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Cord Blood Stem Cells

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells


One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133+ selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stem cell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133+ and CD34+ cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients.

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  1. 1

    Gluckman E, Rocha V, Chevret S . Results of unrelated umbilical cord blood hematopoietic stem cell transplantation. Rev Clin Exp Hematol 2001; 5: 87–99.

  2. 2

    Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, Yoder M et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89: 4109–4113.

  3. 3

    Gluckman E, Rocha V, Arcese W, Michel G, Sanz G, Chan KW et al. Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Hematol 2004; 32: 397–407.

  4. 4

    Kurtzberg J, Laughlin M, Graham ML, Smith C, Olson JF, Halperin EC et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–166.

  5. 5

    Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, Pasquini R et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997; 337: 373–381.

  6. 6

    Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: 3828–3832.

  7. 7

    Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, Wagner JE et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344: 1815–1822.

  8. 8

    Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM, Rubinstein P . Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000; 96: 2717–2722.

  9. 9

    Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–1577.

  10. 10

    de Lima M, St John LS, Wieder ED, Lee MS, McMannis J, Karandish S et al. Double-chimaerism after transplantation of two human leucocyte antigen mismatched, unrelated cord blood units. Br J Haematol 2002; 119: 773–776.

  11. 11

    Barker JN, Weisdorf DJ, Wagner JE . Creation of a double chimera after the transplantation of umbilical-cord blood from two partially matched unrelated donors. N Engl J Med 2001; 344: 1870–1871.

  12. 12

    Barker JN, Weisdorf DJ, Defor TE, Blazar BR, Miller JS, Wagner JE . Rapid and complete donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood 2003; 102: 1915–1919.

  13. 13

    Barker JN, Weisdorf DJ, Defor TE, Blazar BR, McGlave PB, Miller JS et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–1347.

  14. 14

    Guenechea G, Gan OI, Dorrell C, Dick JE . Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001; 2: 75–82.

  15. 15

    Lemischka IR, Jordan CT . The return of clonal marking sheds new light on human hematopoietic stem cells. Nat Immunol 2001; 2: 11–12.

  16. 16

    Hogan CJ, Shpall EJ, Keller G . Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice. Proc Natl Acad Sci USA 2002; 99: 413–418.

  17. 17

    Williams DA . Ex vivo expansion of hematopoietic stem and progenitor cells – robbing Peter to pay Paul? Blood 1993; 81: 3169–3172.

  18. 18

    McNiece IK, Almeida-Porada G, Shpall EJ, Zanjani E . Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol 2002; 30: 612–616.

  19. 19

    McNiece I, Jones R, Bearman SI, Cagnoni P, Nieto Y, Franklin W et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 2000; 96: 3001–3007.

  20. 20

    Shimizu Y, Ogawa M, Kobayashi M, Almeida-Porada G, Zanjani ED . Engraftment of cultured human hematopoietic cells in sheep. Blood 1998; 91: 3688–3692.

  21. 21

    Tisdale JF, Hanazono Y, Sellers SE, Agricola BA, Metzger ME, Donahue RE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998; 92: 1131–1141.

  22. 22

    Abkowitz JL, Taboada MR, Sabo KM, Shelton GH . The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. Stem Cells 1998; 16: 288–293.

  23. 23

    Von Drygalski A, Alespeiti G, Ren L, Adamson JW . Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential. Stem Cells Dev 2004; 13: 101–111.

  24. 24

    Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ . Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 1996; 87: 30–37.

  25. 25

    Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ . Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol 1995; 23: 461–469.

  26. 26

    Holyoake TL, Alcorn MJ, Richmond L, Farrell E, Pearson C, Green R et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997; 19: 1095–1101.

  27. 27

    Piacibello W, Sanavio F, Severino A, Dane A, Gammaitoni L, Fagioli F et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999; 93: 3736–3749.

  28. 28

    Lewis ID, Almeida-Porada G, Du J, Lemischka IR, Moore KA, Zanjani ED et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001; 97: 3441–3449.

