Live-animal imaging of native haematopoietic stem and progenitor cells

Abstract

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3,4,5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Generation and characterization of Mds1GFP/+Flt3Cre (MFG) mice.
Fig. 2: Steady-state localization and oxygen levels around MFG-HSCs and HSPCs.
Fig. 3: Increased motility, expansion, and localization of activated MFG-HSC s.
Fig. 4: Heterogeneity of bone remodelling stages governs expansion of MFG-HSCs (Mds1GFP/+Flt3Cre mice) and HSPCs (Mds1GFP/+ mice).

Data availability

The GEO accession number for GFP cells is GSE115908. The GEO accession number for LSK cells used for overlay has been published previously (GSE90742)18.

References

  1. 1.

    Sun, J. et al. Clonal dynamics of native haematopoiesis. Nature 514, 322–327 (2014).

  2. 2.

    Busch, K. & Rodewald, H. R. Unperturbed vs. post-transplantation hematopoiesis: both in vivo but different. Curr. Opin. Hematol. 23, 295–303 (2016).

  3. 3.

    Lo Celso, C. et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457, 92–96 (2009).

  4. 4.

    Lo Celso, C., Lin, C. P. & Scadden, D. T. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nat. Protoc. 6, 1–14 (2011).

  5. 5.

    Sipkins, D. A. et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973 (2005).

  6. 6.

    Cao, X. et al. Irradiation induces bone injury by damaging bone marrow microenvironment for stem cells. Proc. Natl Acad. Sci. USA 108, 1609–1614 (2011).

  7. 7.

    Acar, M. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015).

  8. 8.

    Chen, J. Y. et al. Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 530, 223–227 (2016).

  9. 9.

    Gazit, R. et al. Fgd5 identifies hematopoietic stem cells in the murine bone marrow. J. Exp. Med. 211, 1315–1331 (2014).

  10. 10.

    Zhang, Y. et al. PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function. Blood 118, 3853–3861 (2011).

  11. 11.

    Métais, J. Y. & Dunbar, C. E. The MDS1-EVI1 gene complex as a retrovirus integration site: impact on behavior of hematopoietic cells and implications for gene therapy. Mol. Ther. 16, 439–449 (2008).

  12. 12.

    Oguro, H., Ding, L. & Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102–116 (2013).

  13. 13.

    Pietras, E. M. et al. Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions. Cell Stem Cell 17, 35–46 (2015).

  14. 14.

    Boyer, S. W., Schroeder, A. V., Smith-Berdan, S. & Forsberg, E. C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells. Cell Stem Cell 9, 64–73 (2011).

  15. 15.

    Buza-Vidas, N. et al. FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells. Blood 118, 1544–1548 (2011).

  16. 16.

    Cabezas-Wallscheid, N. et al. Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy. Cell 169, 807–823.e819 (2017).

  17. 17.

    Zilionis, R. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat. Protocols 12, 44–73 (2017).

  18. 18.

    Rodriguez-Fraticelli, A. E. et al. Clonal analysis of lineage fate in native haematopoiesis. Nature 553, 212–216 (2018).

  19. 19.

    Sanjuan-Pla, A. et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature 502, 232–236 (2013).

  20. 20.

    Guo, G. et al. Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire. Cell Stem Cell 13, 492–505 (2013).

  21. 21.

    Kunisaki, Y. et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643 (2013).

  22. 22.

    Nombela-Arrieta, C. et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat. Cell Biol. 15, 533–543 (2013).

  23. 23.

    Lassailly, F., Foster, K., Lopez-Onieva, L., Currie, E. & Bonnet, D. Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells. Blood 122, 1730–1740 (2013).

  24. 24.

    Coutu, D. L., Kokkaliaris, K. D., Kunz, L. & Schroeder, T. Multicolor quantitative confocal imaging cytometry. Nat. Methods 15, 39–46 (2018).

  25. 25.

    Takubo, K. & Suda, T. Roles of the hypoxia response system in hematopoietic and leukemic stem cells. Int. J. Hematol. 95, 478–483 (2012).

  26. 26.

    Parmar, K., Mauch, P., Vergilio, J. A., Sackstein, R. & Down, J. D. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc. Natl Acad. Sci. USA 104, 5431–5436 (2007).

