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Showing 3 results for Hematopoietic Stem Cells
TGF-b downregulation by RNAi technique in ex vivo-expanded HSCs on 3D DBM scaffold 
Hashemi Zs, Forouzandeh Moghadam M, Soleimani M, Hafizi M, Amirizadeh N,
Volume 70, Issue 2 (5-2012)
Abstract

Background: Bone Marrow Transplantations (BMT) are limited by low CD34+ cell counts in umbilical cord blood (UCB) and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells (HSCs) by enhancing their self-renewal activity on demineralized bone matrix (DBM) scaffold coated with mesenchymal progenitor cells (MPCs) and unrestricted somatic stem cells (USSCs) was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed.

Methods: USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting (MACS) method and were transfected by siRNA against TGFbR2 in two-dimensional (2D) and three-dimensional (3D) cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated.

Results: Ex vivo expansion of CD34+ cells was significantly enhanced (41±0.7 folds) by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay.

Conclusion: The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer.


Expansion of umbilical cord blood hematopoietic stem cell on biocompatible nanofiber scaffolds: brief report
Fatemeh Eskandari , Masoud Soleimani , Nasim Kalantari , Mehdi Azad , Amir Allahverdi ,
Volume 72, Issue 11 (2-2015)
Abstract
Background: Hematopoietic stem cell transplantation (HSCT) is a therapeutic approach in treatment of hematologic malignancies and incompatibility of bone marrow. Umbilical cord blood (UCB) known as an alternative for hematopoietic stem/ progenitor cells (HPSC) for in allogenic transplantation. The main hindrance in application of HPSC derived from umbilical cord blood is the low volume of collected samples. So, ex vivo expansion of HPSCs is the useful approach to overcome this restriction. Synthetic biomaterials such as nanofibers is used to produce synthetic niches. The aim of this study was the ex vivo expansion of hematopoietic stem cells on biocompatible nanofiber scaffolds. Methods: This study was done at Tarbiat Modares University from November 2012 to June 2013 and was a research study. Umbilical cord blood CD133+ hematopoietic stem cells were separated using MidiMacs (positive selection) system by means of monocolonal antibody (microbeads) CD133. Flow cytometry was used to assess the purity of cells. Cell culture was done on plate (2 Dimensional) and fibronectin conjougated polyether sulfone nanofiber scaffold (3 Dimensional). Colony assay test was used to asses the ability of colonization of cells. Results: Cell count analysis revealed the expansion of hematopoietic stem cells in cell culture plate (2D environment) and on nanofiber scaffold (3D environment) after 2 weeks. Expansion of cells in 2D environment was greater than 3D condition. Colony assay test revealed that the colonization ability of cells decreased after 2 weeks, but this decrease was lower in scaffold culture than plate culture. Conclusion: This study demonstrated that umbilical cord blood CD133+ hematopoietic stem cells can expand on fibronectin conjugated polyether sulfone scaffold and we can use this system for expanding of cells in vitro situation.
Gene cloning and expression of soluble thrombopoietin functional domain by harnessing Rosetta-gami expression system
Mohammad Moradi , Kamran Atarodi , Mahshid Mohammadipour , Kamran Mousavi Hosseini ,
Volume 76, Issue 6 (9-2018)
Abstract
Background: Thrombopoietin (TPO) is an important cytokine that has a critical role in regulating hematopoietic stem cells (HSCs) proliferation and megakaryocyte differentiation. Because of scares amount of this protein in human plasma, in many biotechnological centers around the world, recombinant production of this protein has been carried out. This study was aiming to gene cloning and expression of recombinant thrombopoietin.
Methods: This research is an experimental laboratory study carried out in Blood Transfusion Research Center, Tehran, Iran, from July 2016 to August 2017. At the beginning HepG2 cell line was cultured and RNA extraction was performed. Extracted RNA was used as template for cDNA synthesis and subsequently the synthesized cDNA was adopted to isolate TPO gene through polymerase chain reaction (PCR) reaction using designed primers. After isolating the TPO sequence from HepG2 cell line, the designated sequence was inserted into pET32 vectors. Recombinant plasmid was amplified by meriting from DH5α replicating system. The amplified plasmids were sequenced via chain termination method. Next step was transforming the recombinant plasmid into Rosetta-gami bacteria to express the recombinant protein. In order to induce protein expression, an appropriate amount of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to growth media, then bacterial lysate of expression host was prepared and assayed via polyacrylamide gel electrophoresis and western blotting test.
Results: After sequencing of recombinant plasmid, it was confirmed that TPO sequence has been successfully colonized in adopted vector. Subsequent to induction of recombinant protein, total cell protein analysis affirmed that recombinant protein has been expressed in its soluble form at cytoplasmic condition. Location of expected recombinant protein band on polyacrylamide gel and reaction of recombinant protein with His-tag monoclonal antibody at western blotting was asserting that expressed protein is the one of interest.
Conclusion: Rosetta-gami bacteria has capability of expressing recombinant thrombopoietin in its soluble form. By harnessing this method of recombinant protein expression, it would be possible to take advantage of high throughout bacterial expression system which would not produce inclusion body and its product doesn’t need further processing and refolding.

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