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

Nature Protocols volume 12, pages 2169–2188 (2017)Cite this article

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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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Figure 1: Potential applications of humanized ossicle BM xenotransplantation.
Figure 2: BM-MSC isolation, propagation, and phenotypic characterization.
Figure 3: Humanized ossicle niche formation.
Figure 4: In vivo ossicle formation.
Figure 5: Flow-cytometric analysis layout for human engraftment.
Figure 6: Sorting schemes for normal and leukemic hematopoietic cell populations.

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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.

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Authors and 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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  1. Andreas Reinisch
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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.

Corresponding authors

Correspondence to Andreas Reinisch or Ravindra Majeti.

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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.

Integrated supplementary information

Supplementary Figure 1 Illustration of pHPL production.

(a) To prepare platelet concentrates from whole blood donations, four units of blood group (BG) O and one unit of BG AB whole blood donations should be separated into plasma, buffy coat (BF), and red blood cells by centrifugation. (b) One unit of platelet concentrate is generated by combining four buffy coat units, (all BG O) and one BG AB plasma. The combined product should be centrifuged again to separate leukocytes, which will thereafter be depleted through inline-filtration (as described in more detail in BOX1). These steps should be repeated until 10 units are collected. (c) Platelets within the concentrates are lysed by freezing and thawing, and the resulting human platelet lysates (HPL) are pooled to generate 2 – 3 liters of pooled HPL. After aliquoting into smaller storage bags, a second freeze/thaw cycle and one additional centrifugation step should be performed to guarantee maximal lysis and depletion of platelet fragments. The resulting product can be stored in 50mL tubes, ready to use for media preparation.

Supplementary Figure 2 Gating strategy to identify BM-MNCs and BM-MSCs.

(a) Flow-cytometric contour plot showing gating of mononuclear cells (MNCs, red box) within total nucleated cells (TNC, black box). Cell size (FSC-A, x-axis) and cellular granularity (SSC-A, y-axis) allow for the discrimination of lymphocytes (red), monocytes (green) and granulocytes (blue) as illustrated in (b). MNCs are comprised of lymphocytes and monocytes whereas TNCs additionally include granulocytes. (c) Left plot: Mesenchymal stromal cells (MSCs) are gated (black box) based on cell size (FSC-A, x-axis) and cellular granularity (SSC-A, y-axis) and can be clearly separate from contaminating debris (lower left). Right plot: Live cells (black box) are defined by absence of 7-aminoactinomycin D (7-AAD) staining. Arrow indicates that only cells defined by gate in FSC-A versus SSC-A plot are analyzed for 7-AAD.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2. (PDF 539 kb)

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. (MP4 25576 kb)

Ossicle BM transplantation

Video showing direct intraossicle transplantation of human hematopoietic cells. (MP4 21890 kb)

Ossicle BM aspiration

Video demonstrating aspiration of hematopoietic cells directly from a humanized BM-ossicle niche. (MP4 23885 kb)

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Reinisch, A., Hernandez, D., Schallmoser, K. et al. Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice. Nat Protoc 12, 2169–2188 (2017). https://doi.org/10.1038/nprot.2017.088

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