Organ-specific ECM arrays for investigating cell-ECM interactions during stem cell differentiation.

Pluripotent stem cells are promising source of cells for tissue engineering, regenerative medicine and drug discovery applications. The process of stem cell differentiation is regulated by multi-parametric cues from the surrounding microenvironment, one of the critical one being cell interaction with extracellular matrix (ECM). The ECM is a complex tissue-specific structure which is an important physiological regulator of stem cell function and fate. Recapitulating this native ECM microenvironment niche is best facilitated by decellularized tissue/organ derived ECM, which can faithfully reproduce the physiological environment with high fidelity toin vivocondition and promote tissue-specific cellular development and maturation. Recognizing the need for organ specific ECM in a 3D culture environment in driving phenotypic differentiation and maturation of hPSCs, we fabricated an ECM array platform using native-mimicry ECM from decellularized organs (namely pancreas, liver and heart), which allows cell-ECM interactions in both 2D and 3D configuration. The ECM array was integrated with rapid quantitative imaging for a systematic investigation of matrix protein profiles and sensitive measurement of cell-ECM interaction during hPSC differentiation. We tested our platform by elucidating the role of the three different organ-specific ECM in supporting induced pancreatic differentiation of hPSCs. While the focus of this report is on pancreatic differentiation, the developed platform is versatile to be applied to characterize any lineage specific differentiation.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering.