Selection of an appropriate 3D context when studying stem cell models

In this study, we were able to identify for the first time a mutation-specific phenotype in MODY1 β-like cells facilitated by growing the cells in two different 3D environments and comparing the cellular and proteomic readouts to the 2D background. We were also able to suggest two specific molecular mechanisms whereby the two different 3D culture effects could be mediated. Each of these specific molecular mechanisms showed a dependency on the type of 3D context chosen. The cellular confinement in the alginate context helped to identify a metabolic phenotype with lower levels of glycolytic proteins, potentially affecting glucose sensing. The structural scaffolding in the AggreWell 3D context helped to identify a structural collagen-associated phenotype with irregular clusters, a unique proteome with lower levels of structural ECM proteins (laminins, collagens) as well as a functional readout.

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic β-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic β-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.

The two chosen 3D environments challenge the differentiating pancreatic β-like cells in unique ways, including the levels of oxygen delivery, nutrient supply, and cell-to-cell contact. The 3D cell aggregation/AggreWell context is preferred by several investigators in generating pancreatic β-like cells because it mimics the in vivo pancreatic islets in size, level of cell-to-cell contact, and the self-organized compact spheroid arrangement. In the 3D alginate encapsulation context, the cells are immobilized and lack cell-to-cell contact; however, the alginate gel-pore network still allows transport of oxygen and diffusion of nutrients and waste products. Furthermore, alginate encapsulation is suitable for future therapeutic transplantation and can also protect the cells in bioreactor culture systems.

In conclusion, our findings warrants a careful consideration and selection of an appropriate 3D context when studying stem cell models. Our study also provides a patient-specific diabetes model that can be further explored for potential new drug targets that can be used in precision diabetes medicine.