Introduction and Background 

3D bioprinting, the process by which cells are combined with bioinks and printed into 3D structures, has become a popular method for engineering tissues, where the specific properties of the bioink used determine how well the tissue can mimic the physiological properties of the desired tissue type. The properties of the bioink chosen should be compatible with the desired tissue type, including its biocompatibility, printability, and the ability to deliver certain factors needed to induce a desired behaviour from the seeded cells. Hydrogels are often used for their helpful characteristics, including viscosity and their ability to handle the shear stress generated during the printing process. Since the discovery of human induced pluripotent stem cells (hiPSCs) in 2007, scientists have been able to study many diseases from patient-derived hiPSC lines.  Much of the research done with hiPSC models, however, is done on 2D models, limiting the translation of the findings to the complex 3D nature of human tissues such as the brain.

This study thus focuses on using a novel fibrin-based bioink to print hiPSC-derived neural aggregates using the Aspect Biosystems RX1 printer, which has the ability to protect the cells within the bioink from shear stress, increasing cell viability. In a recent study, the research group showed that they could control the delivery of guggulsterone to 3D bioprinted tissues using microspheres that were incorporated in the fibrin bioink to engineer mature neural tissues similar to those found in the brain. Therefore, with the goal to promote the differentiation of hiPSCs into neural tissues, the research group aimed to use guggulsterone – an anti-cancer drug – to differentiate hiPSCs into dopaminergic neurons using guggulsterone-releasing microspheres. To conduct this study, dome-shaped constructs were printed layer-by-layer to produce six-layer constructs with a 1 cm diameter. Three different sets of constructs were generated, one being a construct composed of hiPSC-derived NPCs (neural progenitor cells) within a bioink containing guggulsterone microspheres (GM), and two controls, one of which is composed of NPCs and treated with guggulsterone in media (SG), and one composed of unloaded microspheres and NPCs (UM). Using these three treatment groups, they wanted to determine the cell viability and analyze their expression markers associated with neural differentiation, with a focus on dopaminergic neurons.

Results

Following the 3D printing of each of the three treatment groups, the constructs showed a homogenous distribution of NPCs and microspheres in phase contrast images, in addition to phase microscopy showing that the desired dome shape was maintained. After day 1 and 7, cell viability was measured, which showed high viability 1-day post-printing, with no statistical significance between the groups. On day 7, all of the groups maintained high levels of viability, with the GM group exhibiting the highest level of viability (GM - 98%, UM - 94%, SG - 91%). 

Immunocytochemistry (ICC) was performed on all three groups for multiple cellular markers associated with neuronal differentiation. At day 15, all constructs stained positive for TUJ1 (an immature neuronal marker) and FOXA2 (a midbrain-type dopamine neuron marker), and on day 30, TUJ1 was expressed in all constructs, in addition to TH (an enzyme expressed by dopaminergic neurons) being expressed in the GM and SG groups. 

To quantify the ratio of cells expressing a variety of biomarkers, including TUJ1, TH, glial fibrillary acidic protein (GFAP) and O4, flow cytometry was performed. On day 30, the GM group showed the highest expression of TUJ1, TH, and GFAP, as well as the GM and UM groups showed similar levels of O4 expression. This data suggests that the presence of guggulsterone microspheres promoted the greatest differentiation of bioprinted neural progenitor cells within the engineered tissue.

Quantitative polymerase chain reaction (qPCR) was performed to determine the gene expression levels of TUBB3 (gene coding for TUJ1 protein), NR4A2 (dopaminergic neurotransmitter phenotype gene), TH RNA, and PAX6 RNA (a neural progenitor marker). Analysis of the qPCR data showed that both tissues loaded with microspheres (GM and UM) showed increased expression levels of TUBB3 and TH RNA, and decreased levels of NR4A2. Lastly, the GM group showed the greatest expression of PAX6 RNA.

Conclusion and Next Steps

This study showed that the controlled release of guggulsterone from loaded microspheres can increase the survival of NPCs as well as aid in their differentiation into mature tissues displaying neural phenotypes, including dopaminergic neurons. This method shows promise for increasing cell viability in cell-specific tissues, which is currently one of the main challenges in transplantation. Next steps in this research include increasing the efficiency of dopaminergic neurons in bioprinted tissues via the incorporation of additional microspheres, like those loaded with retinoic acid and purmorphamine, to enhance maturation and growth. 

For more information: https://doi.org/10.3389/fbioe.2020.00057