Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
권호 표지

Ex vivo Expansion and Clonal Maintenance of CD34+ Selected Cells from Cord Blood and Peripheral Blood

제대혈 및 말포혈로부터 분리한 CD34 양성 세포의 체외 증폭 및 클론 유지

  • Kim, Soon Ki (Hematology-Oncology, Department of Pediatrics, Inha University of Hospital, Inha Medical College) ;
  • Ghil, Hye Yoon (Clinical Research Center, Inha University of Hospital, Inha Medical College) ;
  • Song, Sun U. (Clinical Research Center, Inha University of Hospital, Inha Medical College) ;
  • Choi, Jong Weon (Department of Laboratory Medicine, Inha University of Hospital, Inha Medical College) ;
  • Park, Sang Kyu (Department of Pediatrics§, College of Medicine, Ulsan University)
  • 김순기 (인하대학교 의과대학 소아과학교실) ;
  • 길혜윤 (인하대학교병원 중앙연구소) ;
  • 송순욱 (인하대학교병원 중앙연구소) ;
  • 최종원 (인하대학교병원 진단검사의학교실) ;
  • 박상규 (울산대학교 의과대학 소아과학교실)
  • Received : 2005.03.14
  • Accepted : 2005.05.16
  • Published : 2005.08.15
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose : Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. Methods : The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. Results : The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. Conclusion : The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

목 적 : 조직적합 항원의 불일치로 인하여 골수이식을 할 수 없는 경우에 점점 더 제대혈이 사용되고 있다. 그러나 제대혈의 조혈모세포의 수가 적기 때문에 이를 증가시킬 대책이 필요한 바, 여러 성장인자를 조합하여 체외증폭하여 말초혈의 체외증폭과 비교하였다. 방 법 : 저자들은 제대혈 및 말초혈로부터 분리한 CD34+ 세포를 혈청이 아닌 배양체에서 체외 증폭하여 비교하였다. Miltenyi 방법으로 분리한 CD34+는 조혈성장인자들과 함께 체외 증폭 시켰다. 증폭 당일, 4일 후, 7일 후 및 14일에 증폭된 세포를 가지고 burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) 및 colony-forming units of megakaryocytes (CFU-Mk)의 생성 능력을 알아보았다. 결 과 : 말초혈에 비하여 제대혈로부터 분리한 CD34+ 세포의 증폭 능력이 2배로 컸다. 체외에서7일 및 14일 동안 증폭된 제대혈이 더 많은 BFU-E를 생성하였고, 4일 및 7일 동안 증폭된 제대혈이 더 많은 CFU-Mk를 생성하였다. 결 론 : MGDF, FL 및 IL-3를 포함한 성장인자의 자극 하에서 제대혈의 체외 증폭이 더 많은 BFU-E 및 CFU-Mk를 생성하였으므로, 이를 이용한 체외 증폭을 시도하는 것의 가능성을 시사하고 있다.

Keywords

Acknowledgement

Supported by : Korea Research Foundation

References

  1. Gluckman E, Broxmeyer HE, Auerbach AD, Friedman HS, Douglas GW, Devergie A, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-8 https://doi.org/10.1056/NEJM198910263211707
  2. Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E. Allogeneic sibling umbilical cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346:214-9 https://doi.org/10.1016/S0140-6736(95)91268-1
  3. Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR, et al. Outcomes among 562 recipients of plancental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565-76 https://doi.org/10.1056/NEJM199811263392201
  4. 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;344:1815-22 https://doi.org/10.1056/NEJM200106143442402
  5. Sanz GF, Saavedra S, Planelles D, Senent L, Cervera J, Barragan E, et al. Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies. Blood 2001;98:2332-8. https://doi.org/10.1182/blood.V98.8.2332
  6. Nishihira H, Kato K, Isoyama K, Takahashi TA, Kai S, Kato S, et al. The Japanese cord blood bank network experience with cord blood transplantation from unrelated donors for haematological malignancies : an evaluation of graft-versus-host disease prophylaxis.Nishihira H, Kato K, Isoyama K, Takahashi TA, Kai S, Kato S, et al. The Japanese cord blood bank network experience with cord blood transplantation from unrelated donors for haematological malignancies : an evaluation of graft-versus-host disease prophylaxis. Br J Haematol 2003; 120:516-222003; 120:516-22 https://doi.org/10.1046/j.1365-2141.2003.04115.x
  7. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, et al. Acute Leukemia Working Party of European Blood and Marrow Transplant Group; Eurocord-Netcord Registry. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004;351:2276-85 https://doi.org/10.1056/NEJMoa041469
  8. Muench MO, Barcena A. Megakaryocyte growth and development factor is a potent growth factor for primitive hematopoietic progenitors in the human fetus. Pediatr Res 2004;55:1050-6. https://doi.org/10.1203/01.pdr.0000127020.00090.51
  9. Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 1994;369:568-71 https://doi.org/10.1038/369568a0
  10. Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, et al. Humoral regulator of megakaryocytopoiesis. Nature 1994;369:571-4 https://doi.org/10.1038/369571a0
  11. Terada Y, Hato F, Sakamoto C, Hasegawa T, Suzuki K, Nakamae H, Ohta K, Yamane T, Kitagawa S, Hino M. Thrombopoietin stimulates ex vivo expansion of mature neutrophils in the early stages of differentiation. Ann Hematol 2003;82:671-6 https://doi.org/10.1007/s00277-003-0729-7
  12. Debili N, Wendling F, Katz A, Guichard J, Breton-Gorius J, Hunt P, et al. The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 1995;86:2516-25
  13. Young JC, Bruno E, Luens KM, Wu S, Baker M, Murray LJ. Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34＋ progenitor cells from single CD34＋Thy-1＋Lin- primitive progenitor cells. Blood 1996;88:1619-31
  14. Schattner M, Lefebvre P, Mingolelli SS, Goolsby CL, Rademaker A, White JG, et al. Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes. Stem Cells 1996;14:207-14 https://doi.org/10.1002/stem.140207
  15. Moore MAS, Hoskins I. Ex vivo expansion of cord bloodderived stem cells and progenitors. Blood Cells 1994;20:468-81
  16. Li K, Li CK, Chuen CK, Tsang KS, Fok TF, James AE, et al. Preclinical ex vivo expansion of G-CSF-mobilized peripheral blood stem cells : effects of serum-free media, cytokine combinations and chemotherapy. Eur J Haematol 2005;74:128-35 https://doi.org/10.1111/j.1600-0609.2004.00343.x
  17. Almici C, Carlo-Stella C, Wagner JE, Rizzoli V. Umbilical cord blood as a source of hematopoietic stem cells : from research to clinical application. Haematologica 1995;80:473- 9
  18. Bertolini F, Battaglia M, Pedrazzoli P, Da Prada GA, Lanza A, Soligo D, et al. Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral progenitor cell transplant recipients. Blood 1997; 89:2679-88
  19. Shpall EJ, Quinones R, Giller R. Transplantation of adult and pediatric cancer patients with cord blood progenitors expanded ex vivo [abstract]. Blood 2000;98:851a. https://doi.org/10.1182/blood.V98.3.851
  20. Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells : results of a phase 1 trial using the AastromReplicell System. Blood 2003;101:5061-7 https://doi.org/10.1182/blood-2001-12-0290
  21. Miltenyi S, Muller W, Weichel W, and Radbruch A. High gradient magnetic cell separation with MACS. Cytometry 1990;11:231-8. https://doi.org/10.1002/cyto.990110203
  22. Kim SK, Koh SK, Song SW, Shin SH, Choi GS, Kim, WC, et al. Ex vivo expansion and clonality of CD34＋ selected cells from bone marrow and cord blood in a serum-free media. Molecules and Cells 2002;14:367-73
  23. Cairo MS, Wagner JE. Placental and/or umbilical cord blood : an alternative source of hematopoietic stem cells for transplantation. Blood 1997;90:4665-78
  24. 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 highdose chemotherapy in patients with breast cancer. Blood 2000;96:3001-7
  25. Paquette RL, Dergham ST, Karpf E, Wang HJ, Slamon DJ, Souza L, Glaspy JA. Culture conditions affect the ability of ex vivo expanded peripheral blood progenitor cells to accelerate hematopoietic recovery. Exp Hematol 2002;30:374-80 https://doi.org/10.1016/S0301-472X(02)00770-1
  26. Hirao A, Kawano Y, Takaue Y, Suzue T, Abe T, Sato J, et al. Engraftment potential of peripheral and cord blood stem cells evaluated by a long-term culture system. Exp Hematol 1994;22:521-6
  27. Traycoff CM, Abboud MR, Laver J, Brandt JE, Hoffman R, Law P, et al. Evaluation of the in vitro behavior of phenotypically defined populations of umbilical cord blood hematopoietic progenitor cells. Exp Hematol 1994;22:215-22
  28. Seoh JY, Woo SY, Im SA, Kim YJ, Park HY, Lee S, et al. Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocytecolony stimulating factor during ex vivo expansion of human cord blood CD34＋ cells. Br J Haematol 1999;107: 176-85 https://doi.org/10.1046/j.1365-2141.1999.01677.x
  29. Lyman SD. Biology of flt3 ligand and receptor. Int J Hematol 1995;62:63-73 https://doi.org/10.1016/0925-5710(95)00389-A
  30. McKenna HJ, de Vries P, Brasel K, Lyman SD, Williams DE. Effect of flt3 ligand on the ex vivo expansion of human CD34＋ hematopoietic progenitor cells. Blood 1995; 86:3413-20
  31. Bernstein ID, Andrews RG, Zsebo KM. Recombinant human stem cell factor enhances the formation of colonies by CD34＋and CD34＋lin- cells, and the generation of colonyforming cell progeny from CD34＋lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood 1991;77:2316-21
  32. Thomas SJ, Lamping CP, Ziegler BL. Phenotype analysis of hematopoietic CD34＋ cell populations derived from human umbilical cord blood using flow cytometry and cDNApolymerase chain reaction. Blood 1994;83:2103-14
  33. Lefebvre P, Winter JN, Kahn LE, Giri JG, Cohen I. Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium. J Hematotherapy 1999;8:199-208 https://doi.org/10.1089/106161299320479
  34. Goussetis E, Theodosaki M, Paterakis G, Stefanaki K, Tsecoura C, Papassarandis C, et al. Kinetics of quiescent cord blood stem/progenitor cells with high proliferative potential in stem-cell expansion culture. Cytotherapy 2003; 5:500-8 https://doi.org/10.1080/14653240310003602