  27. 27.

    Spencer, J. A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014).

  28. 28.

    Morrison, S. J., Wright, D. E. & Weissman, I. L. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc. Natl Acad. Sci. USA 94, 1908–1913 (1997).

  29. 29.

    Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

  30. 30.

    Yeh, S. A., Wilk, K., Lin, C. P. & Intini, G. In vivo 3D histomorphometry quantifies bone apposition and skeletal progenitor cell differentiation. Sci. Rep. 8, 5580 (2018).

  31. 31.

    Rashidi, N. M. et al. In vivo time-lapse imaging shows diverse niche engagement by quiescent and naturally activated hematopoietic stem cells. Blood 124, 79–83 (2014).

  32. 32.

    Adams, G. B. et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439, 599–603 (2006).

  33. 33.

    Kollet, O. et al. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat. Med. 12, 657–664 (2006).

  34. 34.

    Medaglia, C. et al. Spatial reconstruction of immune niches by combining photoactivatable reporters and scRNA-seq. Science 358, 1622–1626 (2017).

  35. 35.

    Rodríguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

  36. 36.

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  37. 37.

    Kalajzic, Z. et al. Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters. Bone 31, 654–660 (2002).

  38. 38.

    Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).

  39. 39.

    Esipova, T. V. et al. Two new “protected” oxyphors for biological oximetry: properties and application in tumor imaging. Anal. Chem. 83, 8756–8765 (2011).

  40. 40.

    Lebedev, A. Y. et al. Dendritic phosphorescent probes for oxygen imaging in biological systems. ACS Appl. Mater. Interfaces 1, 1292–1304 (2009).

  41. 41.

    Esipova, T. V., Rivera-Jacquez, H. J., Weber, B., Masunov, A. E. & Vinogradov, S. A. Two-photon absorbing phosphorescent metalloporphyrins: effects of π-extension and peripheral substitution. J. Am. Chem. Soc. 138, 15648–15662 (2016).

  42. 42.

    Lo Celso, C., Wu, J. W. & Lin, C. P. In vivo imaging of hematopoietic stem cells and their microenvironment. J. Biophotonics 2, 619–631 (2009).

  43. 43.

    Bixel, M. G. et al. Flow dynamics and HSPC homing in bone marrow microvessels. Cell Rep. 18, 1804–1816 (2017).

  44. 44.

    Itkin, T. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 532, 323–328 (2016).

  45. 45.

    Mondor, I. et al. Clonal proliferation and stochastic pruning orchestrate lymph node vasculature remodeling. Immunity 45, 877–888 (2016).

  46. 46.

    Manolagas, S. C. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21, 115–137 (2000).

  47. 47.

    Weinstein, R. S. et al. Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J. Clin. Invest. 109, 1041–1048 (2002).

  48. 48.

    Ollion, J., Cochennec, J., Loll, F., Escudé, C. & Boudier, T. TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization. Bioinformatics 29, 1840–1841 (2013).

  49. 49.

    Matic, I. et al. Quiescent bone lining cells are a major source of osteoblasts during adulthood. Stem Cells 34, 2930–2942 (2016).

Download references

Acknowledgements

We thank Camargo laboratory members for discussions, D. Scadden and N. Severe for discussions and help with long bone staining, L. Kunz for generating initial Matlab scripts facilitating preliminary long bone data analysis and R. Matthieu of the Stem Cell Program Flow Cytometry facility for FACS assistance. This study was supported by awards from the National Institute of Health (HL128850-01A1 and P01HL13147 to F.D.C., R01EB017274, R01CA194596, R01DK115577, and R24DK103074 to C.P.L., R01EB018464, R24NS092986, EB018464 and NS092986 to S.A.V., R01CA175761 to A.S.P., and NIDKK-supported Cooperative Centers of Excellence in Hematology at BCH U54DK110805). F.D.C. is a Leukemia and Lymphoma Society Scholar and a Howard Hughes Medical Institute Scholar.

