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

Abstract

Here a novel technique for engineering neural tissue consisting of motor neurons by combining human‐induced pluripotent stem cells (hiPSCs) with small molecules releasing microspheres is demonstrated. First, the small molecule purmorphamine (puro) is successfully encapsulated into poly ε‐caprolactone (PCL) microspheres using a single emulsion oil‐in‐water (o/w) method for the first time with an efficiency of (84% ± 2.12%). These microspheres release 91% ± 1.7% of the encapsulated puro in a controlled fashion over 46 days. Puro microspheres, along with previously characterized retinoic acid (RA) releasing microspheres, are then incorporated into hiPSC aggregates to engineer neural tissue. The combination of puro and RA microspheres promotes hiPSC differentiation as indicated by the expression of multiple neural markers, including the neuronal marker β‐tubulin III (βT‐III), and the transcription factor Olig2 (7.69 ± 8.38%) on day 28. These tissues express the motor neuron marker HB9 (24.85 ± 4.51%) on day 35, and the mature motor neuron marker ChaT (12.35 ± 4.17%) on day 60. These engineered tissues can be used for regenerative medicine applications such as treating spinal cord injury (SCI), disease modeling, and drug screening.

3D Bioprinting Human Induced Pluripotent Stem Cell-Derived Neural Tissues Using a Novel Lab-on-a-Printer Technology

“…Media changes were performed every third day by replacing 750 μL with fresh media from day 1 to day 15. Table 1 [27]. The bioprinted constructs were maintained at 37°C with 5% CO2…”

“…Media changes were performed every third day by replacing 750 µL with fresh media from day 1 to day 15. Table 1 [27]. The bioprinted constructs were maintained at 37 • C with 5% CO 2 On day 7, the constructs (n = 3) were degraded by removing culture media followed by incubation with a solution of 25 mM of Sodium Citrate (Sigma, St. Louis, MO, USA).…”

“…The bioprinted constructs were degraded as previously mentioned using a solution of 25 mM of sodium citrate, 0.125% of trypsin-EDTA, and FBS. The resulted cell suspension was processed as previously reported [27,29]. Briefly, the cell suspension was washed three times with PBS by centrifuging at 300 g for 5 min.…”

“…Moreover, the small molecule, CHIR99021, activates the Wnt pathway, enhancing neural patterning of pluripotent stem cells [25]. In addition, the combination of RA and puro promote pluripotent stem cell differentiation into motor neurons [25][26][27]. Cylindrical constructs were printed using the RX1™ bioprinter with seven layers and a 1 cm diameter in a process that took under 5 min to generate four constructs.…”

3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres

“…One promising strategy for promoting such differentiation requires treating these hiPSCs with small molecule morphogens (Zhang et al, 2012). Our lab has extensively explored the use of small molecule morphogens encapsulated in microspheres, which degrade over time to slowly release the drug in a controlled manner, as a means to direct neural differentiation in an autonomous fashion (Gomez et al, 2015;Agbay et al, 2018;De La Vega et al, 2018b). Guggulsterone, an anti-cancer drug, is a potent agent for differentiating both human embryonic stem cells and hiPSCs into dopaminergic neurons, the cellular population affected by Parkinson's disease (Gonzalez et al, 2013;Robinson et al, 2015).…”