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Comprehensive Hematopoietic Stem Cell Isolation Methods

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Stem Cells and Aging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 976))

Abstract

The use of flow cytometry has been critical in establishing methods to isolate and characterize hematopoietic stem cells (HSCs) and their progenitors. For more than 30 years, researchers have been uncovering novel markers that when used in combination significantly enhance the purification of HSCs from murine and human bone marrow. The complex interface between HSCs, the lymphohematopoietic system, and their niches, has made identification of HSC markers critical to understanding their biological nature, more so than other adult stem cell populations. Here we review the phenotypic markers and strategies used to purify HSCs, the appropriateness of using these markers for comparisons of HSC function at different stages of ontogeny, and their utility in defining the lineage bias in the HSC compartment.

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References

  1. Siminovitch L, McCulloch EA, Till JE (1963) The distribution of colony-forming cells among spleen colonies. J Cell Physiol 62:327–336

    Article  PubMed  CAS  Google Scholar 

  2. Snippert HJ, Clevers H (2011) Tracking adult stem cells. EMBO Rep 12:113–122

    Article  PubMed  CAS  Google Scholar 

  3. Yin T, Li L (2006) The stem cell niches in bone. J Clin Invest 116:1195–1201

    Article  PubMed  CAS  Google Scholar 

  4. Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I et al (2011) Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 333:218–221

    Article  PubMed  CAS  Google Scholar 

  5. Spangrude GJ, Heimfeld S, Weissman IL (1988) Purification and characterization of mouse hematopoietic stem cells. Science 241:58–62

    Article  PubMed  CAS  Google Scholar 

  6. Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi S, Kunisada T et al (1991) Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 174:63–71

    Article  PubMed  CAS  Google Scholar 

  7. Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y et al (1992) In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood 80:3044–3050

    PubMed  CAS  Google Scholar 

  8. Boswell HS, Wade PM Jr, Quesenberry PJ (1984) Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential. J Immunol 133:2940–2949

    PubMed  CAS  Google Scholar 

  9. Berman JW, Basch RS (1985) Thy-1 antigen expression by murine hematopoietic precursor cells. Exp Hematol 13:1152–1156

    PubMed  CAS  Google Scholar 

  10. Williams DE, Boswell HS, Floyd AD, Broxmeyer HE (1985) Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen. J Immunol 135:1004–1011

    PubMed  CAS  Google Scholar 

  11. Szilvassy SJ, Lansdorp PM, Humphries RK, Eaves AC, Eaves CJ (1989) Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability. Blood 74:930–939

    PubMed  CAS  Google Scholar 

  12. Ikuta K, Weissman IL (1992) Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their ­generation. Proc Natl Acad Sci USA 89:1502–1506

    Article  PubMed  CAS  Google Scholar 

  13. Uchida N, Weissman IL (1992) Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin− Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 175:175–184

    Article  PubMed  CAS  Google Scholar 

  14. Spangrude GJ, Brooks DM (1992) Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1-negative subset. Blood 80:1957–1964

    PubMed  CAS  Google Scholar 

  15. Morrison SJ, Weissman IL (1994) The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1:661–673

    Article  PubMed  CAS  Google Scholar 

  16. Osawa M, Hanada K, Hamada H, Nakauchi H (1996) Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273:242–245

    Article  PubMed  CAS  Google Scholar 

  17. Adolfsson J, Borge OJ, Bryder D, Theilgaard-Monch K, Astrand-Grundstrom I et al (2001) Upregulation of Flt3 expression within the bone marrow Lin(−)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15:659–669

    Article  PubMed  CAS  Google Scholar 

  18. Christensen JL, Weissman IL (2001) Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA 98: 14541–14546

    Article  PubMed  CAS  Google Scholar 

  19. Yang L, Bryder D, Adolfsson J, Nygren J, Mansson R et al (2005) Identification of Lin(−)Sca1(+)kit(+)CD34(+)Flt3− short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 105:2717–2723

    Article  PubMed  CAS  Google Scholar 

  20. Sitnicka E, Bryder D, Theilgaard-Monch K, Buza-Vidas N, Adolfsson J et al (2002) Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 17:463–472

    Article  PubMed  CAS  Google Scholar 

  21. Adolfsson J, Mansson R, Buza-Vidas N, Hultquist A, Liuba K et al (2005) Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121:295–306

    Article  PubMed  CAS  Google Scholar 

  22. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121

    Article  PubMed  CAS  Google Scholar 

  23. Balazs AB, Fabian AJ, Esmon CT, Mulligan RC (2006) Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 107:2317–2321