Author information

C.C. and F.D.C. designed experiments relevant to the animal models. J.A.S. and C.P.L. designed experiments relevant to live animal calvaria bone marrow imaging and fixed calvaria imaging. S.-C.A.Y. and C.P.L. designed experiments relevant to imaging of bone cavity types in the calvaria and tibia. K.D.K. and T.S. designed experiments relevant to femur staining and imaging. C.C, A.R., A.S.P., Y.Z. and S.R. generated the mouse models. C.C. performed all animal-related experiments and relevant data analysis. R.A.C. and R.P. supervised and performed the bioinformatics analysis, respectively. J.A.S. and N.S. performed the live animal calvaria imaging experiments, fixed calvaria imaging, and relevant data analysis. R.T. performed part of the live animal calvaria imaging experiments and relevant data analysis. T.V.E. and S.A.V. generated the pO2 probe and performed relevant characterization. K.D.K. performed the long bone imaging experiments and data analysis. S.-C.A.Y. performed the imaging experiments and analysis of bone cavity types and cell proliferation. S.H.O. and G.G. designed the fluidigm experiments. G.G. performed the fluidigm experiments and related analysis. C.C., J.A.S., S.-C.A.Y., C.P.L. and F.D.C. wrote the manuscript. C.P.L. and F.D.C. supervised the project and gave final approval.

Correspondence to Charles P. Lin or Fernando D. Camargo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Elisa Laurenti, Cristina Lo Celso and Hanna Mikkola for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of HSPC (Mds1GFP/+) mice demonstrates normal haematopoiesis, HSC frequency, cell cycle and stimuli recovery response.

a, Targeting strategy for the generation of Mds1GFP//+ mice. b, Eight-to-twelve-week-old Mds1GFP/+ mice (n = 9) show similar bone marrow cellularity to control mice (Mds1+/+; n = 7); mean ± s.d. c, eight-to-twelve-week-old Mds1GFP/+ mice (n = 14) have similar peripheral blood parameters to Mds1+/+ control mice (n = 11); mean ± s.d. d, Eight-to-twelve-week-old Mds1GFP/+ mice (n = 7) showed similar frequencies of CD150+CD48LSK (LT-HSCs), CD150CD48LSK (ST-HSCs) and CD150CD48+LSK (MPPs) to control mice (Mds1+/+; n = 4); mean ± s.d. e, Cell cycle analysis of SLAM cells from Mds1GFP/+ (n = 3) and wild-type (Mds1+/+; n = 2) mice in native conditions. Indicated value per gate represents mean ± s.d. f, Dynamics of recovery of white blood cells (WBC), lymphocytes (LY) and red blood cells (RBC) upon 5-FU treatment in Mds1GFP/+ and control (Mds1+/+) mice. Mean ± s.d., n = 4 mice. Source data

Extended Data Fig. 2 Flow cytometric analysis of Mds1GFP/+ expression.

a, GFP+ cells are not present in any mature cellular subpopulations. Data shown are from one representative experiment that was repeated three times. b, c, Mds1GFP/+ cells are not present in the CD45 bone marrow compartment or in mesenchymal (integrin-αV and PDGFRα) or endothelial (CD31 and VE-cadherin) bone marrow niche components. The experiment was performed once. d, Flow cytometry analysis reveals an inverse correlation between MDS1–GFP expression and FLT3 staining in LINSCA+CKIT+ cells. The experiment was performed twice with similar results.

Extended Data Fig. 3 Generation of the MFG (Mds1GFP/+ Flt3Cre) mice results in restriction of GFP expression to LT-HSCs.

a, Schematic of genetic strategy to restrict GFP expression to LT-HSC compartment. b, Analysis of bone marrow from Flt3CreR26LSL-Tom mice shows Flt3Cre-driven activity in compartments downstream of LT-HSCs. n = 4 mice; mean ± s.d. c, Further characterization of the CKIT+SCA1GFP+ cells from MFG mice. CD41+CD150+ cells represent  megakaryocyte progenitors. The experiment was performed twice with similar results. d, Flow characterization of MFG cell in marrow isolated from multiple bones. The experiment was performed three times with similar results. e, MFG-HSCs are predominantly found within the CD34Flt3CD150+ bone marrow fraction. The experiment was performed twice with similar results. f, MFG-HSCs are predominantly found within the SCA1highEPCR+ bone marrow fraction. The experiment was performed once. g, Cell cycle analysis of SLAM cells that are either GFP+ or GFP in MFG mice. Pooled data from three mice. Source data

