Supplementary Figure 1

Transcription

1 DOI: 1.138/ncb273 Supplementary Figure 1 a 1x DAPI field images 4x contoured cells 2µm 5µm b Tissue map Dot plot Histogram Y X antigen X DAPI antigen X Figure S1 Laser Scanning Cytometry of bone marrow microenvironments. (a) Workflow of global LSC-based analysis of BM populations in femoral cavities. Whole longitudinal, 5 µm-thick cryosections of femoral bones are scanned using the icys research cytometer. Low-resolution digital images of entire femoral cavities stained with the nuclear marker DAPI, are generated through scanning with a 45 nm laser and a low magnification objective lens (1x, upper panel). Dynamic scanning areas are defined to cover the entire BM cavity, and are subsequently scanned with lasers of choice (45nm, 488nm, 561nm, 633nm) using a 4x objective lens and a step size of.25 mm, to acquire high-resolution field images in different fluorescent channels. Images are assembled to generate multicolor mosaic images of the entire BM cavity. Lower panel depicts two grayscale field images (right) and a composite image of one BM region generated in the DAPI channel. Automatic image segmentation of DAPI + nuclei is performed with the icys analysis software to generate contours around single nuclei, which define cellular events. Positional information in a XY Cartesian coordinate system is recorded for each individual cell enabling the generation of tissue maps (b) The specific signal in every fluorescent channel is quantified on a per cell basis and can be displayed and analyzed in dot-plot scattergrams and histogram charts. 1

2 3D confocal c-kit Laminin Figure S2 Perivascular accumulation of c-kit + progenitors in the BM. Representative 3D projection of optical image stacks of thick femoral slices wholemount stained for c-kit (green) and Laminin (red). Multiple c-kit + cells are shown in direct contact with Laminin + vessels (see also Supplementary Video 1). 2

3 Supplementary Figure 3 a Lin - CD48 - c-kit + 1.1% 84.1% CD48 c-kit Sca-1 CD % CD3, Ter-119, B22, Gr CD15 b CFU-C/5 cells d Freq. of population (%) WBMC LSK Lin- CD48 - c-kit + c Freq. of BMC (%) MET Metaphysis DIA Lin - c-kit + SK Lin - CD48 - CD41 lo/- c-kit + Lin - c-kit + SK Lin - CD48 - CD41 lo/- c-kit >5-1 Distance to bone (µm) e 4 Diaphysis Lin - c-kit + SK Freq. of population (%) Lin - CD48 - CD41 lo/- c-kit >5-1 Distance to bone (µm) Figure S3 Quantification of distribution of HSPCs in the bone marrow. (a) Flow cytometry analysis of the expression of Sca-1 and CD15 of Lineage - CD48 - c-kit + cells. Although expression of CD41 can be detected in HSPCs by FACS, our immunofluorescence analysis of BM sections CD41 labeled exclusively megakaryocytes and platelets, which express much higher levels than HSPCs. Thus, by LSC we gated on the CD41 lo/- population to exclude both megakaryoctes and platelets from our HSPC analysis. (b) CFU-C content of Lin - CD48 - c-kit + cells compared to that of Lin - Sca-1 + c-kit + (LSK) cells and whole BM cells (WBMC). (c) Absolute frequencies of HSPC subsets in the metaphysis and diaphysis of femoral BM as measured by LSC. (d and e) Histograms of the distances Lin - c-kit + of SK and Lin - CD48 - CD41 lo/- c-kit + HSPC populations to nearest bone surfaces in the metaphysis (d) and diaphysis (e) of femoral cryosections. 3