    Article  PubMed  CAS  Google Scholar 

  24. Kent DG, Copley MR, Benz C, Wohrer S, Dykstra BJ et al (2009) Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood 113:6342–6350

    Article  PubMed  CAS  Google Scholar 

  25. Cheshier SH, Morrison SJ, Liao X, Weissman IL (1999) In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci USA 96:3120–3125

    Article  PubMed  CAS  Google Scholar 

  26. Foudi A, Hochedlinger K, Van Buren D, Schindler JW, Jaenisch R et al (2009) Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27:84–90

    Article  PubMed  CAS  Google Scholar 

  27. Ploemacher RE, Brons NH (1988) Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention. J Cell Physiol 136:531–536

    Article  PubMed  CAS  Google Scholar 

  28. Ploemacher RE, Brons NH (1988) In vivo proliferative and differential properties of murine bone marrow cells separated on the basis of rhodamine-123 retention. Exp Hematol 16:903–907

    PubMed  CAS  Google Scholar 

  29. Zijlmans JM, Visser JW, Kleiverda K, Kluin PM, Willemze R et al (1995) Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-­repopulating ability. Proc Natl Acad Sci USA 92:8901–8905

    Article  PubMed  CAS  Google Scholar 

  30. Peng K, Visser AJ, van Hoek A, Wolfs CJ, Sanders JC et al (1990) Analysis of time-resolved fluorescence anisotropy in lipid-protein systems. I. Application to the lipid probe octadecyl rhodamine B in interaction with bacteriophage M13 coat protein incorporated in phospholipid bilayers. Eur Biophys J 18:277–283

    Article  PubMed  CAS  Google Scholar 

  31. Visser JW, de Vries P (1988) Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling. Blood Cells 14:369–384

    PubMed  CAS  Google Scholar 

  32. Mulder AH, Visser JW (1987) Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence. Exp Hematol 15:99–104

    PubMed  CAS  Google Scholar 

  33. Kim M, Cooper DD, Hayes SF, Spangrude GJ (1998) Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 91:4106–4117

    PubMed  CAS  Google Scholar 

  34. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806

    Article  PubMed  CAS  Google Scholar 

  35. Goodell MA (2005) Stem cell identification and sorting using the Hoechst 33342 side population (SP). Curr Protoc Cytom, Chapter 9: Unit9 18

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  37. Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM (1997) Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp Hematol 25:293–297

    PubMed  CAS  Google Scholar 

  38. Jordan CT, McKearn JP, Lemischka IR (1990) Cellular and developmental properties of fetal hematopoietic stem cells. Cell 61:953–963

    Article  PubMed  CAS  Google Scholar 

  39. Jordan CT, Astle CM, Zawadzki J, Mackarehtschian K, Lemischka IR et al (1995) Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp Hematol 23:1011–1015

    PubMed  CAS  Google Scholar 

  40. Orlic D, Fischer R, Nishikawa S, Nienhuis AW, Bodine DM (1993) Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 82:762–770

    PubMed  CAS  Google Scholar 

  41. Sanchez MJ, Holmes A, Miles C, Dzierzak E (1996) Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity 5:513–525

    Article  PubMed  CAS  Google Scholar 

  42. Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM et al (1997) Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7:335–344

    Article  PubMed  CAS  Google Scholar 

  43. Ito T, Tajima F, Ogawa M (2000) Developmental changes of CD34 expression by murine hematopoietic stem cells. Exp Hematol 28:1269–1273

    Article  PubMed  CAS  Google Scholar 

  44. Matsuoka S, Ebihara Y, Xu M, Ishii T, Sugiyama D et al (2001) CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages. Blood 97:419–425

    Article  PubMed  CAS  Google Scholar 

  45. Uchida N, Dykstra B, Lyons K, Leung F, Kristiansen M et al (2004) ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood 103:4487–4495

    Article  PubMed  CAS  Google Scholar 

  46. Kim I, He S, Yilmaz OH, Kiel MJ, Morrison SJ (2006) Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors. Blood 108:737–744

    Article  PubMed  CAS  Google Scholar 

  47. Randall TD, Weissman IL (1997) Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 89:3596–3606

    PubMed  CAS  Google Scholar 

  48. Sato T, Laver JH, Ogawa M (1999) Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94:2548–2554

    PubMed  CAS  Google Scholar 

  49. Higuchi Y, Zeng H, Ogawa M (2003) CD38 expression by hematopoietic stem cells of newborn and juvenile mice. Leukemia 17:171–174

