RRC ID 44662
Author Saka K, Kawahara M, Teng J, Otsu M, Nakauchi H, Nagamune T.
Title Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells.
Journal J Biotechnol
Abstract The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.
Volume 168(4)
Pages 659-65
Published 2013-12-1
DOI 10.1016/j.jbiotec.2013.09.012
PII S0168-1656(13)00403-3
PMID 24070902
MeSH Amino Acid Motifs / genetics Gene Expression Regulation Genetic Engineering* Hematopoietic Stem Cells / cytology Hematopoietic Stem Cells / metabolism* Humans Mutation Receptors, Thrombopoietin / genetics* Receptors, Thrombopoietin / immunology Receptors, Thrombopoietin / metabolism Recombinant Fusion Proteins / genetics* Recombinant Fusion Proteins / immunology Recombinant Fusion Proteins / metabolism STAT3 Transcription Factor / biosynthesis STAT3 Transcription Factor / genetics STAT5 Transcription Factor / biosynthesis STAT5 Transcription Factor / genetics Signal Transduction Thrombopoietin / immunology Thrombopoietin / metabolism
IF 3.163
Times Cited 2
Human and Animal Cells