Skip to main content
SpringerLink
Log in

High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning Machine Bioreactors

  • Protocol
  • First Online:
Bioreactors in Stem Cell Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2436))

Abstract

Human pluripotent stem cells (PSCs) are known to differentiate into almost all the blood lineage cells in vitro and hold a great promise for studying human early hematopoietic development and have a huge potential in the treatment of hematological disorders. Although several methods of hematopoietic stem/progenitor cell (HSPC) differentiation have been developed, the HSPC yields achieved using these strategies are not yet available for clinical application. Recently, bioreactor-based devices and biochemical factors synergistically have been used to induce hematopoietic differentiation and showed a potential role in hematopoiesis. This chapter describes a protocol for using a random positioning machine bioreactor to culture human PSCs and the large-scale production of HPCs. Techniques for characterizing the differentiated cells and assessing the efficiency of hematopoietic differentiation in the bioreactor with immunostaining and flow cytometry are also presented.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA (2001) Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 98(19):10716–10721

    Article  CAS  Google Scholar 

  2. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M (2003) Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102(3):906–915

    Article  CAS  Google Scholar 

  3. Vodyanik MA, Bork JA, Thomson JA, Slukvin II (2005) Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105(2):617–626

    Article  CAS  Google Scholar 

  4. Vo LT, Daley GQ (2015) De novo generation of HSCs from somatic and pluripotent stem cell sources. Blood 125(17):2641–2648

    Article  CAS  Google Scholar 

  5. Choi KD, Vodyanik M, Slukvin II (2011) Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc 6(3):296–313

    Article  CAS  Google Scholar 

  6. Wang H, Liu C, Liu X, Wang M, Wu D, Gao J, Su P, Nakahata T, Zhou W, Xu Y et al (2018) MEIS1 regulates Hemogenic endothelial generation, Megakaryopoiesis, and Thrombopoiesis in human pluripotent stem cells by targeting TAL1 and FLI1. Stem Cell Rep 10(2):447–460

    Article  CAS  Google Scholar 

  7. Huang X, Gschweng E, Van Handel B, Cheng D, Mikkola HK, Witte ON (2011) Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells. Blood 117(7):2157–2165

    Article  CAS  Google Scholar 

  8. Wang CY, Tang XM, Sun XM, Miao ZC, Lv YX, Yang YL, Zhang HD, Zhang PB, Liu Y, Du LY et al (2012) TGF beta inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy. Cell Res 22(1):194–207

    Article  Google Scholar 

  9. Kennedy M, Awong G, Sturgeon CM, Ditadi A, LaMotte-Mohs R, Zuniga-Pflucker JC, Keller G (2012) T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep 2(6):1722–1735

    Article  CAS  Google Scholar 

  10. Woll PS, Morris JK, Painschab MS, Marcus RK, Kohn AD, Biechele TL, Moon RT, Kaufman DS (2008) Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells. Blood 111(1):122–131

    Article  CAS  Google Scholar 

  11. Sturgeon CM, Ditadi A, Awong G, Kennedy M, Keller G (2014) Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32(6):554–561

    Article  CAS  Google Scholar 

  12. Wang Y, Gao J, Wang H, Wang M, Wen Y, Guo J, Su P, Shi L, Zhou W, Zhou J (2019) R-spondin2 promotes hematopoietic differentiation of human pluripotent stem cells by activating TGF beta signaling. Stem Cell Res Ther 10(1):136

    Article  Google Scholar 

  13. Ran D, Shia WJ, Lo MC, Fan JB, Knorr DA, Ferrell PI, Ye Z, Yan M, Cheng L, Kaufman DS et al (2013) RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells. Blood 121(15):2882–2890

    Article  CAS  Google Scholar 

  14. Ramos-Mejia V, Navarro-Montero O, Ayllon V, Bueno C, Romero T, Real PJ, Menendez P (2014) HOXA9 promotes hematopoietic commitment of human embryonic stem cells. Blood 124(20):3065–3075

    Article  CAS  Google Scholar 

  15. Wang H, Wang M, Wang Y, Wen Y, Chen X, Wu D, Su P, Zhou W, Shi L, Zhou J (2020) MSX2 suppression through inhibition of TGFbeta signaling enhances hematopoietic differentiation of human embryonic stem cells. Stem Cell Res Ther 11(1):147

    Article  CAS  Google Scholar 

  16. North TE, Goessling W, Peeters M, Li PL, Ceol C, Lord AM, Weber GJ, Harris J, Cutting CC, Huang P et al (2009) Hematopoietic stem cell development is dependent on blood flow. Cell 137(4):736–748

    Article  CAS  Google Scholar 

  17. Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA et al (2015) Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis. J Exp Med 212(5):665–680

    Article  CAS  Google Scholar 

  18. Kwan W, Cortes M, Frost I, Esain V, Theodore LN, Liu SY, Budrow N, Goessling W, North TE (2016) The central nervous system regulates embryonic HSPC production via stress-responsive glucocorticoid receptor signaling. Cell Stem Cell 19(3):370–382

    Article  CAS  Google Scholar 

  19. Bourgine PE, Klein T, Paczulla AM, Shimizu T, Kunz L, Kokkaliaris KD, Coutu DL, Lengerke C, Skoda R, Schroeder T et al (2018) In vitro biomimetic engineering of a human hematopoietic niche with functional properties. Proc Natl Acad Sci U S A 115(25):E5688–E5695

    Article  CAS  Google Scholar 

  20. Lundin V, Sugden WW, Theodore LN, Sousa PM, Han A, Chou S, Wrighton PJ, Cox AG, Ingber DE, Goessling W et al (2020) YAP regulates hematopoietic stem cell formation in response to the biomechanical forces of blood flow. Dev Cell 52(4):446–460. e445

    Article  CAS  Google Scholar 

  21. Yang Y, Liu C, Lei X, Wang H, Su P, Ru Y, Ruan X, Duan E, Feng S, Han M et al (2016) Integrated biophysical and biochemical signals augment Megakaryopoiesis and Thrombopoiesis in a three-dimensional rotary culture system. Stem Cells Transl Med 5(2):175–185

    Article  CAS  Google Scholar 

  22. Cao X, Yakala GK, van den Hil FE, Cochrane A, Mummery CL, Orlova VV (2019) Differentiation and functional comparison of monocytes and macrophages from hiPSCs with peripheral blood derivatives. Stem Cell Rep 12(6):1282–1297

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work has been funded by the Chinese Manned Space Station Application Project (YYWT-0901-EXP-15, to XL), CAS Key Technology Talent Program (to XL), and the NSFC Grant (31471287, to XL).

Author information

Authors and Affiliations

  1. Center for Energy Metabolism and Reproduction, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Xiaohua Lei, Chiyuan Ma, Yue Xiong & Jian V. Zhang

  2. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Yujing Cao & Enkui Duan

Authors
  1. Xiaohua Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chiyuan Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yujing Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jian V. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Enkui Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaohua Lei .

Editor information

Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lei, X., Ma, C., Cao, Y., Xiong, Y., Zhang, J.V., Duan, E. (2021). High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning Machine Bioreactors. In: Turksen, K. (eds) Bioreactors in Stem Cell Biology. Methods in Molecular Biology, vol 2436. Humana, New York, NY. https://doi.org/10.1007/7651_2021_412

Download citation

  • DOI: https://doi.org/10.1007/7651_2021_412

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2017-5

  • Online ISBN: 978-1-0716-2018-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation