATGCGGCTGATAGAAC Px7 5 AAGTTCGGGAAGAAAGAGGACGAC 3 5 GAGGTCGGGTTCTGATTCCACATC myf5 5 GTCAACCAAGCTTTCGAGACG 3 5 CGGAGCTTTTATCTGCAGCAC MyoD 5 ACATAGACTTGACAGGCCCCGA 3 5 AGACCTTCGATGTAGCGGATGG Tie2 5 CCGTGGACAGGGGAGATAAT 3 5 CCACTACACCTTTCTTTACA Flk1 5 TCTGTGGTTCTGCGTGGAGA 3 5 GTATCATTTCCAACCACCCT vwf 5 GCGGCAATAGGACAAACAC 3 5 GATGAAGATGGGAGCGATG Runx2 5 GAAATGCCTCCGCTGTTATG 3 5 AGGTGAAACTCTTGCCTCGTC 3 90 PDGFRα 5 GACGAGTGTCCTTCGCCAAAGTG 3 5 CAAAATCCGACCAAGCACGAGG PDGFRβ 5 GCAGCGACACTCCAACAAGCA 3 5 TCACTCTCCCCAGTCAGGTTCAAG CD68 5 AGGTCCAGGGAGGTTGTGA 3 5 CCGCCATGTAGTCCAGGTAG F4/80 5 AAGCATCCGAGACACACACA 3 5 GGCAAGACATACCAGGGAGA OB-cd 5 GGACTCTCAGGGACAACCAA 3 5 TCTGGGTCTTTAGCCTTCACC C/EBPα 5 GCCGAGATAAAGCCAAACAAC 3 5 GACCCGAAACCATCCTCTG C/EBPβ 5 GCCAAGAAGACGGTGGACAA 3 5 ACAAGTTCCGCAGGGTGCT PPARγ 5 TTGCTGAACGTGAAGCCCATCGAGG 3 5 GTCCTTGTAGATCTCCTGGAGCAG leptin 5 TCCTGTGGCTTTGGTCCTATC 3 5 ATACCGACTGCGTGTGTGAA diponectin 5 GCCGTTCTCTTCACCTACGA 3 5 ACTTGGTCTCCCACCTCCA 3 92 osteoclcin 5 AAGCAGGAGGGCAATAAGGT 3 5 ATGCGTTTGTAGGCGGTCTT osteopontin 5 CTCCTTGCGCCACAGAATG 3 5 CTCGCTCTCTGCATGGTCTC αsma 5 GAGAAGCCCAGCCAGTCG 3 5 CTCTTGCTCTGGGCTTCA SM22 5 TAATGGCTTTGGGCAGTTTG 3 5 TGCAGTTGGCTGTCTGTGAA smmhc 5 GCAGAAGGCTCAGACCAAAG 3 5 TATCCAGAATGCCCAGGAAG Clponin1 5 AAACCCCACGACATCTTTGA 3 5 GCATACTTGACTCCCACATTGA GAPDH 5 CCTGGAGAAACCTGCCAAGTATG 3 5 AGAGTGGGAGTTGCTGTTGAAGTC 3 133

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