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Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells

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Erythropoiesis

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

Abstract

Multipotent hematopoietic stem cells differentiate into an ensemble of committed progenitor cells that produce the diverse blood cells essential for life. Physiological mechanisms governing hematopoiesis, and mechanistic aberrations underlying non-malignant and malignant hematologic disorders, are often very similar in mouse and man. Thus, mouse models provide powerful systems for unraveling mechanisms that control hematopoietic stem/progenitor cell (HSPC) function in their resident microenvironments in vivo. Ex vivo systems, involving the culture of HSPCs generated in vivo, allow one to dissociate microenvironment-based and cell intrinsic mechanisms, and therefore have considerable utility. Dissecting mechanisms controlling cellular proliferation and differentiation is facilitated by the use of primary cells, since mutations and chromosome aberrations in immortalized and cancer cell lines corrupt normal mechanisms. Primary erythroid precursor cells can be expanded or differentiated in culture to yield large numbers of progeny at discrete maturation stages. We described a robust method for isolation, culture, and analysis of primary mouse erythroid precursor cells and their progeny.

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References

  1. Dzierzak E, Philipsen S (2013) Erythropoiesis: development and differentiation. Cold Spring Harb Perspect Med 3(4):a011601. doi:10.1101/cshperspect.a011601

    Article  PubMed  PubMed Central  Google Scholar 

  2. Dzierzak E, de Pater E (2016) Regulation of blood stem cell development. Curr Top Dev Biol 118:1–20. doi:10.1016/bs.ctdb.2016.01.001

    Article  CAS  PubMed  Google Scholar 

  3. Tober J, Maijenburg MW, Speck NA (2016) Taking the leap: Runx1 in the formation of blood from endothelium. Curr Top Dev Biol 118:113–162. doi:10.1016/bs.ctdb.2016.01.008

    Article  CAS  PubMed  Google Scholar 

  4. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL (1995) The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A 92(22):10302–10306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM (1996) The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 87(8):3500–3507

    CAS  PubMed  Google Scholar 

  6. Zhang J, Socolovsky M, Gross AW, Lodish HF (2003) Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system. Blood 102(12):3938–3946. doi:10.1182/blood-2003-05-1479

    Article  CAS  PubMed  Google Scholar 

  7. Coffman RL, Weissman IL (1981) B220: a B cell-specific member of the T200 glycoprotein family. Nature 289(5799):681–683

    Article  CAS  PubMed  Google Scholar 

  8. Fleming TJ, Fleming ML, Malek TR (1993) Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J Immunol 151(5):2399–2408

    CAS  PubMed  Google Scholar 

  9. Khandros E, Thom CS, D’Souza J, Weiss MJ (2012) Integrated protein quality-control pathways regulate free alpha-globin in murine beta-thalassemia. Blood 119(22):5265–5275. doi:10.1182/blood-2011-12-397729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. DeVilbiss AW, Sanalkumar R, Hall BD, Katsumura KR, de Andrade IF, Bresnick EH (2015) Epigenetic determinants of erythropoiesis: role of the histone methyltransferase SetD8 in promoting erythroid cell maturation and survival. Mol Cell Biol 35(12):2073–2087. doi:10.1128/MCB.01422-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. McIver SC, Kang YA, DeVilbiss AW, O’Driscoll CA, Ouellette JN, Pope NJ, Camprecios G, Chang CJ, Yang D, Bouhassira EE, Ghaffari S, Bresnick EH (2014) The exosome complex establishes a barricade to erythroid maturation. Blood 124(14):2285–2297. doi:10.1182/blood-2014-04-571083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hewitt KJ, Kim DH, Devadas P, Prathibha R, Zuo C, Sanalkumar R, Johnson KD, Kang YA, Kim JS, Dewey CN, Keles S, Bresnick EH (2015) Hematopoietic signaling mechanism revealed from a stem/progenitor cell cistrome. Mol Cell 59(1):62–74. doi:10.1016/j.molcel.2015.05.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gao X, Wu T, Johnson KD, Lahvic JL, Ranheim EA, Zon LI, Bresnick EH (2016) GATA factor-G-protein-coupled receptor circuit suppresses hematopoiesis. Stem Cell Rep 6(3):368–382. doi:10.1016/j.stemcr.2016.01.008

    Article  CAS  Google Scholar 

  14. McIver SC, Katsumura KR, Davids E, Liu P, Kang Y-A, Yang D, Bresnick EH (2016) Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis. eLife 5:e17877. doi:10.7554/eLife.17877

    Article  PubMed  PubMed Central  Google Scholar 

  15. Hewitt KJ, Katsumura KR, Matson DR, Devadas P, Tanimura N, Hebert AS, Coon JJ, Kim JS, Dewey CN, Keles S, Hao S, Paulson RF, Bresnick EH (2017) GATA factor-regulated Samd14 enhancer confers red blood cell regeneration and survival in severe anemia. Dev Cell 42(3):213–225.e4. https://doi.org/10.1016/j.devcel.2017.07.009.7

    Article  CAS  PubMed  Google Scholar 

  16. Mehta C, Johnson KD, Gao X, Ong IM, Katsumura KR, McIver SC, Ranheim EA, Bresnick EH (2017) Integrating enhancer mechanisms to establish a hierarchical blood development program. Cell Rep (In Press)

    Google Scholar 

  17. Lee HY, Gao X, Barrasa MI, Li H, Elmes RR, Peters LL, Lodish HF (2015) PPAR-alpha and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal. Nature 522(7557):474–477. doi:10.1038/nature14326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Williams DA, Bunn HF, Sieff C, Zon LI (2003) Hematopoiesis. In: Handin RI, Lux SE, Stossel TP (eds) Blood: principles and practice of hematology, 2nd edn. Lippincott Williams and Wilkins, Philadelphia, PA, pp 147–208

    Google Scholar 

  19. McGarry MP, Protheroe CA, Lee JJ (2010) Mouse hematology: a laboratory manual. Cold Spring Harbor Laboratory Press, Woodbury, NY

    Google Scholar 

  20. Broxmeyer HE (1984) Colony assays of hematopoietic progenitor cells and correlations to clinical situations. Crit Rev Oncol Hematol 1(3):227–257

    Article  CAS  PubMed  Google Scholar 

  21. Gregory CJ, Eaves AC (1978) Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biologic properties. Blood 51(3):527–537

    CAS  PubMed  Google Scholar 

  22. Munugalavadla V, Kapur R (2005) Role of c-Kit and erythropoietin receptor in erythropoiesis. Crit Rev Oncol Hematol 54(1):63–75. doi:10.1016/j.critrevonc.2004.11.005

    Article  PubMed  Google Scholar 

  23. Muta K, Krantz SB, Bondurant MC, Dai CH (1995) Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation. Blood 86(2):572–580

    CAS  PubMed  Google Scholar 

  24. Sui X, Krantz SB, You M, Zhao Z (1998) Synergistic activation of MAP kinase (ERK1/2) by erythropoietin and stem cell factor is essential for expanded erythropoiesis. Blood 92(4):1142–1149

    CAS  PubMed  Google Scholar 

  25. Wu H, Klingmuller U, Acurio A, Hsiao JG, Lodish HF (1997) Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation. Proc Natl Acad Sci U S A 94(5):1806–1810. doi:10.1160/TH08-08-0556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wu H, Liu X, Jaenisch R, Lodish HF (1995) Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83(1):59–67

    Article  CAS  PubMed  Google Scholar 

  27. Wu H, Klingmuller U, Besmer P, Lodish HF (1995) Interaction of the erythropoietin and stem-cell-factor receptors. Nature 377(6546):242–246. doi:10.1038/377242a0

    Article  CAS  PubMed  Google Scholar 

  28. Hattangadi SM, Wong P, Zhang L, Flygare J, Lodish HF (2011) From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications. Blood 118(24):6258–6268. doi:10.1182/blood-2011-07-356006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Haas N, Riedt T, Labbaf Z, Bassler K, Gergis D, Frohlich H, Gutgemann I, Janzen V, Schorle H (2015) Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver. Cell Death Differ 22(5):790–800. doi:10.1038/cdd.2014.172

    Article  CAS  PubMed  Google Scholar 

  30. Pop R, Shearstone JR, Shen Q, Liu Y, Hallstrom K, Koulnis M, Gribnau J, Socolovsky M (2010) A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol 8(9):e1000484. doi:10.1371/journal.pbio.1000484

    Article  PubMed  PubMed Central  Google Scholar 

  31. Chen K, Liu J, Heck S, Chasis JA, An X, Mohandas N (2009) Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc Natl Acad Sci U S A 106(41):17413–17418. doi:10.1073/pnas.0909296106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Dunning K, Safo AO (2011) The ultimate Wright-Giemsa stain: 60 years in the making. Biotech Histochem 86(2):69–75. doi:10.3109/10520295.2010.515496

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The lineage negative cell isolation and culture protocols were adapted from those originally provided by M.J. Weiss. This work was supported by NIH grants DK50107 and DK68634.

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Authors and Affiliations

  1. Department of Cell and Regenerative Biology, Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, 4009 WIMR, 1111 Highland Ave, Madison, WI, 53705, USA

    Skye C. McIver, Kyle J. Hewitt, Xin Gao, Charu Mehta & Emery H. Bresnick

  2. UW-Madison Blood Research Program, University of Wisconsin, Madison, WI, 53705, USA

    Skye C. McIver, Kyle J. Hewitt, Xin Gao, Charu Mehta, Jing Zhang & Emery H. Bresnick

  3. McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI, 53705, USA

    Jing Zhang

Authors
  1. Skye C. McIver
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  2. Kyle J. Hewitt
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  3. Xin Gao
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  4. Charu Mehta
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  5. Jing Zhang
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  6. Emery H. Bresnick
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Corresponding author

Correspondence to Emery H. Bresnick .

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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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McIver, S.C., Hewitt, K.J., Gao, X., Mehta, C., Zhang, J., Bresnick, E.H. (2018). Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells. 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_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7428-3_4

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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

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