Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Indian Journal of Hematology and Blood Transfusion Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Delaney C, Ratajczak MZ, Laughlin MJ (2010) Strategies to enhance umbilical cord blood stem cell engraftment in adult patients. Expert Rev Hematol 3:273–283

    Article  PubMed Central  PubMed  Google Scholar 

  2. Broxmeyer HE (2010) Umbilical cord transplantation: epilogue. Semin Hematol 47:97–103

    Article  PubMed Central  PubMed  Google Scholar 

  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 100:1611–1618

    CAS  PubMed  Google Scholar 

  4. Dahlberg A, Delaney C, Bernstein ID (2011) Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 23:234–245

    Google Scholar 

  5. Patricia D, Conrad SG (1998) Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation. J Leuk Biol 64:147–155

    Google Scholar 

  6. Oguro H, Iwama A (2007) Life and death in hematopoietic stem cells. Curr Opin Immunol 19:503–509

    Article  CAS  PubMed  Google Scholar 

  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 82:7–18

    PubMed Central  CAS  PubMed  Google Scholar 

  8. Cohen O, Kimchi A (2001) DAP-kinase from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ 8:6–15

    Article  CAS  PubMed  Google Scholar 

  9. Gozuacik Devrim, Kimchi Adi (2006) DAPk protein family and cancer. Autophagy 2:74–79

    Article  CAS  PubMed  Google Scholar 

  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 35:712–723

    Article  CAS  PubMed  Google Scholar 

  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 9:841–848

    Article  CAS  PubMed  Google Scholar 

  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 60:892–895

    CAS  PubMed  Google Scholar 

  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 10:3441–3454

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 125:1566–1574

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 23:245–257

    Article  CAS  PubMed  Google Scholar 

  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 36:71–79

    Article  CAS  Google Scholar 

  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 26:53–62

    CAS  PubMed  Google Scholar 

  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 87:69–80

    Article  CAS  PubMed  Google Scholar 

  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 15:11–20

    Article  CAS  PubMed  Google Scholar 

  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 85:212–225

    Article  PubMed  Google Scholar 

  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 33:828–835

    Article  PubMed  Google Scholar 

  22. Wagner Wolfgang, Saffrich Rainer, Anthony D (2008) The stromal activity of mesenchymal stromal cells. Transfus Med Hemother 35:185–193

    Article  PubMed Central  PubMed  Google Scholar 

  23. Song Yifang, Bahnson Alfred, Hall Nathan (2010) Stem cell traits in long-term co-culture revealed by time-lapse imaging. Leukemia 24:153–161

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Wagner W, Saffrich R, Wirkner U (2005) Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction. Stem Cells 23:1180–1191

    Article  CAS  PubMed  Google Scholar 

  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):813–823

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 28:141–145

    Article  PubMed Central  PubMed  Google Scholar 

  27. Jones A, Baylin B (2007) The epigenomics of cancer. Cell 128:683–692

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33

    Article  CAS  PubMed  Google Scholar 

  29. Issa Jean-Pierre (2010) Epigenetic changes in the myelodysplastic syndrome. Hematol Oncol Clin North Am 24:317–330

    Article  PubMed Central  PubMed  Google Scholar 

  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 45:181–185

    Article  CAS  PubMed  Google Scholar 

  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 32:74–81

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine, IBTO Bldg. Hemmat Exp Way, Next to the Milad Tower, P.O. Box 14665-1157, Tehran, Iran

    Naser Amirizadeh, Arezoo Oodi, Roya Mehrasa & Mahin Nikougoftar

Authors
  1. Naser Amirizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arezoo Oodi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roya Mehrasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mahin Nikougoftar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Naser Amirizadeh.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Amirizadeh, N., Oodi, A., Mehrasa, R. et al. 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. Indian J Hematol Blood Transfus 32, 67–77 (2016). https://doi.org/10.1007/s12288-015-0545-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12288-015-0545-y

Keywords

Search

Navigation