  29. 29

    Guenechea G, Segovia JC, Albella B, Lamana M, Ramirez M, Regidor C et al. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells. Blood 1999; 93: 1097–1105.

  30. 30

    Traycoff CM, Cornetta K, Yoder MC, Davidson A, Srour EF . Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp Hematol 1996; 24: 299–306.

  31. 31

    Muench MO, Moore MA . Accelerated recovery of peripheral blood cell counts in mice transplanted with in vitro cytokine-expanded hematopoietic progenitors. Exp Hematol 1992; 20: 611–618.

  32. 32

    Holyoake TL, Freshney MG, McNair L, Parker AN, McKay PJ, Steward WP et al. Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow. Blood 1996; 87: 4589–4595.

  33. 33

    Zhai QL, Qiu LG, Li Q, Meng HX, Han JL, Herzig RH et al. Short-term ex vivo expansion sustains the homing-related properties of umbilical cord blood hematopoietic stem and progenitor cells. Haematologica 2004; 89: 265–273.

  34. 34

    Tian H, Huang S, Gong F, Tian L, Chen Z . Karyotyping, immunophenotyping, and apoptosis analyses on human hematopoietic precursor cells derived from umbilical cord blood following long-term ex vivo expansion. Cancer Genet Cytogenet 2005; 157: 33–36.

  35. 35

    Purdy MH, Hogan CJ, Hami L, McNiece I, Franklin W, Jones RB et al. Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation. J Hematother 1995; 4: 515–525.

  36. 36

    Briddell RA, Kern BP, Zilm KL, Stoney GB, McNiece IK . Purification of CD34+ cells is essential for optimal ex vivo expansion of umbilical cord blood cells. J Hematother 1997; 6: 145–150.

  37. 37

    McNiece IK, Stoney GB, Kern BP, Briddell RA . CD34+ cell selection from frozen cord blood products using the Isolex 300i and CliniMACS CD34 selection devices. J Hematother 1998; 7: 457–461.

  38. 38

    Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002; 8: 368–376.

  39. 39

    McNiece I, Kubegov D, Kerzic P, Shpall EJ, Gross S . Increased expansion and differentiation of cord blood products using a two-step expansion culture. Exp Hematol 2000; 28: 1181–1186.

  40. 40

    Lemischka IR, Moore KA . Stem cells: interactive niches. Nature 2003; 425: 778–779.

  41. 41

    Fuchs E, Tumbar T, Guasch G . Socializing with the neighbors: stem cells and their niche. Cell 2004; 116: 769–778.

  42. 42

    Hackney JA, Charbord P, Brunk BP, Stoeckert CJ, Lemischka IR, Moore KA . A molecular profile of a hematopoietic stem cell niche. Proc Natl Acad Sci USA 2002; 99: 13061–13066.

  43. 43

    Etheridge SL, Spencer GJ, Heath DJ, Genever PG . Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells 2004; 22: 849–860.

  44. 44

    Kadereit S, Deeds LS, Haynesworth SE, Koc ON, Kozik MM, Szekely E et al. Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(−) early progenitors cultured over human MSCs as a feeder layer. Stem Cells 2002; 20: 573–582.

  45. 45

    Rattis FM, Voermans C, Reya T . Wnt signaling in the stem cell niche. Curr Opin Hematol 2004; 11: 88–94.

  46. 46

    Zhang J, Niu C, Ye L, Huang H, He X, Tong WG et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003; 425: 836–841.

  47. 47

    Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL . Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol 1998; 176: 57–66.

  48. 48

    Yildirim S, Boehmler AM, Kanz L, Mohle R . Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005; 36: 71–79.

  49. 49

    Breems DA, Blokland EA, Siebel KE, Mayen AE, Engels LJ, Ploemacher RE . Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells. Blood 1998; 91: 111–117.

  50. 50

    Brandt JE, Galy AH, Luens KM, Travis M, Young J, Tong J et al. Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line. Exp Hematol 1998; 26: 950–961.

