Patient-derived xenografts (PDXs) can be improved by implantation of a humanized niche. Nevertheless, the overall complexity of the current protocols, as well as the use of specific biomaterials and procedures, limits the wider adoption of this approach. Here, we identify the essential minimum steps required to create the humanized scaffolds and achieve successful acute myeloid leukemia (AML) engraftment. We compared seven biomaterials, which included both published and custom-designed materials. The highest level of bone marrow niche was achieved with extracellular matrix gels and custom collagen fiber, both of which allowed for a simple non-surgical implantation. The biomaterial selection did not influence the following AML infiltration. Regarding xenotransplantation, standard intravenous administration produced the most robust engraftment, even for two out of four otherwise non-engrafting AML samples. In contrast, direct intra-scaffold xenotransplantation did not offer any advantage. In summary, we demonstrate that the combination of an injectable biomaterial for scaffold creation plus an intravenous route for AML xenotransplantation provide the most convenient and robust approach to produce AML PDX using a humanized niche.

Central European Institute of Technology Brno Institute of Technology Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno Czech Republic

Department of Orthopedic Surgery Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pathology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pathology University Hospital Brno Czech Republic

Department of Physiology Faculty of Medicine Masaryk University Brno Czech Republic

Orthopedic Clinic University Hospital Brno Czech Republic

Zobrazit více v PubMed

Reinisch A, Etchart N, Thomas D, Hofmann NA, Fruehwirth M, Sinha S, et al. Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood. 2015;125:249–260. PubMed PMC

Lee J, Li M, Milwid J, Dunham J, Vinegoni C, Gorbatov R, et al. Implantable microenvironments to attract hematopoietic stem/cancer cells. Proc Natl Acad Sci USA. 2012;109:19638–19643. PubMed PMC

Chen Y, Jacamo R, Shi YX, Wang RY, Battula VL, Konoplev S, et al. Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment. Blood. 2012;119:4971–4980. PubMed PMC

Groen RW, Noort WA, Raymakers RA, Prins HJ, Aalders L, Hofhuis FM, et al. Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood. 2012;120:e9–e16. PubMed

Antonelli A, Noort WA, Jaques J, de Boer B, de Jong‐Korlaar R, Brouwers‐Vos AZ, et al. Establishing human leukemia xenograft mouse models by implanting human bone marrow‐like scaffold‐based niches. Blood. 2016;128:2949–2959. PubMed

Abarrategi A, Foster K, Hamilton A, Mian SA, Passaro D, Gribben J, et al. Versatile humanized niche model enables study of normal and malignant human hematopoiesis. J Clin Invest. 2017;127:543–548. PubMed PMC

Mian SA, Abarrategi A, Kong KL, Rouault‐Pierre K, Wood H, Oedekoven CA, et al. Ectopic humanized mesenchymal niche in mice enables robust engraftment of myelodysplastic stem cells. Blood Cancer Discov. 2021;2:135–145. PubMed PMC

Vaiselbuh SR, Edelman M, Lipton JM, Liu JM. Ectopic human mesenchymal stem cell‐coated scaffolds in NOD/SCID mice: an in vivo model of the leukemia niche. Tissue Eng Part C Methods. 2010;16:1523–1531. PubMed

Reinisch A, Hernandez DC, Schallmoser K, Majeti R. Generation and use of a humanized bone‐marrow‐ossicle niche for hematopoietic xenotransplantation into mice. Nat Protoc. 2017;12:2169–2188. PubMed PMC

Bourgine PE, Fritsch K, Pigeot S, Takizawa H, Kunz L, Kokkaliaris KD, et al. Fate distribution and regulatory role of human mesenchymal stromal cells in engineered hematopoietic bone organs. iScience. 2019;19:504–513. PubMed PMC

Passaro D, Abarrategi A, Foster K, Ariza‐McNaughton L, Bonnet D. Bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging. J Vis Exp. 2017;e55914. 10.3791/55914 PubMed DOI PMC

Fritsch K, Pigeot S, Feng X, Bourgine PE, Schroeder T, Martin I, et al. Engineered humanized bone organs maintain human hematopoiesis in vivo. Exp Hematol. 2018;61:45–51.e5. PubMed

Martine LC, Holzapfel BM, McGovern JA, Wagner F, Quent VM, Hesami P, et al. Engineering a humanized bone organ model in mice to study bone metastases. Nat Protoc. 2017;12:639–663. PubMed

Prosecka E, Rampichova M, Vojtova L, Tvrdik D, Melcakova S, Juhasova J, et al. Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix. J Biomed Mater Res A. 2011;99:307–315. PubMed

