Pluripotent stem cells are unique in their abilities for self renewal and capacity to differentiate into organ-specific mature cell types, rendering them attractive for the treatment of degenerative diseases. Currently, human pluripotent stem cell (hPSC) based regenerative therapies are transitioning from two dimensional (2D) adherent differentiation towards three dimensional (3D) platforms which more adequately resemble native cellular environments. Such 3D aggregation has the potential to create multicellular ‘organoids’ relevant for studying advanced organ development, disease physiology, and drug testing. Specifically, over the last few years, hPSCs have been used to generate gut, retinal, and liver organoids. A majority of these current methods employed for cellular aggregation rely on external pressures or the confinement of cells within three dimensional artificial scaffolds to enforce aggregation. These approaches however differ from spontaneously organizing structures formed by the inherent forces and interactions of only the integrated cell population. In this regard, we have developed a novel hydrogel platform which not only promotes spontaneous aggregation of hESC derived pancreatic progenitor cells (hESC-PPs) into robust, mechanically stable, controlled spheroids but also render them free from embedding or forced cellular interactions.
Pre-differentiated hESC-PP cells seeded on the pre-formed hydrogel spontaneously aggregated within 14 hours producing one aggregate per well of a 96 well plate. These hESC-PP aggregates were tunable for size and cellular composition, robust enough for mechanical manipulation, and viable over extended culture in situ. Additionally, this system allowed for the efficient application of functional assays on individual aggregates, enabling high throughput analysis in 3D. Self-organized hESC-PP aggregates exhibited significant enhancement in relevant PP gene expression, along with two-fold increase in overall cell population positive for Pdx-1, a key PP protein marker. Furthermore, a significant portion of the Pdx-1 population co-expressed NKX6.1 protein marker, an early marker for potential endocrine maturation. Moreover, self-organization also improved the efficiency of chemically driven hESC-PP maturation towards beta-like cells compared to conventional cultures. An endothelial presence during in vivo islet development is crucial for extra cellular matrix and microvascular development. Therefore co-seeding hESC-PPs with endothelial cells on the hydrogel resulted in self-organized multicellular pancreatic ‘organoids’ to support pancreatic development. Interestingly, the endothelialization of the resulting organoid also induced spontaneous maturation into beta-like cells in the absence of specific chemical induction. In conclusion, our hydrogel platform has direct application not only to improve pancreatic tissue engineering, but also has the flexibility to be further adapted to multiple cell types and other tissue engineering applications.
1. Lancaster, M.A. and J.A. Knoblich, Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 2014. 345(6194): p. 1247125.
2. Stendahl, J.C., D.B. Kaufman, and S.I. Stupp, Extracellular matrix in pancreatic islets: relevance to scaffold design and transplantation. Cell Transplant, 2009. 18(1): p. 1-12.
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