Directed differentiation of human induced pluripotent stem cells into neuronal subtypes by small molecule releasing microspheres

摘要

Introduction: Previous microsphere research has demonstrated the potential of microsphere-based drug delivery for directing the differentiation of human induced pluripotent stem cells (hiPSCs) for neural tissue engineering applications. Cell replacement therapies using hiPSCs are ideal for conditions where patients experience loss or damage of neurons such as in Parkinson's disease (dopaminergic neurons) (DNs) and spinal cord injury (motor neurons) (MNs). The steroid guggulsterone has been shown to efficiently derive DNs from hiPSCs while MNs can be derived from a combination of the small molecules retinoic acid (RA) and purmorphamine. The aim of this study is to produce guggulsterone and purmorphamine encapsulated poly(e-caprolactone) (PCL) microspheres and then incorporate these drug releasing microspheres into hiPSC aggregates where they can promote the differentiation of hiPSCs into different neuronal subtypes. Materials and Methods: A water-in-oil emulsion was used to fabricate the microspheres. Encapsulation efficiencies were calculated by measuring light absorbance of the extracted drug at 240 nm for guggulsterone and 319 nm for purmorphamine. In vitro drug release studies over 44 and 28 days were done in triplicate. Guggulsterone microspheres will be incorporated with neural aggregates to induce differentiation into DNs while a mixture of previously encapsulated RA microspheres and purmorphamine microspheres will be incorporated to generate MNs. For incorporation of microspheres into neural aggregates, appropriate combinations of microspheres will be added to hiPSC suspensions and centrifuged into AggreWell?800 inserts (STEMCELL Technologies). The resulting differentiation will be determined by flow cytometry to detect viability, pluripotency, and neuronal marker expression while immunocytochemistry will be used to visualize the resulting differentiation. Finally, gene expression profiling will be done using next-generation sequencing techniques (Illumina, MiSeq~?) on neurons derived from the drug delivery systems. Results and Discussion: Guggulsterone-loaded and purmorphamine-loaded microspheres were successfully prepared at an encapsulation efficiency of 31.6 ± 4.8% and 84.7 ±2.3%, respectively. The average diameter of microspheres was 14.8 ± 5.9 μm for guggulsterone and 5.5 ± 2.6 μm for purmorphamine. Figure 1. Scanning electron micrographs of (A) guggulsterone microspheres and (B) purmorphamine microspheres showing a spherical and smooth morphology. Purmorphamine microspheres had a cumulative release of -25% over 28 days. Figure 2. Cumulative release of purmorphamine over 28 days. Sample size n = 3. After 12 days, cell viability for the purmorphamine microsphere incorporated aggregates (～73%) was comparable to the negative control (～70%) suggesting low cytotoxic effects from the microspheres. These microsphere aggregates showed decreased SSEA-4 pluripotency marker expression (～27%) compared to the negative control (～45%) as expected. Conclusions: We have successfully fabricated guggulsterone and purmorphamine PCL microspheres using a single emulsion technique and shown that the purmorphamine microspheres can be incorporated in hiPSC aggregates, influencing differentiation. Such combinations of small molecules releasing microspheres with hiPSC aggregates represent a novel strategy for engineering neural tissue.