Mian SA, Ariza‐McNaughton L, Anjos‐Afonso F, Guring R, Jackson S, Kizilors A, et al. Influence of donor–recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation. HemaSphere. 2024;8:e80. PubMed PMC

Wunderlich M, Brooks RA, Panchal R, Rhyasen GW, Danet‐Desnoyers G, Mulloy JC. OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood. 2014;123:e134–e144. PubMed PMC

Amend SR, Valkenburg KC, Pienta KJ. Murine hind limb long bone dissection and bone marrow isolation. J Vis Exp. 2016;e53936. 10.3791/53936 PubMed DOI PMC

Kuznetsov SA, Krebsbach PH, Satomura K, Kerr J, Riminucci M, Benayahu D, et al. Single‐colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J Bone Miner Res. 1997;12:1335–1347. PubMed

Prins HJ, Rozemuller H, Vonk‐Griffioen S, Verweij VG, Dhert WJ, Slaper‐Cortenbach IC, et al. Bone‐forming capacity of mesenchymal stromal cells when cultured in the presence of human platelet lysate as substitute for fetal bovine serum. Tissue Eng Part A. 2009;15:3741–3751. PubMed

Prosecká E, Rampichová M, Litvinec A, Tonar Z, Králíčková M, Vojtová L, et al. Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte‐rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo. J Biomed Mater Res A. 2015;103:671–682. PubMed

Mazzoni E, Mazziotta C, Iaquinta MR, Lanzillotti C, Fortini F, D'Agostino A, et al. Enhanced osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells by a hybrid Hydroxylapatite/collagen scaffold. Front Cell Dev Biol. 2021;8:610570. PubMed PMC

Akhir HM, Teoh PL. Collagen type I promotes osteogenic differentiation of amniotic membrane‐derived mesenchymal stromal cells in basal and induction media. Biosci Rep. 2020;40:BSR20201325. PubMed PMC

Lobo SE, Glickman R, da Silva WN, Arinzeh TL, Kerkis I. Response of stem cells from different origins to biphasic calcium phosphate bioceramics. Cell Tissue Res. 2015;361:477–495. PubMed PMC

Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH. Identification of multiple active growth factors in basement membrane matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res. 1992;202:1–8. PubMed

Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10:1886–1890. PubMed

Lindner U, Kramer J, Behrends J, Driller B, Wendler N‐O, Boehrnsen F, et al. Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement‐membrane extracellular matrix proteins. Cytotherapy. 2010;12:992–1005. PubMed

Reinisch A, Thomas D, Corces MR, Zhang X, Gratzinger D, Hong W‐J, et al. A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat Med. 2016;22:812–821. PubMed PMC

King GN, King N, Cruchley AT, Wozney JM, Hughes FJ. Recombinant human bone morphogenetic protein‐2 promotes wound healing in rat periodontal fenestration defects. J Dent Res. 1997;76:1460–1470. PubMed

Kim J, Kim IS, Cho TH, Lee KB, Hwang SJ, Tae G, et al. Bone regeneration using hyaluronic acid‐based hydrogel with bone morphogenic protein‐2 and human mesenchymal stem cells. Biomaterials. 2007;28:1830–1837. PubMed

de la Guardia RD, Velasco‐Hernandez T, Gutiérrez‐Agüera F, Roca‐Ho H, Molina O, Nombela‐Arrieta C, et al. Engraftment characterization of risk‐stratified AML in NSGS mice. Blood Adv. 2021;5:4842–4854. PubMed PMC

von Bonin M, Wermke M, Cosgun KN, Thiede C, Bornhauser M, Wagemaker G, et al. In vivo expansion of Co‐transplanted T cells impacts on tumor Re‐initiating activity of human acute myeloid leukemia in NSG mice. PLoS One. 2013;8:e60680. PubMed PMC

Woiterski J, Ebinger M, Witte KE, Goecke B, Heininger V, Philippek M, et al. Engraftment of low numbers of pediatric acute lymphoid and myeloid leukemias into NOD/SCID/IL2Rcgammanull mice reflects individual leukemogenecity and highly correlates with clinical outcome. Int J Cancer. 2013;133:1547–1556. PubMed

Herudkova Z, Culen M, Folta A, Jeziskova I, Cerna J, Loja T, et al. Clonal hierarchy of main molecular lesions in acute myeloid leukaemia. Br J Haematol. 2020;190:562–572. PubMed

Scotti C, Piccinini E, Takizawa H, Todorov A, Bourgine P, Papadimitropoulos A, et al. Engineering of a functional bone organ through endochondral ossification. Proc Natl Acad Sci USA. 2013;110:3997–4002. PubMed PMC