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In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood

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Book cover Erythropoiesis

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

Abstract

Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.

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Acknowledgments

We would like to thank the St. Louis Cord Blood Bank (SLCBB, St. Louis, MO) for providing us with fresh umbilical cord blood.

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Authors and Affiliations

  1. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA

    Anna Kovilakath, Safa Mohamad, Farrah Hermes & Joyce A. Lloyd

  2. Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA

    Shou Zhen Wang, Gordon D. Ginder & Joyce A. Lloyd

  3. Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA

    Gordon D. Ginder

Authors
  1. Anna Kovilakath
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  2. Safa Mohamad
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  3. Farrah Hermes
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  4. Shou Zhen Wang
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  5. Gordon D. Ginder
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  6. Joyce A. Lloyd
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Corresponding author

Correspondence to Joyce A. Lloyd .

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Editors and Affiliations

  1. Department of Human & Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, USA

    Joyce A. Lloyd

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Kovilakath, A., Mohamad, S., Hermes, F., Wang, S.Z., Ginder, G.D., Lloyd, J.A. (2018). In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood. In: Lloyd, J. (eds) Erythropoiesis. Methods in Molecular Biology, vol 1698. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7428-3_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7428-3_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7427-6

  • Online ISBN: 978-1-4939-7428-3

  • eBook Packages: Springer Protocols

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