Incorporation of Retinoic Acid Releasing Microspheres into Pluripotent Stem Cell Aggregates for Inducing Neuronal Differentiation

Abstract

Pluripotent stem cells (PSCs) can form any specialized cell type found in the body making them an excellent tool for regenerative medicine applications. Directed differentiation of PSCs into specific phenotypes can be accomplished by introducing specific chemical cues such as the small molecule retinoic acid (RA). Expressed in the developing nervous system, RA can induce differentiation of PSCs into neural phenotypes including neurons. In this study, we encapsulated all-trans RA within poly (ɛ-caprolactone) (PCL) microspheres to generate controlled morphogen release over 28 days. RA/PCL microspheres less than ~10 µm in diameter were readily incorporated within the interstitial sites of human induced pluripotent stem cell (hiPSC) aggregates. After 5 days of culture, the microspheres did not induce cytotoxic effects and the hiPSC aggregates containing microspheres showed a decrease in the pluripotency marker SSEA-4. After 7 days of culture on laminin surfaces, aggregates expressed the neuronal marker TUJ1 and displayed extended neurite outgrowth. This approach provides consistent RA delivery throughout the aggregate and could be an effective strategy for differentiating cells in vivo.Overall, our results demonstrate that it is possible to combine hiPSC aggregates with RA/PCL microspheres for neural tissue engineering applications.

Engineering Neural Tissue from Human Pluripotent Stem Cells Using Novel Small Molecule Releasing Microspheres

“…Compared to previous methods, this protocol avoids selection steps and allows for the sustained release of puro, lowering the amount of drug required. The EE of puro into the microspheres (84%) was higher than previously reported of other encapsulated drugs like RA (60.0%) and guggulsterone (42.4%), and similar to Clonazepam (72.6–95.1%) and tamoxifen . The average microsphere diameter size was 3.4 ± 1.17 µm, similar in size to unloaded and RA‐loaded microspheres with diameters of 2.24 ± 1.04 µm and 3.59 ± 2.48 µm, respectively .…”

“…The EE of puro into the microspheres (84%) was higher than previously reported of other encapsulated drugs like RA (60.0%) and guggulsterone (42.4%), and similar to Clonazepam (72.6–95.1%) and tamoxifen . The average microsphere diameter size was 3.4 ± 1.17 µm, similar in size to unloaded and RA‐loaded microspheres with diameters of 2.24 ± 1.04 µm and 3.59 ± 2.48 µm, respectively . The amount and size of microspheres serve as important parameters for the sustained delivery of morphogens when combined with cell aggregates.…”

“…Analysis determined that the encapsulation efficiency (EE) of puro into the microspheres was 84 ± 2.12%. In comparison, 4 µg mg −1 (w/w, RA/PCL) RA‐loaded microspheres were previously reported to have an average diameter of 3.59 ± 2.48 µm with an EE of 60.9 ± 1.9% . 16% of the total puro was released in an initial burst of drug release on Day 1 followed by slow, continuous release over the remaining 45 d. 91 ± 1.7% of encapsulated puro was released by day 46 (Figure d).…”

“…The amount and size of microspheres serve as important parameters for the sustained delivery of morphogens when combined with cell aggregates. In particular, Carpenedo et al reported that smaller microspheres in a range of 2–5 µm allow for a better incorporation within the cell aggregates, while Gomez et al reported that an excessive numbers of microspheres disrupts cell‐to‐cell interactions required for cell survival, proliferation, and differentiation . The total amount of microspheres per cell aggregate used in this study was optimized previously based on aggregate formation, survival, and differentiation (Figure S1, Supporting Information) .…”

“…Morphogen‐loaded microspheres enable localized drug delivery in a temporally controlled manner for homogeneous and organized stem cell differentiation inside of aggregates . Our group previously demonstrated that applying such a strategy by using retinoic acid (RA)‐loaded microspheres led to high levels of neuronal differentiation for hiPSC‐derived cell aggregates . Such homogeneous cell populations serve as a potential platform for in vivo studies to promote recovery and regeneration of cells affected by neurological disorders such as spinal cord injury (SCI) …”