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A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells

Abstract

Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases.

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Figure 1: Human HSPCs and primary acute leukemia engraft robustly in human BM-MSC-derived ossicles.
Figure 2: Primary AML blasts preferentially engraft humanized ossicle niches, as compared to mouse BM.
Figure 3: Humanized ossicle niche transplantation reveals an increased LIC frequency in AML.
Figure 4: Humanized ossicle niches facilitate robust engraftment of APL cells, which are probably derived from lineage-committed LICs.
Figure 5: Humanized ossicle niches facilitate robust engraftment of MF, which derives from LICs in the HSC compartment.

References

  1. Doulatov, S., Notta, F., Laurenti, E. & Dick, J.E. Hematopoiesis: a human perspective. Cell Stem Cell 10, 120–136 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Wunderlich, M. et al. AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24, 1785–1788 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Nicolini, F.E., Cashman, J.D., Hogge, D.E., Humphries, R.K. & Eaves, C.J. NOD/SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration. Leukemia 18, 341–347 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Rongvaux, A. et al. Development and function of human innate immune cells in a humanized mouse model. Nat. Biotechnol. 32, 364–372 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Willinger, T. et al. Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung. Proc. Natl. Acad. Sci. USA 108, 2390–2395 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Goyama, S., Wunderlich, M. & Mulloy, J.C. Xenograft models for normal and malignant stem cells. Blood 125, 2630–2640 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Sarry, J.E. et al. Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice. J. Clin. Invest. 121, 384–395 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Sanchez, P.V. et al. A robust xenotransplantation model for acute myeloid leukemia. Leukemia 23, 2109–2117 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Patel, S. et al. Successful xenografts of AML3 samples in immunodeficient NOD/shi-SCID IL2Rγ−/− mice. Leukemia 26, 2432–2435 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Kim, D., Park, C.Y., Medeiros, B.C. & Weissman, I.L. CD19-CD45low/− CD38high/CD138+ plasma cells enrich for human tumorigenic myeloma cells. Leukemia 26, 2530–2537 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Kim, D. et al. Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells. Leukemia 26, 2538–2545 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Pang, W.W. et al. Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes. Proc. Natl. Acad. Sci. USA 110, 3011–3016 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Medyouf, H. et al. Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit. Cell Stem Cell 14, 824–837 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Mendelson, A. & Frenette, P.S. Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat. Med. 20, 833–846 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ishikawa, F. et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat. Biotechnol. 25, 1315–1321 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Schepers, K., Campbell, T.B. & Passegué, E. Normal and leukemic stem cell niches: insights and therapeutic opportunities. Cell Stem Cell 16, 254–267 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Reinisch, A. et al. Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood 125, 249–260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Saito, Y. et al. Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells. Sci. Transl. Med. 2, 17ra9 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Song, J. et al. An in vivo model to study and manipulate the hematopoietic stem cell niche. Blood 115, 2592–2600 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pettway, G.J. et al. Anabolic actions of PTH (1-34): use of a novel tissue engineering model to investigate temporal effects on bone. Bone 36, 959–970 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Zhang, J. et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425, 836–841 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Klco, J.M. et al. Functional heterogeneity of genetically defined subclones in acute myeloid leukemia. Cancer Cell 25, 379–392 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lapidot, T. et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645–648 (1994).

    Article  CAS  PubMed  Google Scholar 

  24. Bonnet, D. & Dick, J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3, 730–737 (1997).

    Article  CAS  PubMed  Google Scholar 

  25. Grimwade, D. & Enver, T. Acute promyelocytic leukemia: where does it stem from? Leukemia 18, 375–384 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Kralovics, R. et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N. Engl. J. Med. 352, 1779–1790 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Baxter, E.J. et al. Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365, 1054–1061 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Klampfl, T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N. Engl. J. Med. 369, 2379–2390 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Nangalia, J. et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N. Engl. J. Med. 369, 2391–2405 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. James, C. et al. The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity. Blood 112, 2429–2438 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Ishii, T. et al. Behavior of CD34+ cells isolated from patients with polycythemia vera in NOD/SCID mice. Exp. Hematol. 35, 1633–1640 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Wang, X. et al. Spleens of myelofibrosis patients contain malignant hematopoietic stem cells. J. Clin. Invest. 122, 3888–3899 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Dameshek, W. Some speculations on the myeloproliferative syndromes. Blood 6, 372–375 (1951).

