title: Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34+ cells.
abstract: Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche.
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Eun-Ju
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Cheang Ly
authors.initials: CL
authors.lastname: Be
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Vinson
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Riches
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Friederike
authors.initials: F
authors.lastname: Fehr
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Gardiner
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Gengenbach
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia; Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Winkler
authors.affiliation: Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia; Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3168, Australia. Electronic address: david.haylock@csiro.au.
authors.forename: David
authors.initials: D
authors.lastname: Haylock
mesh_terms: D000595:Amino Acid Sequence; D018952:Antigens, CD34 / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D055664:Electrochemical Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D016130:Immunophenotyping; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D056951:Photoelectron Spectroscopy; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
chemical_list: D018952:Antigens, CD34; D055767:Immobilized Proteins; D008024:Ligands; D010455:Peptides; D013438:Sulfhydryl Compounds; C515728:thrombopoietin mimetic peptide
keywords: Haematopoietic stem cell; Ligand immobilisation; Oxime; Self-assembled monolayers; Thrombopoietin