title: Comparison of TGFbR2 down-regulation in expanded HSCs on MBA/DBM scaffolds coated by UCB stromal cells.
abstract: Bone marrow transplants (BMTs) are mainly limited by a low number of CD34(+) cells. The transforming growth factor-beta (TGF-β) pathway downregulation is a key factor that increases cell self-renewal. In nature, hematopoietic stem cells (HSCs) are in a microenvironment, surrounded by cells in a three-dimensional (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 (UCB)-CD34(+) cells were then ex vivo expanded in them and transfected by small interfering RNA (siRNA) against TGFbR2, a type 2 receptor in the TGF-β pathway. Finally, quantitative real-time 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 transfection.
authors.affiliation: Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran, z.hashemi87@gmail.com.
authors.forename: Zahra Sadat
authors.initials: ZS
authors.lastname: Hashemi
authors.forename: Mehdi Forouzandeh
authors.initials: MF
authors.lastname: Moghadam
authors.forename: Masoud
authors.initials: M
authors.lastname: Soleimani
mesh_terms: D064591:Allografts / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D001857:Bone Matrix / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D055153:Stem Cell Niche / Q000502:physiology*; D017154:Stromal Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D018952:Antigens, CD34; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II