Extended Data Fig. 4 Additional characterization of MFG-HSCs.

a, b, InDrops scRNA-seq analysis of MFG+ cells in comparison to multiple populations of HSC and MPPs. MFG cells (46 cells) are predominantly found in areas where Mecom (purple, n = 742 cells), but not Flt3 (orange, n = 1,111 cells) is expressed. Teal, MPP2; purple, MPP3; light green, MPP4; grey, other cells; bright green, Mds1GFP/+Flt3Cre cells. Gradient colour demonstrates normalized read counts. Each dot represents an individual cell. MFG-HSCs represent cells from a single mouse, the rest of the cells represent cells from a separate single mouse. c, d, Heatmaps (c) and spring plot map (d) showing expression levels of previously published ‘dormant’ HSC genes in scRNA-seq data from LTHSC and MFG cell populations. For the spring plot analysis: MFG, n = 46 cells; CD34, n = 2,380 cells (teal); each dot represents an individual cell. MFG-HSCs represent cells from a single mouse; the rest of the cells represent cells from a separate single mouse. e, Single-cell transcriptional fluidigm profile of MFG-HSCs demonstrates that they cluster together with LT-HSCs. f, Summary of transplants with 3, 7, or 15 MFG or SLAM HSCs together with 100,000 bone marrow cells, analysed 4 months after transplantation. HSC frequencies were calculated using ELDA software (see Methods). g, Engraftment analysis following secondary transplantations using whole bone marrow from one primary recipient of 25 MFG+ HSCs. Experiment shown is representative of three independent experiments. h, Percentage chimaerism at 4, 8, 12, 16 and 20 weeks in primary recipients transplanted with 25 SLAM cells sorted on the basis of GFP expression isolated from Mds1GFP/+Flt3Cre mice (n = 12 GFP mice, n = 5 GFP+ mice). Our data demonstrate that GFP+ cells within the SLAM compartment are more functionally enriched. Each line represents an individual mouse. Source data

Extended Data Fig. 5 Multicolour quantitative deep-tissue confocal imaging of complete femoral sections from MFG (Mds1GFP/+Flt3Cre) mice.

a, Identification of C-KIT+GFP+MFG-HSCs using multicolour quantitative deep-tissue confocal imaging of full bone femoral sections. Pictures are 10-μm xy projections of one area of interest. n = 3 mice. The experiment was performed three times with similar results. b, Example of one full-bone femoral section with colour-coded visualization of HSCs based on their distance from bone. Yellow squares represent individual HSCs in proximity to cortical or trabecular bone, whereas green dots represent individual HSCs located more than 10 μm away. The picture represents data from an individual mouse. The experiment was performed three times with similar results (d). c, Example of full-bone femoral section (only Col.1 and DAPI staining are shown). The experiment was performed three times with similar results. d, Colour-coded visualization of HSCs based on their distance to bone. Yellow squares represent individual HSCs in proximity to cortical or trabecular bone, whereas green dots represent individual HSCs located more than 10 μm away. This picture represents an independent mouse from b. The experiment was performed three times with similar results. e, Quantification of absolute number and anatomical location of C-KIT+GFP+MFG-HSCs per individual experiment. (N = 3 mice) f, Spatial distribution of HSCs (circles) and random dots (triangles) relative to Col.1 marking bone surfaces (left panel) and CD105+ vasculature (sinusoids, right panel) (n = 3 mice). P values were calculated using two-tailed Kolmogorov–Smirnov (distance distributions, left panel P = 0.1516, right panel P > 0.9999) and one-tailed Mann–Whitney (first bin of histograms, left, HSCs: 8.56 ± 5.74, RDs: 6.88 ± 1.94, P = 0.50; right, HSCs: 67.52 ± 10.99, random dots: 68.53 ± 3.51, P = 0.35) tests. Data points with mean ± s.d. (red for HSCs, blue for random dots). NS, not significant. Epi: epiphysis, meta: metaphysis, dia: diaphysis. Source data

Extended Data Fig. 6 Synthesis, structure and characterization of phosphorescent probe Oxyphor PtG4.

The structure of Oxyphor PtG4 is almost identical to that of the previously published probe Oxyphor PdG439, but it contains Pt instead of Pd at the core of the porphyrin (1: Pt tetra-meso-3,5-dicarboxyphenyl-tetrabenzoporphyrin). a, Synthesis of Oxyphor PtG4. First, eight carboxyl groups on the porphyrin 1 were amended with 4-amino-ethylbutyrate linkers. Upon hydrolysis of the terminal esters in the resulting porphyrin 2, eight aryl-glycine dendrons (H2N-AG2(OBu)4) were coupled to the resulting porphyrin-octacarboxylic acid, giving dendrimer 3. The butyl esters on the latter were hydrolysed under mild basic conditions, and the resulting free carboxylic acid groups were amidated with mono-methoxypolyethyleneglycol amine (MeO-PEG-NH2, Av MW 1000), giving the target probe Oxyphor PtG4. MALDI–TOF (m/z) was used to confirm the identity of the intermediate products as well as of the target probe molecule. Structure 2 (C116H124N12O24Pt, calculated at MW 2,263.85) was found 2,264.48 [M]+; structure 3 (C468H540N60O120Pt, calculated at MW 9,114.76) was found at 9,115.68 [M+H]+ and Oxyphor PtG4 (C1780H3196N92O792Pt, calculated at MW 40,538) was found at 35,952. For Oxyphor PtG4 we identified an additional peak at MW 66,123.6 which is probably due to the presence of dimeric species formed during the ionization process. b, Linear (one photon) absorption (green) and emission spectra (red) of PtG4 in 50 mM phosphate buffer solution (pH 7.2, λex = 623 nm; photophysical constants in PBS, 22 °C: e(623) ~ 90,000 M−1 cm−1 (molar extinction coefficient), φphos(deox) ~ 0.07 (phosphorescence quantum yield in deoxygenated solution), τair = 16 μs (phosphorescence decay time on air), tdeox = 47 ms (phosphorescence decay time in deoxygenated solution). c, Phosphorescence oxygen quenching plot of Oxyphor PtG4. The calibration was performed as previously described39. The experimental points were fitted to an arbitrary double-exponential form and the obtained parametric equation was used to convert the phosphorescence lifetimes obtained in in vivo experiments to pO2 values. d, Two-photon absorption spectrum of PtG4 in deoxygenated dimethylacetamide (DMA, 22 °C). e, Arbitrarily scaled one- (green line) and two-photon (blue line) absorption spectra of PtG4. The two-photon absorption (2PA) spectra of PtG4 and of the reference compounds were measured by the relative phosphorescence method, as previously described41. The laser source was a Ti:Sapphire oscillator (80 MHz rep. rate) with tunability range of 680–1,300 nm (Insight Deep See, Spectra Physics). All optical spectroscopic experiments and oxygen titrations were performed at least three times, giving highly reproducible results. f, Representative intravital images of an HSPC (green, left image), MFG-HSC (green, right image), vasculature (grey, Rhodamine-B-dextran 70 kDa), and autofluorescence (blue) overlaid with localized oxygenation measurements. White arrows, GFP cells. Black arrow, colour representing 10 mm Hg. Coloured squares represent individual localized oxygen measurement areas. Images represent data from two independent experiments for each mouse model. Scale bars, ~50 µm. Source data

Extended Data Fig. 7 Increased motility and expansion of activated MFG-HSCs.

a, Schematic illustration of protocol for activating bone marrow HSCs using Cy/GCSF. b, Flow cytometry analysis of Cy/GCSF-treated MFG mice (n = 3 mice). Data show Lineage cells. Mean ± s.d. c, Number of GFP+ cells identified per calvaria in untreated and Cy/GCSF-treated Mds1GFP/+Flt3Cre mice (n = 5 and 4 mice, respectively). Red bars indicate mean. P was calculated using two-tailed Mann–Whitney test. d, Cell cycle analysis of MFG+ cells from Cy/GCSF-treated mice. Three mice were pooled together to acquire the displayed data. e, Graph showing in vivo motility measurements of HSPCs (n = 66 cells) and MFG-HSCs (n = 30 cells) at steady-state and of activated MFG-HSCs (n = 142 cells) in the calvaria. Red bars indicate mean. P were calculated using two-tailed Mann–Whitney test. f, g, Distance from MFG+ cells to the endosteum (n = 24 and 12 cells for untreated and Cy/GCSF-treated, respectively) and to the nearest vessel (n = 20 and 17 cells for untreated and Cy/GCSF-treated, respectively), after treatment with Cy/GCSF. Red bars indicate mean. P values calculated using two-tailed unpaired t-test. Source data

Extended Data Fig. 8 Characterization of MFG-HSCs upon activation.

a, Bone marrow analysis of HSPC (Mds1GFP/+) PBS control (n = 1 mouse) and HSPC (Mds1GFP/+) 5-FU-treated mice (n = 2 mice, value represents mean), 17 days after treatment. Data show marked expansion of HSPCs even after recovery of blood (Extended Data Fig. 1e). b, Graph showing in vivo motility measurements of MFG-HSCs at days 4 (n = 14 cells) and 20 (n = 13 cells) after 5-FU treatment. Red bar represents mean. Compare to untreated Mds1GFP/+Flt3Cre mice in Fig. 3a and Extended Data Fig. 7e. P was calculated using two-tailed Mann–Whitney test. c, Representative map of the locations of MFG-HSCs in the calvaria on day 20 after 5-FU treatment (n = 2 mice). Scale barm ~500 µm. d, Generation of Mds1CreER/+Rosa26Confetti/+ mice. e, Schematic illustration of Cy/GCSF treatment protocol for multicoloured Mds1CreER/+Rosa26Confetti/+ labelling and activation. Low tamoxifen dosage (2 mg) was used to restrict recombination and expression of fluorescence in LT-HSCs that express higher levels of Mds1. f, Detailed flow cytometry analysis of MPP3/4 cells, ST-HSCs and LT-HSCs with differential colour labelling upon treatment of Mds1CreER/+Rosa26Confetti/+ mice shows labelling enriched in but not fully restricted to LT-HSCs. The experiment was performed once. g, 2D maps of the 3D locations of activated and labelled HSPCs in the fixed calvaria of control (left top, tamoxifen only, n = 2 mice) and induced mice (left bottom, tamoxifen + Cy/GCSF, n = 3 mice) along with maximum intensity projection (MIP) images (right top and bottom) of the Mds1-labelled cells (red, green, and blue). Scale bars for graphical map and MIP images, ~200 µm and 50 µm, respectively. h, Colour purity of cell clusters (original colours) compared to randomized colours (10,000 cycles) in three independent experiments (n = 3 mice). P values calculated using two-tailed unpaired t-test. Bar graphs with error bars represent mean and s.d., respectively. Source data

Extended Data Fig. 9 Validating bone cavity types using 2.3Col1–GFP (mature osteoblasts) and cathepsin K-activated fluorescent agent (osteoclasts).

a, A montage of multiple z-stacks, displayed as the maximum intensity projection, showing double staining of bone marrow cavities in the calvarium. b, The same area as in a, showing the locations of 2.3Col1–GFP osteoblasts in areas of the old bone front that has not been eroded (n = 3 mice). c, Quantification of 2.3Col1–GFP pixels in D-type (n = 10 regions), M-type (n = 16 regions) and R-type cavities (n = 18 regions). Mean ± s.d. d, A montage of multiple z-stacks, displayed as the maximum intensity projection, showing the double staining pattern (blue and red), 2.3Col1–GFP cells (green), osteoclasts (white), and bone marrow vasculature (purple). White arrows, osteoclast clusters. n = 3 mice. e, A zoomed-in region from d (box A), showing correlation between 2.3Col1–GFP cells and the remaining dye 1 (blue) in a D-type cavity, and abundant cathepsin K+ osteoclasts in the R-type region where dye 1 was eroded. f, Examples of an M-type region from d (box B). In this region, dye 1 was eroded to some extent in spite of the presence of abundant 2.3Col1–GFP cells in the cavity. The corresponding cathepsin K panel shows the co-existence of several cathepsin K+ osteoclasts. g, Quantification of cathepsin K+ pixels in D-type (n = 11 regions), M-type (n = 33 regions), and R-type (n = 10 regions) cavities based on maximum intensity projection of montaged z-stacks. Compared to c, cathepsin K coverage shows a larger spread because it does not stain the cell body uniformly. Instead it frequently shows a punctate staining pattern, which is likely to represent lysosomes and endosomes. *P < 0.0189; **P = 0.0015; ****P < 0.0001; two-sided Mann–Whitney test. Mean ± s.d. Source data

Extended Data Fig. 10 Cell distribution in D-, M- and R-type cavities before and after Cy/GCSF treatment.

n = 4 mice per group. Graphs show the fractions of MDS or MFG cells distributed in D-, M- and R-type cavities at the steady state and after Cy/GCSF treatment. The fraction is calculated by the total cells found in each cavity type divided by the total cells found in the calvaria of that mouse. a, The fractions of MFG cells increased in M-type cavities but decreased in D-type cavities after Cy/GCSF treatment. Mean ± s.d. Non-treated groups: 24.5 ± 12.8, 54.3 ± 12.6 and 21.3 ± 15.6 in D-, M- and R-type cavities, respectively. Treated groups: 0.5 ± 1.0, 96.0 ± 4.7 and 3.5 ± 4.4 in D-, M- and R-type cavities, respectively. **P = 0.0096; *** P = 0.0008. b, The fractions of cells decreased in D-type cavities but remained the same in M- and R-type cavities. Mean ± s.d. Non-treated groups: 20.5 ± 5.6, 66.5 ± 2.4 and 13.3 ± 3.6 in D-, M- and R-type cavities, respectively. Treated groups: 6.8 ± 2.5, 75.0 ± 9.6 and 18.8 ± 8.9 in D-, M- and R-type cavities, respectively. **P = 0.004; unpaired, two-tailed t-test.

Extended Data Fig. 11 Heterogeneous bone remodelling in bone marrow cavities of tibia metaphysis.

A mechanically thinned metaphysis was imaged from the bone surface, labelled by sequential calcium staining. ac, En face views of D-, M- and R-type cavities from tibia metaphysis. df, xz cross-section views from annotated white lines in Supplementary Video 15 show bone marrow cavities of varied remodelling stages similar to mouse calvaria.

Extended Data Table 1 Activated MFG-HSCs (Mds1GFP/+Flt3Cre mice) are characterized by increased motility and various cellular interactions between GFP cells
Extended Data Table 2 Summary table of findings from live imaging of native HSCs versus transplanted HSCs

Supplementary information

Supplementary Information

This file contains supplementary files 1-3. Supplementary file 1: Flow cytometry schematic of cell surface markers used for the identification of MPP3/4, MPP2, ST-HSC, LT-HSC and megakaryocyte progenitors. In summary these populations are identified by the next combination of cell surface markers: MPP3/4: Lin-ckit+Sca+CD48+CD150-, MPP2: Lin-ckit+Sca+CD48+CD150+, ST-HSC: Lin-ckit+Sca+CD48-CD150+, LT-HSC: Lin-ckit+Sca+CD48-CD150+, megakaryocyte progenitors: Lin-ckit+Sca-/lowFcgR-CD41+CD150+. Supplementary file 2: Flow cytometry schematic of cell surface markers used for the identification of various mature bone marrow populations. In summary these populations are identified by the next combination of cell surface markers: pre/pro B cells: B220+IgM-, Immature B cells: B220highIgM+, Mature B cells: B220interIgM+, Neutrophils: Mac1+Ly6G+, Monocytes: Mac1+Ly6G-, T cells: CD4+CD8a+, Granulocytes: B220-CD4-CD8a-Ly6G+, Erythroid: Ter119+. Supplementary file 3: Flow cytometry schematic of cell surface markers used for the identification of endothelial cells within the bone marrow. To ensure identification of this population lineage depletion is performed prior to flow cytometry analysis. In summary this population is identified by the next combination of cell surface markers: Lin-CD45-CD31+VECadherin+.

