Guggulsterone-releasing microspheres direct the differentiation of human induced pluripotent stem cells into neural phenotypes

Abstract

Parkinson's disease (PD), a common neurodegenerative disorder, results from the loss of motor function when dopaminergic neurons (DNs) in the brain selectively degenerate. While pluripotent stem cells (PSCs) show promise for generating replacement neurons, current protocols for generating terminally differentiated DNs require a complicated cocktail of factors. Recent work demonstrated that a naturally occurring steroid called guggulsterone effectively differentiated PSCs into DNs, simplifying this process. In this study, we encapsulated guggulsterone into novel poly-ε-caprolactone-based microspheres and characterized its release profile over 44 d in vitro, demonstrating we can control its release over time. These guggulsterone-releasing microspheres were also successfully incorporated in human induced pluripotent stem cell-derived cellular aggregates under feeder-free and xeno-free conditions and cultured for 20 d to determine their effect on differentiation. All cultures stained positive for the early neuronal marker TUJ1 and guggulsterone microsphere-incorporated aggregates did not adversely affect neurite length and branching. Guggulsterone microsphere incorporated aggregates exhibited the highest levels of TUJ1 expression as well as high Olig 2 expression, an inhibitor of the STAT3 astrogenesis pathway previously known as a target for guggulsterone in cancer treatment. Together, this study represents an important first step towards engineered neural tissues consisting of guggulsterone microspheres and PSCs for generating DNs that could eventually be evaluated in a pre-clinical model of PD.

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

“…Scanning electron microscopy showed that puro‐encapsulated microspheres possessed an average diameter of 3.4 ± 1.17 µm and exhibited a smooth rounded morphology ( Figure 1 a) similar to unloaded microspheres (average diameter previously reported as 2.24 ± 2.04 µm) (Figure b) . The size distribution for the puro‐loaded microspheres is shown in Figure c.…”

“…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.…”

“…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) . The microspheres used (puro, RA, and unloaded) all fell within the ideal size range for incorporation into cell aggregates.…”

The role of biomaterials in overcoming barriers to regeneration in the central nervous system

“…Integration of more mature neurons into the existing circuitry could have beneficial effects on behavioural outcomes. Agbay et al followed early reports that guggulsterone can promote differentiation of pluripotent stem cells into dopaminergic neurons and encapsulated the drug into poly-ε-caprolactone (PCL) microparticles, realising it over 44 days in vitro [5]. Guggulsterone microspheres promoted the highest levels of TUJ1 expression in human pluripotent stem-cell derived aggregates, suggesting that co-delivery of guggulsterone microspheres and stem cells could be a potential treatment strategy for Parkinson's disease.…”