Protocol | Published:

Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice

Nature Protocols volume 12, pages 21692188 (2017) | Download Citation

Abstract

Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function. The ossicles, therefore, allow for accelerated and superior engraftment of primary patient-derived acute myeloid leukemia (AML) and other hematologic malignancies such as myelofibrosis (MF) in mice. The humanized ossicles are formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). Human hematopoietic cells can subsequently be transplanted directly into the ossicle marrow space or by intravenous injection. Using this method, a humanized engraftable BM microenvironment can be formed within 6–10 weeks. Engraftment of human hematopoietic cells can be evaluated by flow cytometry 8–16 weeks after transplantation. This protocol describes a robust and reproducible in vivo methodology for the study of normal and malignant human hematopoiesis in a more physiologic setting.

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Acknowledgements

We acknowledge the Tissue Bank of the Division of Hematology at Stanford University and the patients for donating their samples. We acknowledge F. Zhao for lab management, M. Stafford for technical help with ossicle generation, and D. Strunk for providing critical reagents, advice, and intellectual support for the initial development of the protocol. A.R. was supported by an Erwin-Schroedinger Research Fellowship (Austrian Science Fund). D.C.H. is a California Institute for Regenerative Medicine (CIRM) scholar. R.M. is a New York Stem Cell Foundation Robertson Investigator and Leukemia and Lymphoma Society Scholar. This research was supported by the Leukemia and Lymphoma Society, the New York Stem Cell Foundation, and National Institutes of Health grants R01CA188055 and U01HL099999 to R.M. This work was also supported by funding from the European Union's Horizon 2020 research and innovation programme under grant agreement number 668724 (TECHNOBEAT) to D. Strunk (Paracelsus Medical University of Salzburg, Austria) and grant agreement number 731377 (MUSIC) to K.S.

Author information

Affiliations

  1. Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA.

    • Andreas Reinisch
    • , David Cruz Hernandez
    •  & Ravindra Majeti
  2. Department of Blood Group Serology and Transfusion Medicine, Paracelsus Medical University, Salzburg, Austria.

    • Katharina Schallmoser
  3. Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria.

    • Katharina Schallmoser

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Contributions

A.R. and R.M. conceived and designed the project. A.R. and D.C.H. performed the experimental work. A.R. analyzed all data, K.S. provided critical reagents, and A.R. and R.M. wrote the protocol.

Competing interests

R.M. has an ownership interest (including patents) in Forty Seven Inc. and is a consultant/advisory board member for the same. The other authors declare no competing financial interests.

Corresponding authors

Correspondence to Andreas Reinisch or Ravindra Majeti.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2.

Videos

  1. 1.

    BM-MSC transplantation

    Video demonstrating preparation of the mouse (shaving and skin disinfection) and subsequent injection of BM-MSCs admixed with extracellular matrix into subcutaneous mouse tissue at four different locations. After the procedure, the mouse is placed underneath a warm light source to guarantee quick recovery from anesthesia.

  2. 2.

    Ossicle BM transplantation

    Video showing direct intraossicle transplantation of human hematopoietic cells.

  3. 3.

    Ossicle BM aspiration

    Video demonstrating aspiration of hematopoietic cells directly from a humanized BM-ossicle niche.

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DOI

https://doi.org/10.1038/nprot.2017.088

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