Skip to main content
SpringerLink
Log in

Functional Analysis of Human Hematopoietic Stem Cells In Vivo in Humanized Mice

  • Protocol
  • First Online:
Cell Reprogramming for Immunotherapy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2097))

Abstract

Ex vivo generation and expansion of functional hematopoietic stem cells represents the holy grail of reprogramming and would constitute a major advance in stem cell therapies and generation of blood cellular products. In vivo testing is critical to assure proper cell intrinsic function in an organismal context. Here we describe methods for the generation of human hematopoiesis chimeric mice and evaluation of hematopoietic stem cell function. The choice of mouse model, stem cell source, and transplantation route can be adjusted to suit the desired application.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G, Watanabe T, Akashi K, Shultz LD, Harada M (2005) Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 106(5):1565–1573. https://doi.org/10.1182/blood-2005-02-0516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL, Handgretinger R (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174(10):6477–6489

    Article  CAS  Google Scholar 

  3. Wunderlich M, Chou FS, Link KA, Mizukawa B, Perry RL, Carroll M, Mulloy JC (2010) AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24(10):1785–1788. https://doi.org/10.1038/leu.2010.158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nicolini FE, Cashman JD, Hogge DE, Humphries RK, Eaves CJ (2004) 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

    Article  Google Scholar 

  5. Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A (2011) Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rgamma(null) humanized mice. Blood 117:3076–3086

    Article  CAS  Google Scholar 

  6. Cosgun KN, Rahmig S, Mende N, Reinke S, Hauber I, Schafer C, Petzold A, Weisbach H, Heidkamp G, Purbojo A, Cesnjevar R, Platz A, Bornhauser M, Schmitz M, Dudziak D, Hauber J, Kirberg J, Waskow C (2014) Kit regulates HSC engraftment across the human-mouse species barrier. Cell Stem Cell 15(2):227–238. https://doi.org/10.1016/j.stem.2014.06.001

    Article  CAS  PubMed  Google Scholar 

  7. Rahmig S, Kronstein-Wiedemann R, Fohgrub J, Kronstein N, Nevmerzhitskaya A, Bornhauser M, Gassmann M, Platz A, Ordemann R, Tonn T, Waskow C (2016) Improved human erythropoiesis and platelet formation in humanized NSGW41 mice. Stem Cell Reports 7(4):591–601. https://doi.org/10.1016/j.stemcr.2016.08.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Strowig T, Rongvaux A, Rathinam C, Takizawa H, Borsotti C, Philbrick W, Eynon EE, Manz MG, Flavell RA (2011) Transgenic expression of human signal regulatory protein alpha in Rag2−/−{gamma}c−/− mice improves engraftment of human hematopoietic cells in humanized mice. Proc Natl Acad Sci U S A 108(32):13218–13223. https://doi.org/10.1073/pnas.1109769108

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rongvaux A, Takizawa H, Strowig T, Willinger T, Eynon EE, Flavell RA, Manz MG (2013) Human hemato-lymphoid system mice: current use and future potential for medicine. Annu Rev Immunol 31:635–674. https://doi.org/10.1146/annurev-immunol-032712-095921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Deng K, Pertea M, Rongvaux A, Wang L, Durand CM, Ghiaur G, Lai J, McHugh HL, Hao H, Zhang H, Margolick JB, Gurer C, Murphy AJ, Valenzuela DM, Yancopoulos GD, Deeks SG, Strowig T, Kumar P, Siliciano JD, Salzberg SL, Flavell RA, Shan L, Siliciano RF (2015) Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature 517(7534):381–385. https://doi.org/10.1038/nature14053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Song Y, Rongvaux A, Taylor A, Jiang T, Tebaldi T, Balasubramanian K, Bagale A, Terzi YK, Gbyli R, Wang X, Zhao J, Podoltsev N, Xu M, Neparidze N, Wong E, Torres R, Bruscia EM, Kluger Y, Manz MG, Flavell RA, Halene S (2019) A highly efficient and faithful MDS patient-derived xenotransplantation model for pre-clinical studies. Nat Commun 10(1):366

    Article  Google Scholar 

  12. Takenaka K, Prasolava TK, Wang JC, Mortin-Toth SM, Khalouei S, Gan OI, Dick JE, Danska JS (2007) Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat Immunol 8(12):1313–1323. https://doi.org/10.1038/ni1527

    Article  CAS  PubMed  Google Scholar 

  13. Waskow C, Madan V, Bartels S, Costa C, Blasig R, Rodewald HR (2009) Hematopoietic stem cell transplantation without irradiation. Nat Methods 6(4):267–269. https://doi.org/10.1038/Nmeth.1309

    Article  CAS  PubMed  Google Scholar 

  14. Reinisch A, Thomas D, Corces MR, Zhang X, Gratzinger D, Hong WJ, Schallmoser K, Strunk D, Majeti R (2016) A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat Med 22(7):812–821. https://doi.org/10.1038/nm.4103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rathinam C, Poueymirou WT, Rojas J, Murphy AJ, Valenzuela DM, Yancopoulos GD, Rongvaux A, Eynon EE, Manz MG, Flavell RA (2011) Efficient differentiation and function of human macrophages in humanized CSF-1 mice. Blood 118(11):3119–3128. https://doi.org/10.1182/blood-2010-12-326926

