Erythropoiesis pp 275-284 | Cite as

Growing and Genetically Manipulating Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP) Cell Lines

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)

Abstract

The recently established human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines have equipped red blood cell researchers with valuable in vitro models of erythroid development. Of the three established HUDEP cell lines, HUDEP-2 cells express predominantly adult β-globin and most closely resemble adult erythroid cells. This chapter describes culture protocols for the maintenance and erythroid differentiation of HUDEP-2 cells. Methods to genetically manipulate HUDEP-2 cells using a CRISPR/Cas9 nuclease-based approach are also discussed.

Key words

HUDEP Immortalized erythroid cell line Erythroid differentiation Hemoglobin switching Fetal hemoglobin CRISPR/Cas9 

Notes

Acknowledgments

D.E.B. is supported by NIDDK (K08DK093705, R03DK109232), Burroughs Wellcome Fund, American Society of Hematology, and the Doris Duke Charitable, Charles H. Hood, and Cooley’s Anemia Foundations.

References

  1. 1.
    Tsiftsoglou AS, Vizirianakis IS, Strouboulis J (2009) Erythropoiesis: model systems, molecular regulators, and developmental programs. IUBMB Life 61(8):800–830. doi: 10.1002/iub.226. Review. PubMed PMID: 19621348CrossRefPubMedGoogle Scholar
  2. 2.
    Olivier E, Qiu C, Bouhassira EE (2012) Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood. Stem Cells Transl Med 1(8):604–614. doi: 10.5966/sctm.2012-0059. PubMed PMID: 23197866; PubMed Central PMCID: PMC3659727CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lapillonne H, Kobari L, Mazurier C, Tropel P, Giarratana MC, Zanella-Cleon I, Kiger L, Wattenhofer-Donzé M, Puccio H, Hebert N, Francina A, Andreu G, Viville S, Douay L (2010) Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 95(10):1651–1659. doi: 10.3324/haematol.2010.023556. PubMed PMID: 20494935; PubMed Central PMCID: PMC2948089CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Migliaccio G, Di Pietro R, di Giacomo V, Di Baldassarre A, Migliaccio AR, Maccioni L, Galanello R, Papayannopoulou T (2002) In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients. Blood Cells Mol Dis 28(2):169–180. PubMed PMID: 12064913CrossRefPubMedGoogle Scholar
  5. 5.
    Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, François S, Trugnan G, Peyrard T, Marie T, Jolly S, Hebert N, Mazurier C, Mario N, Harmand L, Lapillonne H, Devaux JY, Douay L (2011) Proof of principle for transfusion of in vitro-generated red blood cells. Blood 118(19):5071–5079. doi: 10.1182/blood-2011-06-362038. PubMed PMID: 21885599; PubMed Central PMCID: PMC3217398CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, Tani K, Nakamura Y (2013) Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS One 8(3):e59890. doi: 10.1371/journal.pone.0059890. PubMed PMID: 23533656; PubMed Central PMCID: PMC3606290CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Münger K, Howley PM (2002) Human papillomavirus immortalization and transformation functions. Virus Res 89(2):213–228. Review. PubMed PMID: 12445661CrossRefPubMedGoogle Scholar
  8. 8.
    Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE (2015) BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527(7577):192–197. doi: 10.1038/nature15521. PubMed PMID: 26375006; PubMed Central PMCID: PMC4644101CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Masuda T, Wang X, Maeda M, Canver MC, Sher F, Funnell AP, Fisher C, Suciu M, Martyn GE, Norton LJ, Zhu C, Kurita R, Nakamura Y, Xu J, Higgs DR, Crossley M, Bauer DE, Orkin SH, Kharchenko PV, Maeda T (2016) Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin. Science 351(6270):285–289. doi: 10.1126/science.aad3312. PubMed PMID: 26816381; PubMedCentral PMCID: PMC4778394CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Traxler EA, Yao Y, Wang YD, Woodard KJ, Kurita R, Nakamura Y, Hughes JR, Hardison RC, Blobel GA, Li C, Weiss MJ (2016) A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 22(9):987–990. doi: 10.1038/nm.4170. PubMed PMID: 27525524CrossRefPubMedGoogle Scholar
  11. 11.
    Addgene. https://www.addgene.org/crispr/cut/. Accessed Nov 2016
  12. 12.
  13. 13.
    Dzierzak E, Philipsen S (2013) Erythropoiesis: development and differentiation. Cold Spring Harb Perspect Med 3(4):a011601. doi: 10.1101/cshperspect.a011601. Review. PubMed PMID: 23545573; PubMed Central PMCID: PMC3684002CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381(Pt 1):307–312. PubMed PMID: 15065984; PubMed Central PMCID: PMC1133789CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Hematology/OncologyBoston Children’s HospitalBostonUSA
  2. 2.Dana-Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell InstituteBostonUSA

Personalised recommendations