Reporting Summary

Video 1

Intravital z-stack video (1 µm/z-step) starting from about 40 µm into the bone of HSPCs (bright green, GFP), blood vessels (red, Angiosense 680EX), auto-fluorescence (blue), and bone (white, second harmonic generation). Examples of an arteriole (red arrows), transitional vessel (green arrows), and sinusoid (blue arrows) are labeled in the video. Scale bar ~50 µm. (n=8 mice).

Video 2

Intravital z-stack video (1 µm/z-step) starting from about 40 µm into the bone of an MFG-HSC (bright green, GFP), blood vessels (red, Angiosense 680EX), auto-fluorescence (blue), and bone (white, second harmonic generation). Scale bar ~50 µm. (n=10 mice).

Video 3

Intravital time-lapse video (30 sec/frame) of an MFG-HSC (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue) from the same field of view as Video 2. Scale bar ~50 µm. (n=8 mice).

Video 4

Intravital time-lapse video (30 sec/frame) of an HSPC (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue). Scale bar ~50 µm. (n=3 mice).

Video 5

Long-term intravital time-lapse video (30 min/frame for 2.5 hrs) of HSPCs (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue). Scale bar ~50 µm. (n=1 mouse).

Video 6

Long-term intravital time-lapse video (30 min/frame for 2.5 hrs) of an MFG-HSC (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue). Scale bar ~50 µm. (n=2 mice).

Video 7

Intravital z-stack video (2 µm/z-step) starting from about 40 µm into the bone of Cy/G-CSF activated MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), auto-fluorescence (blue), and bone (white, second harmonic generation). Scale bar ~50 µm. (n=4 mice).

Video 8

Intravital time-lapse video (20 min/frame for 6 hrs) of Cy/G-CSF activated MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue) from the same field of view as Video 7. Scale bar ~50 µm. (n=4 mice).

Video 9

Intravital z-stack video (2 µm/z-step) starting from about 40 µm into the bone of MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), auto-fluorescence (blue), and bone (white, second harmonic generation) on day 4 after 5-fluorouracil treatment. Scale bar ~50 µm. (n=1 mouse).

Video 10

Intravital time-lapse video (30 min/frame for 2.5hr) of MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue) on day 4 after 5-fluorouracil treatment. Scale bar ~50 µm. (n=1 mouse).

Video 11

Intravital z-stack video (2 µm/z-step) starting from about 40 µm into the bone of MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), auto-fluorescence (blue), and bone (white, second harmonic generation) on day 20 after 5-fluorouracil treatment. Scale bar ~50 µm. (n=2 mice).

Video 12

Intravital time-lapse video (30 min/frame for 2.5hr) of MFG-HSCs (bright green, GFP), blood vessels (red, Angiosense 680EX), and auto-fluorescence (blue) on day 20 after 5-fluorouracil treatment. Scale bar ~50 µm. (n=2 mice).

Video 13

Intravital z-stack video (2 µm/z-step) starting from just above the endosteum into the bone marrow to show clonal proliferation of MFG-HSCs (bright green, GFP), as well as autofluorescent cells (blue), bone (white, second harmonic generation), Dye1 (yellow, tetracycline), and Dye 2 (purple, alizarin red). Scale bar ~50 µm. (n=4 mice).

Video 14

Intravital z-stack video (2 µm/z-step) starting from just above the endosteum into the bone marrow to show an example of cell proliferation in a M-type cavity (Left) compared to a D-type cavity (Right). HSPCs (bright green, GFP), autofluorescent cells (blue), bone (white, second harmonic generation), Dye1 (yellow, tetracycline), and Dye 2 (purple, alizarin red). Scale bar ~50 µm. (n=4 mice).

Video 15

Z-stack video (3 µm/z-step) of a freshly harvested and thinned tibia, imaged from above the endosteum to show distinct bone remodeling activities in the metaphysis. The x-z cross-sections of white lines are demonstrated in Extended data figure 10. Dye1 (blue, tetracycline); Dye2 (red, alizarin red); bone (white, second harmonic generation). Scale bar ~500 µm. (n=4 mice).

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Christodoulou, C., Spencer, J.A., Yeh, S.A. et al. Live-animal imaging of native haematopoietic stem and progenitor cells. Nature 578, 278–283 (2020). https://doi.org/10.1038/s41586-020-1971-z

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.