title: Delivery of definable number of drug or growth factor loaded poly(DL-lactic acid-co-glycolic acid) microparticles within human embryonic stem cell derived aggregates.
abstract: Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13μm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell aggregate. This provides a powerful tool for controlling and investigating differentiation within 3D cell cultures and has applications to drug delivery, drug discovery, stem cell biology, tissue engineering and regenerative medicine.
authors.affiliation: Wolfson Centre for Stem Cells Tissue Engineering and Modelling, Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK.
authors.forename: Omar
authors.initials: O
authors.lastname: Qutachi
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Shakesheff
authors.forename: Lee D K
authors.initials: LD
authors.lastname: Buttery
mesh_terms: D055396:Bone Morphogenetic Protein 2 / Q000008:administration & dosage* / Q000737:chemistry; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004337:Drug Carriers / Q000008:administration & dosage / Q000737:chemistry*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D019821:Simvastatin / Q000008:administration & dosage* / Q000737:chemistry; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D004337:Drug Carriers; C493709:RUNX2 protein, human; D042461:Vascular Endothelial Growth Factor A; D015675:Osteocalcin; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D019821:Simvastatin