4 a Supplementary Figure 4 Nombela-Arrieta and Silberstein c-kit 5K 1K 15K 2K 25K FSC-A Lineage Sca-1 i.v injection LS - K - LKS - LSK PBS anti CD45-PE CD45-PE b CD45-PECy Laminin c **** 6 5 **** % intravascular LSK LKS - LS - K - B22 + Figure S4 HSPCs do not reside in the intravascular compartment of the BM. Labeling of hematopoietic BM intravascular populations by injection of PEconjugated anti-cd45. Representative FACS dot plots (a) and quantification (c) of PE + LSK, LS - K (Lin - c-kit + Sca-1 - ) and LS - K - (Lin - c-kit + Sca-1 - ) cells in the BM of mice after injection of PBS (control) or CD45-PE (upper and lower panels) n=1 ****p<,1. (b) Representative 3D projection of optical image stacks of the BM cavities of CD45-PE injected mice. CD45-PE + cells (red) are exclusively observed inside vascular structures (green) therefore validating the specificity of the method as previously reported. See also Supplementary Video

5 Supplementary Video legends Supplementary Video 1. Interaction of c-kit cells with BM microvasculature. 3D-reconstruction of confocal optical sections of thick bone marrow slices stained for c-kit (green) and the vascular marker Laminin (red). Multiple examples of vessel-adjacent c-kit + cells can be visualized. Supplementary Video 2. In vivo labeling of intravascular populations. The movie depicts a series of sequential optical sections on the z-axis followed by the 3D-reconstruction of a thick BM femoral slice, 2 mins post-injection of CD45-PE. Intravascular populations (blood vessels in green labeled with Laminin) are specifically stained with CD45 (red). Supplementary Video 3. Vascular compartment of the femoral diaphysis. 3D-reconstruction of confocal optical sections of a thick slice of a murine femur stained with the panvascular marker Laminin (green) and the arterial marker Sca-1 (red). Supplementary Video 4. BM sinusoidal microvasculature. 3D-reconstruction of multiphoton optical sections of a femoral diaphysis stained for Laminin (green). The central sinus, as well as an underlying central artery can be visualized running longitudinally across the diaphysis. Bone is visualized in blue as revealed by second harmonic generation. Supplementary Video 5. Arteriolar to sinusoidal transition in endosteal regions of the diaphysis. High-magnification 3D-reconstruction of the endosteal vessels shown in Supplementary Video 4. Arrow depicts the arteriolar to sinusoidal transition in close proximity to endosteal surfaces (bone shown in blue). Supplementary Video 6. Vascular compartment of the femoral metaphysis. 3D-reconstruction of the vascular network of a femoral metaphysis. All BM vascular structures are marked by Laminin (green), while the arterial network is positive for Sca-1 (red). Supplementary Video 7. Endosteal microvascular network of femoral metaphysis. 3D reconstruction of the vascular network of endosteal regions of a femoral metaphysis. Arrow depicts the arteriolar to sinusoidal transition in close proximity to endosteal surfaces (bone shown in blue). Supplementary Table Legends Supplementary Table 1. Complete list and technical specifications of antibodies employed in this study for flow cytometry and immunostaining of BM cryosections. Supplementary Table 2. Detailed strategies for immunofluorescent stainings, including antibody and fluorochrome combinations and laser and filter setups, used for LSC detection and analysis of the different hematopoietic populations in BM sections. Supplementary Tables 3 and 4. Statistics source data for Figures 6b and 6c. 5

6 Protocol for immunofluorescent staining of murine femoral BM cryosections 1) Fix tissues and rehydrate as described in Methods section. Freeze embedded in OCT. 2) Section using a Leica Cryostat and the Cryojane tape transfer system (Leica microsystems) 3) Rehydrate in PBS (2 mins) and.1% Tween2/ PBS (2 mins) 4) Block in PBS/1% donkey serum 5) Endogenous biotin block using Vector Biotin Blocking Kit according to manufacturerʼs instructions 6) Incubate in primary antibodies in PBS /1% donkey serum for 1 hour at room temp or overnight at 4 C (antibodies in Supplementary Table 1). 7) Wash in PBS/.1% Tween2 3x5 minutes 8) Secondary Antibodies: Room temperature for 1 hour in PBS /1% donkey serum 9) Wash in PBS/.1% Tween2 3x5 minutes 1) Tertiary antibody and additional steps: Room temperature for 1 hour in PBS /1% donkey serum 11) Incubation with DAPI.5µM 1.µM (Invitrogen) 12) Wash in PBS/.1% Tween2 3x5 minutes 13) Dehydrate in EtOH gradient: 7%, 85%, 95%, 1%, 3 minutes each 14) Rinse in xylene 2x5 minutes 15) Coverslip with mounting media (Vectashield, Vector labs), store at 4 C until scanned