    Article  PubMed  CAS  Google Scholar 

  50. Spangrude GJ, Brooks DM, Tumas DB (1995) Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 85:1006–1016

    PubMed  CAS  Google Scholar 

  51. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL (1996) The aging of hematopoietic stem cells. Nat Med 2:1011–1016

    Article  PubMed  CAS  Google Scholar 

  52. Pearce DJ, Anjos-Afonso F, Ridler CM, Eddaoudi A, Bonnet D (2007) Age-dependent increase in side population distribution within hematopoiesis: implications for our understanding of the mechanism of aging. Stem Cells 25:828–835

    Article  PubMed  CAS  Google Scholar 

  53. Yilmaz OH, Kiel MJ, Morrison SJ (2006) SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. Blood 107:924–930

    Article  PubMed  CAS  Google Scholar 

  54. Sudo K, Ema H, Morita Y, Nakauchi H (2000) Age-associated characteristics of murine hematopoietic stem cells. J Exp Med 192:1273–1280

    Article  PubMed  CAS  Google Scholar 

  55. Kim M, Moon HB, Spangrude GJ (2003) Major age-related changes of mouse hematopoietic stem/progenitor cells. Ann N Y Acad Sci 996:195–208

    Article  PubMed  Google Scholar 

  56. Li CL, Johnson GR (1992) Rhodamine123 reveals heterogeneity within murine Lin−, Sca-1+ hemopoietic stem cells. J Exp Med 175:1443–1447

    Article  PubMed  CAS  Google Scholar 

  57. Sieburg HB, Cho RH, Dykstra B, Uchida N, Eaves CJ et al (2006) The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets. Blood 107:2311–2316

    Article  PubMed  CAS  Google Scholar 

  58. Muller-Sieburg CE, Cho RH, Karlsson L, Huang JF, Sieburg HB (2004) Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness. Blood 103:4111–4118

    Article  PubMed  CAS  Google Scholar 

  59. Muller-Sieburg CE, Cho RH, Thoman M, Adkins B, Sieburg HB (2002) Deterministic regulation of hematopoietic stem cell self-renewal and differentiation. Blood 100:1302–1309

    PubMed  CAS  Google Scholar 

  60. Sieburg HB, Rezner BD, Muller-Sieburg CE (2011) Predicting clonal self-renewal and extinction of hematopoietic stem cells. Proc Natl Acad Sci USA 108:4370–4375

    Article  PubMed  CAS  Google Scholar 

  61. Beerman I, Bhattacharya D, Zandi S, Sigvardsson M, Weissman IL et al (2010) Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc Natl Acad Sci USA 107:5465–5470

    Article  PubMed  CAS  Google Scholar 

  62. Challen GA, Boles NC, Chambers SM, Goodell MA (2010) Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1. Cell Stem Cell 6:265–278

    Article  PubMed  CAS  Google Scholar 

  63. Morita Y, Ema H, Nakauchi H (2010) Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J Exp Med 207:1173–1182

    Article  PubMed  CAS  Google Scholar 

  64. Matsuzaki Y, Kinjo K, Mulligan RC, Okano H (2004) Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 20:87–93

    Article  PubMed  CAS  Google Scholar 

  65. Robinson SN, Seina SM, Gohr JC, Kuszynski CA, Sharp JG (2005) Evidence for a qualitative hierarchy within the Hoechst-33342 ‘side population’ (SP) of murine bone marrow cells. Bone Marrow Transplant 35:807–818

    Article  PubMed  CAS  Google Scholar 

  66. Kiel MJ, Yilmaz OH, Morrison SJ (2008) CD150− cells are transiently reconstituting multipotent progenitors with little or no stem cell activity. Blood 111:4413–4414, author reply 4414–4415

    Article  PubMed  CAS  Google Scholar 

  67. Weksberg DC, Chambers SM, Boles NC, Goodell MA (2008) CD150− side population cells represent a functionally distinct population of long-term hematopoietic stem cells. Blood 111:2444–2451

    Article  PubMed  CAS  Google Scholar 

  68. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R et al (2010) Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell 6:48–58

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Paula Thomason for her expert editorial assistance in the preparation of the manuscript and Ying Liang for her critical review.

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Correspondence to Kyle Rector .

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Rector, K., Liu, Y., Van Zant, G. (2013). Comprehensive Hematopoietic Stem Cell Isolation Methods. In: Turksen, K. (eds) Stem Cells and Aging. Methods in Molecular Biology, vol 976. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-317-6_1

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  • DOI: https://doi.org/10.1007/978-1-62703-317-6_1

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-316-9

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