Photochemically Activated Notch Signaling Hydrogel Preferentially Differentiates Human Derived Hepatoblasts to Cholangiocytes

Abstract

Cholangiocytes form an intricate network of bile ducts to enable proper liver function; yet, recapitulating human stem cell differentiation to cholangiocytes in vitro requires Notch signaling and soluble ligands do not activate the Notch pathway. To overcome these limitations, jagged1 is immobilized on a chemically defined hyaluronan to specifically differentiate human embryonic stem cell‐derived hepatoblasts to cholangiocytes. Hepatoblasts cultured on the jagged1‐hydrogels upregulate Notch target genes and express key cholangiocyte markers including cystic fibrosis transmembrane conductance regulator. Moreover, cholangiocytes adopt morphological changes that resemble liver biliary structures. To emulate natural biliary system development, a new strategy is developed to achieve spatiotemporal control over the Jagged1–Notch2 interaction: jagged1 is first caged with a photocleavable streptavidin and then it is uncaged photochemically to restore the biological function of Jagged1, which is confirmed with Notch2 activation in a fluorescent reporter cell line. Moreover, the differentiation of human embryonic stem cell‐derived hepatoblasts to cholangiocytes is temporally controlled with photochemical uncaging of this streptavidin‐Jagged1‐immobilized hyaluronan hydrogel. This strategy defines a framework to control protein signaling in time and space and specifically for Notch signaling for ultimate use in regenerative medicine strategies of the liver.

Phenotyping Rare CFTR Mutations Reveal Functional Expression Defects Restored by TRIKAFTATM

“…HEK293 were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, 0.2% SDS, and 0.1% Triton X-100) with protease inhibitor cocktail (Roche, Mannheim, Germany) for 10 min in ice [ 21 ]. Soluble fractions were analyzed by SDS-PAGE on 6% Tris-Glycine gels (Life Technologies, Carlsbad, CA, USA).…”

Designing Hydrogels for 3D Cell Culture Using Dynamic Covalent Crosslinking

“…[ 1 ] Photo‐mediated reactions can enable exogenous control of spatiotemporal cues of the ECM that regulate cellular differentiation. [ 239 ] Farahani et al. demonstrated that photocaged alkoxyamine enables oxime crosslinking upon exposure of light, thus providing a facile method to tune bulk as well as local properties of the hydrogel in the presence of cells.…”

Engineering tissue morphogenesis: taking it up a Notch

“…Early on Varnum-Finney et al found that ligand immobilization can induce Notch receptor activation [41], opening the doors for mimicking native cell-cell interactions by well-defined cell-material interactions. Efforts have since been made to develop Notch-activating platforms, and today Notch-functionalized materials range from tissue-culture plastics [24,25,27,[41][42][43][44][45][46] to 3D matrices [37,[47][48][49][50][51][52][53] and intricate dynamic systems such as magnetic nanoparticles and photo-reversible Notch-activating gels (Figure 2, fourth tier) [54][55][56][57] (Table 1). However, challenges remain for robust control over Notch as well in its systematic use to control tissue morphogenesis.…”

“…Notch also elicits different responses during tissue development as a function of temporal regulation [ 12 , 73 ]. A small number of studies have investigated methods for spatial and/or temporal control of Notch ( Figure 2 , third tier) [ 30 , 40 , 56 , 57 ]. One study utilized printed lines of Dll4 ligands to spatially guide the direction of the endothelial sprouts.…”