  51. 51

    Rafii S, Shapiro F, Pettengell R, Ferris B, Nachman RL, Moore MA et al. Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 1995; 86: 3353–3363.

  52. 52

    Chute JP, Saini AA, Chute DJ, Wells MR, Clark WB, Harlan DM et al. Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow. Blood 2002; 100: 4433–4439.

  53. 53

    Davis TA, Robinson DH, Lee KP, Kessler SW . Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors. Blood 1995; 85: 1751–1761.

  54. 54

    Zhang Y, Li C, Jiang X, Zhang S, Wu Y, Liu B et al. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells. Exp Hematol 2004; 32: 657–664.

  55. 55

    Kanai M, Hirayama F, Yamaguchi M, Ohkawara J, Sato N, Fukazawa K et al. Stromal cell-dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity. Bone Marrow Transplant 2000; 26: 837–844.

  56. 56

    McNiece I, Harrington J, Turney J, Kellner J, Shpall EJ . Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells. Cytotherapy 2004; 6: 311–317.

  57. 57

    in't Anker PS, Noort WA, Kruisselbrink AB, Scherjon SA, Beekhuizen W, Willemze R et al. Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34(+) cells in NOD/SCID mice. Exp Hematol 2003; 31: 881–889.

  58. 58

    Noort WA, Kruisselbrink AB, in't Anker PS, Kruger M, van Bezooijen RL, de Paus RA et al. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice. Exp Hematol 2002; 30: 870–878.

  59. 59

    Maitra B, Szekely E, Gjini K, Laughlin MJ, Dennis J, Haynesworth SE et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004; 33: 597–604.

  60. 60

    Almeida-Porada G, Porada CD, Tran N, Zanjani ED . Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 2000; 95: 3620–3627.

  61. 61

    Angelopoulou M, Novelli E, Grove JE, Rinder HM, Civin C, Cheng L et al. Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 2003; 31: 413–420.

  62. 62

    Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, Uzunel M et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363: 1439–1441.

  63. 63

    Le Blanc K . Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 2003; 5: 485–489.

  64. 64

    Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O . HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 2003; 31: 890–896.

  65. 65

    Gotherstrom C, Ringden O, Westgren M, Tammik C, Le Blanc K . Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells. Bone Marrow Transplant 2003; 32: 265–272.

  66. 66

    Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O . Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003; 57: 11–20.

  67. 67

    Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC . Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 2003; 75: 389–397.

  68. 68

    Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99: 3838–3843.

  69. 69

    Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002; 30: 42–48.

  70. 70

    Neben S, Anklesaria P, Greenberger J, Mauch P . Quantitation of murine hematopoietic stem cells in vitro by limiting dilution analysis of cobblestone area formation on a clonal stromal cell line. Exp Hematol 1993; 21: 438–443.

  71. 71

    Ploemacher RE, van der Sluijs JP, Voerman JS, Brons NH . An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 1989; 74: 2755–2763.

  72. 72

    Kusadasi N, van Soest PL, Mayen AE, Koevoet JL, Ploemacher RE . Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood. Leukemia 2000; 14: 1944–1953.

  73. 73

    Breems DA, Blokland EA, Neben S, Ploemacher RE . Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukemia 1994; 8: 1095–1104.

  74. 74

    Wagner JE, Barker JN, Defor TE, Baker KS, Blazar BR, Eide C et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–1618.

  75. 75

    Barker JN, Wagner JE . Umbilical cord blood transplantation: current practice and future innovations. Crit Rev Oncol Hematol 2003; 48: 35–43.

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We gratefully acknowledge the helpful advice of Michael Thomas, Nirmali Ponweera and Dr Sean O’Connor, Department of Blood and Marrow Transplantation, University of Texas MD Anderson Cancer Center and Dr WE Fibbe, Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands, in the isolation and propagation of MSC. This research was supported by NCI 5R01CA061508-13 (EJS).

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Correspondence to S N Robinson.

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  • cord blood
  • ex vivo expansion
  • mesenchymal stem cell
  • CD34+
  • CD133+
  • cobblestone area-forming cell assay

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