    Article  CAS  PubMed  Google Scholar 

  16. Rongvaux A, Willinger T, Takizawa H, Rathinam C, Auerbach W, Murphy AJ, Valenzuela DM, Yancopoulos GD, Eynon EE, Stevens S, Manz MG, Flavell RA (2011) Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo. Proc Natl Acad Sci U S A 108(6):2378–2383. https://doi.org/10.1073/pnas.1019524108

    Article  PubMed  PubMed Central  Google Scholar 

  17. Willinger T, Rongvaux A, Takizawa H, Yancopoulos GD, Valenzuela DM, Murphy AJ, Auerbach W, Eynon EE, Stevens S, Manz MG, Flavell RA (2011) Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung. Proc Natl Acad Sci U S A 108(6):2390–2395. https://doi.org/10.1073/pnas.1019682108

    Article  PubMed  PubMed Central  Google Scholar 

  18. Saito Y, Ellegast JM, Rafiei A, Song Y, Kull D, Heikenwalder M, Rongvaux A, Halene S, Flavell RA, Manz MG (2016) Peripheral blood CD34+ cells efficiently engraft human cytokine knock-in mice. Blood 128(14):1829–1833. https://doi.org/10.1182/blood-2015-10-676452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Theocharides AP, Rongvaux A, Fritsch K, Flavell RA, Manz MG (2016) Humanized hemato-lymphoid system mice. Haematologica 101(1):5–19. https://doi.org/10.3324/haematol.2014.115212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, Manz MG (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304(5667):104–107. https://doi.org/10.1126/science.1093933

    Article  CAS  PubMed  Google Scholar 

  21. Wunderlich M, Brooks RA, Panchal R, Rhyasen GW, Danet-Desnoyers G, Mulloy JC (2014) OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood 123(24):e134–e144. https://doi.org/10.1182/blood-2014-02-556340

    Article  PubMed  PubMed Central  Google Scholar 

  22. Wolber FM, Leonard E, Michael S, Orschell-Traycoff CM, Yoder MC, Srour EF (2002) Roles of spleen and liver in development of the murine hematopoietic system. Exp Hematol 30(9):1010–1019

    Article  CAS  Google Scholar 

  23. Zhan Y, Zhao Y (2008) Hematopoietic stem cell transplant in mice by intra-femoral injection. Methods Mol Biol 430:161–169. https://doi.org/10.1007/978-1-59745-182-6_11

    Article  PubMed  Google Scholar 

  24. Chung YR, Kim E, Abdel-Wahab O (2014) Femoral bone marrow aspiration in live mice. J Vis Exp (89):e51660. https://doi.org/10.3791/51660

  25. Lepus CM, Gibson TF, Gerber SA, Kawikova I, Szczepanik M, Hossain J, Ablamunits V, Kirkiles-Smith N, Herold KC, Donis RO, Bothwell AL, Pober JS, Harding MJ (2009) Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/gammac−/−, Balb/c-Rag1−/−gammac−/−, and C.B-17-scid/bg immunodeficient mice. Hum Immunol 70(10):790–802. https://doi.org/10.1016/j.humimm.2009.06.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Holyoake TL, Nicolini FE, Eaves CJ (1999) Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol 27(9):1418–1427

    Article  CAS  Google Scholar 

  27. Lan P, Tonomura N, Shimizu A, Wang S, Yang YG (2006) Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood 108(2):487–492. https://doi.org/10.1182/blood-2005-11-4388

    Article  CAS  PubMed  Google Scholar 

  28. Bonig H, Papayannopoulou T (2012) Mobilization of hematopoietic stem/progenitor cells: general principles and molecular mechanisms. Methods Mol Biol 904:1–14. https://doi.org/10.1007/978-1-61779-943-3_1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rongvaux A, Willinger T, Martinek J, Strowig T, Gearty SV, Teichmann LL, Saito Y, Marches F, Halene S, Palucka AK, Manz MG, Flavell RA (2014) Development and function of human innate immune cells in a humanized mouse model. Nat Biotechnol 32(4):364–372. https://doi.org/10.1038/nbt.2858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by the Edward P. Evans and the Frederick Deluca Foundations (to S.H.). Y.S. is supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 81800122). This study was supported by the Animal Modeling Core of the Yale Cooperative Center of Excellence in Hematology (NIDDK U54DK106857).

Author information

Authors and Affiliations

  1. Section of Hematology, Department of Internal Medicine and Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Yuanbin Song, Rana Gbyli, Xiaoying Fu & Stephanie Halene

  2. Department of Laboratory Medicine, Shenzhen Children’s Hospital, Shenzhen, People’s Republic of China

    Xiaoying Fu

Authors
  1. Yuanbin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rana Gbyli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaoying Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephanie Halene
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephanie Halene .

Editor information

Editors and Affiliations

  1. Department of Pathology, Yale School of Medicine, New Haven, CT, USA

    Samuel G. Katz

  2. Department of Pathology, Yale School of Medicine, New Haven, CT, USA

    Peter M. Rabinovich

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Song, Y., Gbyli, R., Fu, X., Halene, S. (2020). Functional Analysis of Human Hematopoietic Stem Cells In Vivo in Humanized Mice. In: Katz, S., Rabinovich, P. (eds) Cell Reprogramming for Immunotherapy. Methods in Molecular Biology, vol 2097. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0203-4_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0203-4_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0202-7

  • Online ISBN: 978-1-0716-0203-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation