Advertisement

Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells

  • Naser AmirizadehEmail author
  • Arezoo Oodi
  • Roya Mehrasa
  • Mahin Nikougoftar
Original Article

Abstract

Expansion of umbilical cord blood-derived CD34+ cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34+ cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34+ cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34+ cells in all three culture conditions after 10 days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p < 0.01). DAPK1gene promoter of expanded CD34+ cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34+ cells. Expression of DAPK1 in hematopoietic cells was increased during 10 days expansion of CD34+ cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Keywords

Hematopoietic stem cells Cord blood expansion Mesenchymal stem cells Apoptosis 

References

  1. 1.
    Delaney C, Ratajczak MZ, Laughlin MJ (2010) Strategies to enhance umbilical cord blood stem cell engraftment in adult patients. Expert Rev Hematol 3:273–283PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Broxmeyer HE (2010) Umbilical cord transplantation: epilogue. Semin Hematol 47:97–103PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Wagner JE, Barker JN, DeFor TE, Baker KS, Blazar BR, Eide C, Goldman A, Kersey J, Krivit W, MacMillan ML, Orchard PJ, Peters C, Weisdorf DJ, Ramsay NK, Davies SM (2002) 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 100:1611–1618PubMedGoogle Scholar
  4. 4.
    Dahlberg A, Delaney C, Bernstein ID (2011) Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 23:234–245Google Scholar
  5. 5.
    Patricia D, Conrad SG (1998) Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation. J Leuk Biol 64:147–155Google Scholar
  6. 6.
    Oguro H, Iwama A (2007) Life and death in hematopoietic stem cells. Curr Opin Immunol 19:503–509CrossRefPubMedGoogle Scholar
  7. 7.
    Alenzi FQ, Alenazi BQ, Ahmad SY, Salem ML, Al-Jabri AA, Wyse RK (2009) The haemopoietic stem cell: between apoptosis and self renewal. Yale J Biol Med 82:7–18PubMedCentralPubMedGoogle Scholar
  8. 8.
    Cohen O, Kimchi A (2001) DAP-kinase from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ 8:6–15CrossRefPubMedGoogle Scholar
  9. 9.
    Gozuacik Devrim, Kimchi Adi (2006) DAPk protein family and cancer. Autophagy 2:74–79CrossRefPubMedGoogle Scholar
  10. 10.
    Hopfer O, Komor M, Koehler IS, Schulze M, Hoelzer D, Thiel E, Hofmann WK (2007) DNA methylation profiling of myelodysplastic syndrome hematopoietic progenitor cells during in vitro lineage-specific differentiation. Exp Hematol 35:712–723CrossRefPubMedGoogle Scholar
  11. 11.
    Majumdar MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson SL (2000) Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res 9:841–848CrossRefPubMedGoogle Scholar
  12. 12.
    Sanchez-Cespedes M, Esteller M, Wu L, Nawroz-Danish H, Yoo GH, Koch WM, Jen J, Herman JG, Sidransky D (2000) Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 60:892–895PubMedGoogle Scholar
  13. 13.
    Xu XL, Yu J, Zhang HY, Sun MH, Gu J, Du X, Shi DR, Wang P, Yang ZH, Zhu JD (2004) Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World J Gastroenterol 10:3441–3454PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Gade P, Singh AK, Roy SK, Reddy SP, Kalvakolanu DV (2009) Down-regulation of the transcriptional mediator subunit Med1 contributes to the loss of expression of metastasis-associated DAPK1 in human cancers and cancer cells. Int J Cancer 125:1566–1574PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Tung SS, Parmar S, Robinson SN, De Lima M, Shpall EJ (2010) Ex vivo expansion of umbilical cord blood for transplantation. Best Pract Res Clin Haematol 23:245–257CrossRefPubMedGoogle Scholar
  16. 16.
    Yildirim S, Boehmler Am, Kanz L, Mohle R (2005) Expansion of cord blood CD34+ hematopoietic progenitor cells in co-culture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transpl 36:71–79CrossRefGoogle Scholar
  17. 17.
    Traycoff CM, Orazi A, Ladd AC (1998) Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells. Exp Hematol 26:53–62PubMedGoogle Scholar
  18. 18.
    Wulf-Goldenberg A, Eckert K, Fichtner I (2008) Cytokine-pretreatment of CD34+ cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice. Eur J Cell Biol 87:69–80CrossRefPubMedGoogle Scholar
  19. 19.
    Alvarado-Moreno A, Chávez-González A, Cérbulo A, Arriaga L, Mayani H (2010) In vitro cell cycle dynamics of primitive hematopoietic cells from human umbilical cord blood. Hematology 15:11–20CrossRefPubMedGoogle Scholar
  20. 20.
    Jang YK, Jung DH, Jung MH, Kim DH, Yoo KH, Sung KW, Koo HH, Oh W, Yang YS, Yang SE (2006) Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells. Ann Hematol 85:212–225CrossRefPubMedGoogle Scholar
  21. 21.
    Da Silva CL, Gonçalves R, Crapnell KB, Cabral JM, Zanjani ED, Almeida-Porada G (2005) A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells. Exp Hematol 33:828–835CrossRefPubMedGoogle Scholar
  22. 22.
    Wagner Wolfgang, Saffrich Rainer, Anthony D (2008) The stromal activity of mesenchymal stromal cells. Transfus Med Hemother 35:185–193PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Song Yifang, Bahnson Alfred, Hall Nathan (2010) Stem cell traits in long-term co-culture revealed by time-lapse imaging. Leukemia 24:153–161PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Wagner W, Saffrich R, Wirkner U (2005) Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction. Stem Cells 23:1180–1191CrossRefPubMedGoogle Scholar
  25. 25.
    Zou Jing, Zou Ping, Wang Jie (2012) Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells. Ann Hematol 91(6):813–823PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Wei Z, Chen N, Guo H, Wang X, Xu F, Ren Q, Lu S, Liu B, Zhang L, Zhao H (2009) Bone marrow mesenchymal stem cells from leukemia patients inhibit growth and apoptosis in serum-deprived K562 cells. J Exp Clin Cancer Res 28:141–145PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Jones A, Baylin B (2007) The epigenomics of cancer. Cell 128:683–692PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33CrossRefPubMedGoogle Scholar
  29. 29.
    Issa Jean-Pierre (2010) Epigenetic changes in the myelodysplastic syndrome. Hematol Oncol Clin North Am 24:317–330PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Greco M, D’Alò F, Scardocci A, Criscuolo M, Fabiani E, Guidi F, Di Ruscio A, Migliara G, Pagano L, Fianchi L, Chiusolo P, Hohaus S, Leone G, Voso MT (2010) Promoter methylation of DAPK1, E-cadherin and thrombospondin-1 in de novo and therapy-related myeloid neoplasms. Blood Cells Mol Dis 45:181–185CrossRefPubMedGoogle Scholar
  31. 31.
    Qian J, Yao DM, Lin J, Wang YL, Han LX, Xu WR, Wu CY (2010) Methylation of DAPK1 promoter: frequent but not an adverse prognostic factor in myelodysplastic syndrome. Int J Lab Hematol 32:74–81CrossRefPubMedGoogle Scholar

Copyright information

© Indian Society of Haematology & Transfusion Medicine 2015

Authors and Affiliations

  • Naser Amirizadeh
    • 1
    Email author
  • Arezoo Oodi
    • 1
  • Roya Mehrasa
    • 1
  • Mahin Nikougoftar
    • 1
  1. 1.Blood Transfusion Research CenterHigh Institute for Education and Research in Transfusion MedicineTehranIran

Personalised recommendations