Controlled BMP4 Delivery within ESC Aggregates via pNIPMAm Microparticles

1. Controlled BMP4 Delivery within ESC Aggregates via pNIPMAm Microparticles Denise D. Sullivan1, ShaliniSaxena2, Jeff C. Gaulding2, L. Andrew Lyon3,4, Todd C. McDevitt1,4 1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University 2School of Materials Science and Engineering, 3School of Chemistry and Biochemistry, 4Petit Institute of Bioengineering & Bioscience, Georgia Institute of Technology * $ * * 400μm Introduction Microparticle Characterization Growth Factor Delivery BMP4 Release BMP4 Delivery to Embryoid Body Strategies to direct differentiation of embryonic stem cell (ESC) aggregates, or embryoid bodies (EBs), often employ an “outside-in” approach by addition of soluble factors to culture medium. For example, soluble BMP4 is regularly added to induce mesoderm differentiation. However, diffusion into EBs is limited. Our lab has previously demonstrated that one method to direct differentiation and potentially overcome diffusion limitations is incorporation of engineered microparticles (MPs), which have been widely used to control delivery of entrapped molecules, within 3D aggregates. Development of synthetic MPs with tunable protein release kinetics may provide further spatiotemporal control over differentiation. Induction of mesoderm differentiation of ESC aggregates via BMP4 MPs was evaluated by the gene expression of mesoderm marker, Brachyury-T, and ESC pluripotency marker Oct4 at days 1, 4, 5 and 6. Treatment Soluble BMP4 Unloaded MP BMP4 MP 0.5 ng/ml 10 ng/ml 10 ng/ml 10 ng/ml 10 ng/ml Bratt-Leal et al. Biomater, 2011 ESC BMP4 MP pNIPAM, a commonly used thermosensitive polymer can be chemically modified by the addition of a methyl group to form pNIPMAm, increasing the transition temperature from 31 to 43 ̊C and allowing the polymer to maintain a swollen hydrated state at physiological temperatures to permit passive diffusion of entrapped soluble factors. Coupling of pNIPMAm microgels to core particles combines the hydrophilic and morphogen-releasing properties of hydrogels with a dense material for incorporation within EBs, thus providing a synthetic biomaterial platform to control morphogen presentation to cells. Day Release of BMP4 from MPs loaded with low (10 ng/mg MP) and high (100 ng/mg MP) amounts of BMP4 was analyzed by collecting supernatant at different time points for 2 weeks. Total release of BMP4 (ng) was directly dependent on the initial loading amount of BMP4 and demonstrated increased release for 7 days. Overall, less than 60% of BMP4 was released at both loading amounts. No MP Soluble BMP4 Unloaded MP Unloaded MP BMP4 MP +Soluble BMP4 Day 1 Day 4 Gaulding et al. ACS Macro Lett, 2013 The objective of this study was to characterize pNIPMAm MPs for controlled delivery of bioactive BMP4 and incorporation in ESC aggregates to enhance mesoderm differentiation. Microparticle Characterization pNIPMAM Microparticle Synthesis SEM Phase Microparticle Incorporation BMP4 MPs demonstrated comparable gene expression of Brachyury-T to soluble BMP4, despite the delivery of 20-fold less total protein (p<0.05 compared to: * Unloaded MP; $ Unloaded MP +Soluble BMP4). Overall, Oct4 expression decreased over the time course as compared to No MP. MPs were synthesized by precipitation polymerization and are a core-shell structure, consisting of microgels composed of 68% pNIPMAm, 2% N,N’ methylene-bisacrylamide, 30% acrylic acid. MP synthesis yields approximately 14.5 million MPs/mg of polymer with an average diameter of 4.6 ± 1.0 μm. Microgel coverage was determined to be >85%. 1 μm 50 μm 1 μm Polysytrene Core Microgel * UV Aggregation Rotary Culture BMP Signaling No MP Phase 1.02% Day 0 Day 1 Day 3 Day 5 Rotary Culture Agarose μ-wells EBs BMP4 Loading ESCs Day of Differentiation EBs were formed by centrifugation of mESCs into μ-wells with 1000 cells/well and transferred to rotary orbital suspension culture (40 rpm) after 18 hr. Nuclei CFP 100 µm MPs Soluble BMP4 Serup et al. Dis Model Mech, 2012. The BMP4 loading capacity was quantified by re-suspending dehydrated MPs in PBS containing BMP4, ranging from 2.5–2,000 ng protein per mg of MPs, for 18 hours at 4̊C. BMP4 MPs were subsequently centrifuged and the supernatant was collected for analysis of BMP4 depletion. 12.8% 3:1 1:1 1:3 No MP Conclusions $ * pNIPMAm MPs can be incorporated within ESC aggregates and deliver bioactive BMP4 to induce similar mesoderm gene expression to soluble BMP4 delivery, yet with 20-fold less total protein. In addition, BMP signaling reporter ESCs demonstrated the extent of BMP signaling within EBs treated with BMP4 loaded MPs was increased over soluble BMP4 delivery, indicating that growth factor loaded pNIPMAm MPs can be used to improve ESC differentiation over soluble delivery. $ Unloaded MP * 0.713% CFP Unloaded MP + Soluble BMP4 Overall, these results suggest that pNIPMAm MPs can be used as a platform to control the release of bioactive BMP4 and to induce a functional cellular response. 13.5% Negatively charged MPs, due to the acrylic acid content, electrostatically bind with BMP4 (pI=8.97). The maximum MP loading of BMP4 at each dose was quantified by ELISA. MPs demonstrated increased growth factor binding capacity with increasing initial growth factor loading concentrations. Average loading efficiency for BMP4 was 55.5 ± 6.2%. * * BMP4 loaded MPs were incorporated in aggregates of BMP signal responsive sentinel ESCs with CFP fluorescence reporter. All ESCs treated with BMP4 (soluble or loaded) express CFP by Day 2. However, ESCs from EBs incorporated with BMP4 loaded MPs expressed significantly more CFP as compared to soluble delivery of BMP4. (p<0.05 compared to: *No MP; $ Unloaded MP). Acknowledgements BMP4 Loaded MP MPs were incorporated in EBs at MP:cell seeding ratios of 1:10, 1:3, 1:1 and 3:1. Maximum MP incorporation was ~80 MPs/EB at a 1:1 ratio. MP incorporation efficiency was <20% for all ratios. (*p<0.05) 18.2% Funding is provided by NIH grant GM088291. DDS is supported by an UNCF/Merck Graduate Science Research Dissertation fellowship and Georgia Tech UCEM fellowship. DDS was previously supported by an NSF Graduate Research Program fellowship. * * * $ $ $ * $ ** FSC * MP:cellratio