RESUMEN
Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Osteopontina/fisiología , Animales , Secuencia de Bases , Células CHO , Línea Celular , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Cricetinae , Cricetulus , Cartilla de ADN/genética , Sangre Fetal/citología , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Técnicas In Vitro , Integrina alfa4beta1/genética , Integrinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteopontina/deficiencia , Osteopontina/genética , Osteopontina/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMEN
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.