Controlled BMP4 Delivery within ESC Aggregates Via Pnipmam Microparticles

Strategies to direct differentiation of embryonic stem cell (ESC) aggregates, or embryoid bodies (EBs), often employ an â??outside-inâ? approach by addition of soluble factors to culture medium. However, diffusion into EBs is limited. One method to direct differentiation and potentially overcome diffusion limitations is incorporation of engineered microparticles (MPs), which have been widely used to control molecular delivery locally, within 3D multicellular aggregates. Poly(n- isopropylmethacrylamide) (pNIPMAm) is a thermoresponsive polymer that exhibits a volume phase transition temperature at
43ºC, 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 core material that enhances incorporation within EBs. The objective of this study was to characterize pNIPMAm MPs for controlled delivery of bioactive BMP4 within EBs to direct ESC differentiation.
Core-shell MPs were constructed with the shell consisting of microgels composed of 68% pNIPMAm, 2% N,Nâ?? methylene- bisacrylamide, 30% acrylic acid and synthesized by precipitation polymerization. Core carboxyl-functionalized polystyrene MPs (~4.5 μm) were conjugated to 4-amino-benzophenone using EDC chemistry. Microgels were coupled to functionalized cores by UV excitation. The BMP4 binding capacity was examined by loading MPs with a range of BMP4 doses (10 â?? 300 ng/mg MPs) over 18 hours at 4ºC. An ELISA was used to quantify passive release of BMP4 from MPs over 12 days. Bioactivity of BMP4-laden MPs was evaluated using an in vitro alkaline phosphatase (ALP) assay to quantify ALP activity of skeletal myoblasts (C2C12s), following treatment with soluble BMP4, â??emptyâ? MPs, or BMP4-laden MPs. MPs were incorporated within EBs by forced aggregation in micro-well inserts at MP:cell seeding ratios of 1:10, 1:3, 1:1 and 3:1. BMP4 was delivered solubly and via MPs to ESC aggregates to evaluate gene expression of pluripotency marker, Oct-4, and mesoderm marker, Brachyury-T.
MP formation produced approximately 14.5 million MPs/mg of polymer with an average diameter of 4.6 ± 1.0 μm. BMP4- laden MPs released less than 25% of BMP4 for all loading amounts. At higher loading amounts (200 and 300 ng/mg MPs),
less than 5% of BMP4 was released from MPs. Results demonstrate that MPs maintain BMP4 bioactivity as treatment with BMP4-laden MPs induced ALP activity similar to delivery of soluble BMP4. However, treatment with â??emptyâ? MPs and soluble BMP4 induced less ALP activity as compared to soluble BMP4 alone, suggesting that MPs actively sequester free BMP4 and prevent interactions with cells. Maximum MP incorporation within EBs was achieved with ~80 MPs per EB at a MP to cell seeding ratio of 1:1. Oct-4 expression decreased over the course of differentiation in all groups. BMP4-laden MPs demonstrated comparable expression of Brachyury-T to soluble BMP4, despite the delivery of 20-fold less total protein from the MPs. These results suggest that pNIPMAm MPs can be used to control the release of bioactive BMP4 and induce ESC mesoderm differentiation.