Comparison of TGFbR2 downregulation in expanded H

Authors: Zahra Sadat Hashemi Mehdi Forouzandeh Moghadam Masoud Soleimani

Publish Date: 2014/12/25

Volume: 51, Issue: 5, Pages: 495-506

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Abstract

Bone marrow transplants BMTs are mainly limited by a low number of CD34+ cells The transforming growth factorbeta TGFβ pathway downregulation is a key factor that increases cell selfrenewal In nature hematopoietic stem cells HSCs are in a microenvironment surrounded by cells in a threedimensional 3D configuration The aim of this study is to investigate the association between a 3D culture and the delivery ratio of downregulation Demineralized bone matrix DBM and mineralized bone allograft MBA scaffolds were coated using unrestricted somatic stem cells USSCs as the feeder layer Umbilical cord blood UCBCD34+ cells were then ex vivo expanded in them and transfected by small interfering RNA siRNA against TGFbR2 a type 2 receptor in the TGFβ pathway Finally quantitative realtime PCR flow cytometry and clonogenic assay were performed In a global comparison we observed that the highest expansion ratio lowest expression level and the highest CD34 marker belonged to the simple 2D culture transfected group This suggests that TGFbR2 downregulation in a 2D culture can be done more effectively The siRNA delivery system and the transfection ratio in an ex vivo environment which mimicks in vivo conditions have low efficiency Genetic modification of the cells needs free 3D spaces to enable better transfectionThe authors would like to thank Dr Tavakoli in the Iranian Tissue Bank for expert technical assistance This study was performed at the University of Tarbiat Modares and supported by the Biotechnology Development Council BDC The authors are thankful to the BDC for their support

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