    Article  CAS  PubMed  Google Scholar 

  34. Majeti, R., Park, C.Y. & Weissman, I.L. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1, 635–645 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Eppert, K. et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat. Med. 17, 1086–1093 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Goardon, N. et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell 19, 138–152 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Takatsuki, H. et al. PML/RARα fusion gene is expressed in both granuloid/macrophage and erythroid colonies in acute promyelocytic leukaemia. Br. J. Haematol. 85, 477–482 (1993).

    Article  CAS  PubMed  Google Scholar 

  38. Turhan, A.G. et al. Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia. Blood 85, 2154–2161 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Haferlach, T. et al. Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12). Br. J. Haematol. 103, 93–99 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Knuutila, S. et al. Cell lineage involvement of recurrent chromosomal abnormalities in hematologic neoplasms. Genes Chromosom. Cancer 10, 95–102 (1994).

    Article  CAS  PubMed  Google Scholar 

  41. Guibal, F.C. et al. Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia. Blood 114, 5415–5425 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Brown, D. et al. A PMLRARα transgene initiates murine acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA 94, 2551–2556 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Grignani, F. et al. PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype. Blood 96, 1531–1537 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Matsushita, H. et al. Establishment of a humanized APL model via the transplantation of PML-RARA-transduced human common myeloid progenitors into immunodeficient mice. PLoS One 9, e111082 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, X. et al. Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells. Blood 116, 5972–5982 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Rongvaux, A. et al. Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo. Proc. Natl. Acad. Sci. USA 108, 2378–2383 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Willinger, T., Rongvaux, A., Strowig, T., Manz, M.G. & Flavell, R.A. Improving human hemato-lymphoid-system mice by cytokine knock-in gene replacement. Trends Immunol. 32, 321–327 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Groen, R.W. et al. Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood 120, e9–e16 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Alvarez, L.L. & Pardo, H.G. Guide for the care and use of laboratory animals — Natl-Res-Council. Psicothema 9, 232–234 (1997).

    Google Scholar 

  50. Rohde, E., Schallmoser, K., Bartmann, C., Reinisch, A & Dirk, S. in Pharmaceutical Manufacturing Handbook: Regulations and Quality (ed. Gad, S.C.) 97–115 (John Wiley & Sons, Inc., 2008).

  51. Schallmoser, K. et al. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng. Part C Methods 14, 185–196 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Dominici, M. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Notta, F. et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 333, 218–221 (2011).

    Article  CAS  PubMed  Google Scholar 

  54. Moraga, I. et al. Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Cell 160, 1196–1208 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Corces-Zimmerman, M.R., Hong, W.J., Weissman, I.L., Medeiros, B.C. & Majeti, R. Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proc. Natl. Acad. Sci. USA 111, 2548–2553 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Boggs, D.R. The total marrow mass of the mouse: a simplified method of measurement. Am. J. Hematol. 16, 277–286 (1984).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge the Hematology Division Tissue Bank and the patients for donating their samples. We acknowledge F. Zhao for lab management; N. Hofmann and B. Luo for technical assistance with ossicle analysis and calreticulin sequencing; and the Stanford Cytogenetics Lab for FISH analysis. A.R. is supported by an Erwin-Schroedinger Research Fellowship (Austrian Science Fund) and D.T. by a CJ Martin Overseas Biomedical Research Fellowship (NHMRC, Australia). R.M. is a New York Stem Cell Foundation Robertson Investigator. This research was supported by the New York Stem Cell Foundation and National Institutes of Health grants R01CA188055 and U01HL099999 to R.M. and by funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 668724 to D.S.

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A.R. and R.M conceived and designed the project. A.R., D.T., M.R.C., W.-J.H., and X.Z. performed the experimental work. A.R., D.T., M.R.C., and D.G. analyzed the data. K.S. and D.S. provided crucial reagents. A.R. and R.M. wrote the manuscript. All authors discussed the results.

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Correspondence to Ravindra Majeti.

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Reinisch, A., Thomas, D., Corces, M. et al. A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat Med 22, 812–821 (2016). https://doi.org/10.1038/nm.4103

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