(J-369) Bioprinting-based Self-assembly of Human Islet-like Cellular Aggregates and Vasculatures

Introduction:: Pancreatic islets are dense cellular aggregates containing hormonal cells such as insulin-secreting beta cells and glucagon-secreting alpha cells, which maintain glucose homeostasis. The metabolic regulation of islets is influenced not just by the interactions between distinct hormone-producing cell types, but also by the complex microenvironments and unique cellular organization. Adhesive and signaling molecules are especially enriched in the microdomains of islets and neighboring vascular networks, enabling communicative interactions that leads to synchronized insulin secretion. In this regard, the current application of in vitro islets derived from stem cells is limited by the lack of physiologically relevant niches and structural cues to understand the key phenomena in matured islets ranging from healthy to diabetic states. Here we suggest two engineering approaches to enhance the maturity of stem cell-derived islets, (1) recreation of the bespoke environment to provide a three-dimensional islet-specific niche with pancreatic tissue-derived extracellular matrix and supplemented basement membrane proteins and (2) inducing self-assembly of islets and vasculatures using bioprinting technology to mimic the spatial arrangement of islet periphery. The engineered peri-islet niche significantly improved beta cell-specificity and glucose responsiveness of stem cell-derived islets through combinational biochemical cues. Geometrically controlled stem cell-derived human islet-like cellular aggregates and vasculatures within the tailored niche reproduced robust formation of vascular networks and adhesion proteins via paracrine and juxtracrine signaling, facilitating metabolic functions of islets. Our engineering perspectives regarding the manipulation of niche properties to recapitulate realistic tissue organization pave the way for investigating the microenvironmental effects on islet development, maturation, and disease modeling.

Materials and Methods:: Characterization of pancreatic islet niche-mimetic bioink

To demonstrate the effects of pancreatic tissue-specific cues on the maturation of stem cell-derived islets, porcine-derived pancreatic tissues were decellularized and prepared as hydrogel forms. The decellularized pancreatic tissue-derived extracellular matrix (pdECM) was characterized using proteomic analysis based on the matrisome database to investigate the main ECM components in the pancreatic microenvironment. In addition, reflecting the basement membrane proteins around the native islets, basement membrane proteins were supplemented to pdECM for further improvement of functional maturity. The efficacy of the developed pdECM based-peri-islet niche-like (PINE) bioink was evaluated by a glucose-stimulated insulin secretion test and gene expression levels related to metabolic functions of encapsulated stem cell-derived islets. In addition, rheological analysis was performed to demonstrate shear-thinning behavior and self-healing process of PINE bioink for the stable localization of the printed cells.

Fabrication and functional validation of human islet-like cellular aggregates (HICAs)-vasculatures

Using in-bath printing method, after sequential printing of polymeric housing structure and PINE bioink supporting bath, endothelial cells were linearly printed, and mixture of stem cell-derived islets and endothelial cells subsequently printed as aggregate form at the proximal region of the linear vasculature. To observe self-assembly of patterned HICAs-vasculatures, insulin, vascular markers, and cell adhesion molecules were stained at each timepoint. Furthermore, we compared gene expression levels and glucose responsiveness of non-printed group, HICAs only group, and HICAs-vasculatures group to evaluate optimal printing design for the maturation. Finally, investigation of physio-mimetic responses of printed HICAs-vasculatures was performed under the hyperglycemia conditions in vitro.

Results, Conclusions, and Discussions:: In this study, we developed a bioprinted HICAs-vasculatures by optimizing niche properties such as biochemical composition and geometrical guidance. This combinational engineering approach aims to address issues with in vitro islet models that lack microenvironmental cues similar to those of in vivo conditions, which hinder the comprehensive understanding of how various interactions (e.g., cell-cell interactions, ECM interactions, paracrine and juxtracrine signaling) can impact the functionality of islets. The developed pdECM-based PINE bioink, reconstituted with the representative basement membrane proteins, enabled functional maturation of stem cell-derived islets via activating integrin signaling. With the tailored pancreatic niche, programmed patterning of islet-specific cellular architecture could control the self-assembly of HICAs and vasculatures, replicating the realistic pancreatic islet periphery. Our bioprinted HICAs-vasculatures reproduced crucial features of matured islets as follows; (1) the structural aspects of highly clustered islet morphogenesis and tight connection with the vascular networks (e.g., abundant expression of adhesive proteins (Cadherins, connexins, EphA/ephrin A)) that can facilitate insulin release with enriched signaling molecules, (2) the functional properties (e.g., stable glucose responsiveness, high expression levels of metabolic genes (GLUT1, glucokinase, insulin, chromogranin A, mafA)), (3) physio-mimetic responses such as upregulation of inflammation and vasoconstrictive proteins under the hyperglycemic conditions and restoration of insulin secretory functions after anti-diabetic drug treatment. Towards a better understanding of islet development, maturation, and diabetic disease modeling, the incorporation of more diverse cellularity into a one-pot system could be a beneficial strategy. Although our system is focusing on the endocrine functions of the pancreatic tissues, the developed HICAs-vasculatures can suggest the translationally relevant applications of islet models with advanced pancreatic-specific cues. Collectively, recreating the complexity of the islet microenvironment in vitro will help understand its effects on the functionality of islets and enhance the translatability of potential diabetic therapeutics.

Acknowledgements (Optional): : This work funded by the National Research Foundation of South Korea (NRF) grant funded by the Ministry of Science and ICT (No. 2021R1A2C2004981).

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