Fibrin hydrogels functionalized with alpha6beta1 ligands for transplantation of neural stem/progenitor cells

Abstract

Event Abstract Back to Event Fibrin hydrogels functionalized with alpha6beta1 ligands for transplantation of neural stem/progenitor cells Joana N. Silva1, 2, Ana R. Bento1, 2, 3, Tiago L. Laundos1, 2, Ana P. Pêgo1, 2, 3, 4 and Isabel Amaral1, 2, 3 1 INEB - Instituto de Engenharia Biomédica, University of Porto, NanoBiomaterials for Targeted Therapies (nBTT) group, Biomaterials for Neuroscience Research Team, Portugal 2 I3S - Instituto de Investigação e Inovação em Saúde, University of Porto, Portugal 3 Faculdade de Engenharia da Universidade do Porto, Portugal 4 Instituto de Ciências Biomédicas Abel Salazar, Portugal Introduction: Transplantation of neural stem/progenitor cells (NSPCs) has emerged as a promising therapy to overcome the limited capacity of the central nervous system to repair. Still, despite their functional benefits, NSPCs directly injected into the lesion site of chronic injuries show low survival and integration of grafted cells. To overcome these limitations, NSPCs are being combined with biodegradable hydrogels which, besides allowing a homogenous distribution of transplanted cells, constitute a more permissive environment for NSPC anchorage, migration, and differentiation. In this study we aimed at developing a fibrin (Fb)-based hydrogel for transplantation of embryonic stem (ES)-derived NSPCs. To enhance Fb specificity towards NSPCs and promote NSPC migration in vivo, Fb was functionalized with ligands for selective binding to α6β1 integrin receptors, an integrin highly expressed by NSCs and involved in NSC migration [1]. Six peptides with reported affinity to α6β1 integrin (P3, HYD1, T1, AG10, N4 and A5G81) were investigated. Methods: NSPCs were derived from a mouse ES cell line (46C) expressing GFP under the promoter of the neural-specific Sox1 gene. NSPCs were seeded on peptide-adsorbed surfaces and cell adhesion (in the presence/absence of mAbs against α6β1 integrin subunits), attachment strength, migration and differentiation along the neuronal lineage were quantitatively assessed. Bi-domain peptides with the selected α6β1 ligands at the C-terminus and the factor XIIIa substrate domain at the N-terminus were bound to Fb gels at different concentrations by the action of factor XIIIa [2]. Peptide binding efficiency was determined using 125I-labeled peptides while structural changes in Fb network were followed by confocal microscopy using FITC-labelled fibrinogen.To assess the effect of immobilized ligands on NSPC migration, neurospheres (NSPC aggregates) were seeded on functionalized Fb gels and radial outgrowth (outgrowth area and maximal outgrowth distance) determined after 72 h of cell culture using automatic image analysis. Unmodified Fb and Fb containing laminin (LN) 111/LN 511 were used as controls. To assess the effect of immobilized ligands on neurite extension, rat E18 DRGs were cultured in functionalized gels and neurite outgrowth quantified after 48h of cell culture. Results & discussion: Among the tested peptides HYD1, T1, and A5G81 were selected for immobilization in Fb due to their higher efficiency in promoting NSPC adhesion mediated through α6β1 integrin. Following incorporation in Fb, soluble LN was not able to promote radial outgrowth from neurospheres, regardless of the LN isoform used. Functionalization of Fb with A5G81, neither. Still, immobilization of HYD1 and T1 in Fb led to significantly higher outgrowth area (2.4-fold and 2.2-fold increase) as well as maximal outgrowth distance, for bi-domain peptide concentrations of 20 µM and 40 µM, respectively. Blocking against α6β1 integrin subunits significantly inhibited cell outgrowth on Fb gels functionalized with HYD1/T1, indicating that HYD1/T1 ability to promote NSC migration on Fb is partially mediated through α6β1 integrin. Finally, HYD1- and T1-functionalized Fb gels led to a 2-fold and 1.9-fold increase in neurite extension from rat E18 DRGs for bi-domain peptide concentrations of 20 µM and 40 µM, respectively. Presently, the in vivo biological performance of HYD1/T1-functionalized Fb is being assessed. The authors would like to acknowledge Prof. Domingos Henrique (Instituto de Medicina Molecular, Lisbon) for providing the ES 46C cell line.; This work was supported by FCT / MEC through National Funds (PTDC/SAU-BMA/118869/2010), and co-financed by the FEDER through COMPETE (FCOMP-01-0124-FEDER-021125), Programa Operacional Regional do Norte (ON.2) under the QREN (NORTE-07-0162-FEDER-00018/000067), and via the PT2020 Partnership Agreement under the 4293 Unit I&D.