Evaluation of DNA Damage and Repair In In Vitro Expanded Cord Blood CD 34 Positive Hematopoietic Stem Cell

Mohammad  Shokouhian; Hossein  Mozdarani; Masoud  Soleimani; Majid  Safa; Mohammad Reza  Rezvany

doi:10.18502/ijml.v10i1.12424

Mohammad Shokouhian Department of Hematology and Blood Transfusion, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran
Hossein Mozdarani Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Masoud Soleimani Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Majid Safa Department of Hematology and Blood Transfusion, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran
Mohammad Reza Rezvany Department of Hematology and Blood Transfusion, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ijml.v10i1.12424

Keywords: Cord blood stem cells, DNA damage, Comet assay, Ku80, KU70

Abstract

Background and Aims: The occurrence of single and double-strand breaks of DNA damage is the major obstacle for proliferation under various environmental factors and, if not repaired, can result in many consequences, including mutation, cell death, and others. So, the present study was conducted to evaluate the damage of DNA and the expression status of DNA repair system genes before and after stem cell proliferation.

Materials and Methods: The MACS method isolated the umbilical cord blood hematopoietic stem cells (UCB-HSCs). In order to investigate cell death, the study of Annexin V/PI was done by flow cytometry. Comet assay made observation and identification of DNA breaks, and the expression of genes normally involved in the repair of DNA breaks was evaluated by real-time polymerase chain reaction.

Results: The average number of stem cells increased by 1.9-fold after three days of proliferation. The apoptotic percentage of cells was negligible (less than 0.2%), and the purity of the CD34⁺ cells was reduced by about one-third in three days (67%). By examining the expression of DNA repair genes, including KU70, KU80, RAD51, and XRCC1, their increased fold change was not significant. In a microscopic examination of stem cells in the comet assay, there was no significant difference between DNA damage before (1.33% ± 0.31) and after (2.08% ± 0.92) replication.

Conclusion: In our investigation, neither DNA damage nor changes in the DNA break repair were observed. However, further studies are required to clarify the DNA break repair by recruiting more UCB-HSCs samples.