7 Supplementary Table 1. Technical specifications of the Antibodies (dilutions, clones and sources) employed in our study Antigen/Specificity Conjugation Concentration Host Clone Source Catalogue # Application CD117/c-kit PECy7 1µg/ml Rat 2B8 BioLegend FC Sca-1/Ly6A/E APCCy7 1µg/ml Rat D7 BioLegend FC Gr-1 APC 1µg/ml Rat RB6-8C5 BioLegend FC CD3 APC 1µg/ml Rat 145-2C11 ebioscience FC B22 APC 1µg/ml Rat RA3-6B2 BioLegend FC Ter-119 APC 1µg/ml Rat Ter-119 ebioscience FC CD34 FITC 1µg/ml Rat RAM34 ebioscience FC CD34 PerCpCy5.5 1µg/ml Rat HM34 BioLegend FC CD48 FITC 2.5µg/ml Rat HM48-1 ebioscience FC CD15 PE 1µg/ml Rat TC15-12F12.2 BioLegend FC CD45 PE injected i.v 1µg Rat 3-F11 BD biosciences FC CD45 PECy7 injected i.v 1µg Rat 3-F11 BioLegend FC B22 PerCpCy5.5 1µg/ml Rat RA3-6B2 BioLegend FC Streptavidin PerCpCy5.5 1µg/ml N/A N/A BD biosciences FC mouse IgG 1 isotype ctrl APC 2µl/test Mouse MOPC-21 BD biosciences FC Ki67 APC 5µl/test Mouse B56 BD biosciences FC mouse IgG isotype ctrl PE.5µg/test Mouse R&D IC2P FC HIF-1a PE.5µg/test Mouse R&D IC1935P FC Pimonidazole FITC 2.5 µg/ml Mouse Hypoxyprobe HP2-1 FC/IC Gr-1 biotin 1µg/ml Rat RB6-8C5 ebioscience FC/IC CD3 biotin 1µg/ml Hamster 145-2C11 ebioscience FC/IC B22 biotin 1µg/ml Rat RA-6B2 ebioscience FC/IC Ter-119 biotin 1µg/ml Rat Ter-119 ebioscience FC/IC CD117/c-kit unconjugated 5µg/ml Goat polyclonal R&D IEO2 IF Sca-1 unconjugated 5µg/ml Rat E BioLegend IF Laminin unconjugated 1µg/ml Rabbit polyclonal Sigma L9393 IF B22 unconjugated 5µg/ml Rat RA-6B2 ebioscience IF Ter-119 unconjugated 5µg/ml Rat Ter-119 ebioscience IF Gr-1 unconjugated 6.25µg/ml Rat RB6-8C5 BD biosciences IF CD48 Biotin 5µg/ml Hamster HM48-1 BioLegend 1342 IF CD41 Biotin 5µg/ml Rat ebiomwreg3 ebioscience IF CD4 unconjugated 5µg/ml Rat H BioLegend 1332 IF CD8 unconjugated 5µg/ml Rat BioLegend 171 IF Pimonidazole DyLight549 1 µg/ml Mouse Hypoxyprobe HP2-1 IF HIF-1a unconjugated 1:5 Rabbit polyclonal Thermoscientific PA IF Endoglin unconjugated.6µg/ml Goat polyclonal R&D AF132 IF rat IgG DyLight µg/ml Donkey polyclonal Jackson immunoresearch * IF rat IgG DyLight µg/ml Donkey polyclonal Jackson immunoresearch * IF rat IgG DyLight µg/ml Donkey polyclonal Jackson immunoresearch * IF goat IgG Biotin 7.5µg/ml Donkey polyclonal Jackson immunoresearch IF goat IgG DyLight µg/ml Donkey polyclonal Jackson immunoresearch * IF rabbit IgG Biotin 7.5µg/ml Donkey polyclonal Jackson immunoresearch IF rabbit IgG DyLight µg/ml Donkey polyclonal Jackson immunoresearch * IF hamster IgG Biotin 7.5µg/ml Goat polyclonal Jackson immunoresearch IF Streptavidin Brilliant Violet 65 5µg/ml N/A N/A BioLegend IF Streptavidin Qdot75.1µM N/A N/A Invitrogen Q1161MP IF Streptavidin AF555 4µg/mL N/A N/A Invitrogen S32355 IF isotype ctrl goat IgG unconjugated 5µg/ml Goat polyclonal Southern Biotech 19-1 IF isotype ctrl Rabbit IgG unconjugated 1µg/ml rabbit polyclonal Southern Biotech IF isotype ctrl Rat IgG unconjugated 2µg/ml rat ebr2a ebioscience IF IF= Imunofluorescence FC= flow cytometry *discontinued by company

8 Supplementary Table 2: Antibodies and strategies used for immunofluorescent staining of BM cryosections Target populations c-kit + progenitors and BM vasculature Lin - c-kit + HSPCs and BM vasculature Sca-1 + c-kit + hematopoietic progenitors and BM vasculature Sca-1 + c-kit + hematopoietic progenitors, pimonidazole and BM vasculature B22 + cells, c-kit + hematopoietic progenitors, pimonidazole and BM vasculature Lin - CD48 - CD41 - c- kit + HSPCs and BM vasculature Primary antibodies Secondary/Tertiary antibodies Laser-Filter set detector- Dye - Donkey anti-goat DyLight /4-DAPI - Donkey anti-rabbit DyLight /5-DyLight549 - Rat anti-cd4 and CD8 - Rat anti-gr-1 - Rat anti-ter119 - Rat anti-sca-1 - Rat anti-sca- - FITC or DyLight549 mouse anti-pim - Rat anti-b22 - FITC or DyLight549 mouse anti-pimo - Goat anti-c-kit - Donkey anti-goat DyLight 488 -Block with excess goat IgG (5-1µg/ml) - Donkey anti-goat biotin - Streptavidin DyLight Donkey anti-rat DyLight549 - Donkey anti-rabbitdylight649 - Donkey anti-goat DyLight Donkey-anti-rat biotin + SA Alexa Donkey anti-rabbit DyLight Donkey anti-goat biotin + Streptavidin Qdot 75 - Donkey anti-rat DyLight549 or Donkey anti-rabbit DyLight649 - Donkey anti-goat biotin + Streptavidin Qdot 75 - Donkey anti-rat DyLight549 or Donkey anti-rabbit DyLight649 - Biotinylated rat anti-b22 - Biotinylated rat anti-ter11 - Biotinylated rat anti-gr-1 - Biotinylated hamster-anti CD3 - Biotinylated hamster anti-cd48 - Biotinylated rat-anti CD41 - Goat antihamster biotin - Donkey anti-rabbit DyLight649 - SA-AF /4-DAPI /5-DyLight /LP-DyLight /4-DAPI /5-DyLight /LP-DyLight /4-DAPI LP-Qdot /5-DyLight /LP-DyLight /4 DAPI LP- Qdot /15-FITC/DyLight /5-DyLight /LP- DyLight /4-DAPI /15-FITC/DyLight /5-SA-AF /LP-DyLight649 c-kit + progenitors, B22 cells and HIF-1α - Rat anti-b22 - Rabbit anti-hif-1α - Donkey anti-rabbit biotin - Donkey anti-goat DyLight488 - SA-AF555 (Invitrogen) - Donkey anti-rat DyLight /4-DAPI /15- DyLight /5-SA-AF /LP-DyLight649 Sca-1 + c-kit + HSPCs, HIF-1α and BM vasculature - Rat anti-sca-1 - Rabbit anti-hif-1α - Donkey anti-goat biotin - Streptavidin Brilliant Violet (BV) 65 - Isolectin B4 AF589 (injected) - Donkey anti-rat DyLight488 - Donkey anti-rabbit DyLight /4-DAPI /5-DyLight /LP-DyLight649