Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

UNLABELLED: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable. SIGNIFICANCE: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Three-Dimensional Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels.

The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extracellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three-dimensional (3D) environment of the ECM has been shown to significantly altered cellular response compared to that of traditional two-dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol % of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol % of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics, and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels, and the mechanical properties were characterized for various weight percent (wt %) formulations of the 5 mol % PR_g-95 mol % E2 peptide-amphiphile mixture. The 0.5 wt % formulations had an elastic modulus of 429.0 ± 21.3 Pa whereas the 1.0 wt % peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus, and the decrease was a function of cell loading. Although both formulations supported cell proliferation, the 0.5 wt % gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt % gels. However, compared to the 0.5 wt % formulations, the 1.0 wt % hydrogels promoted greater increases in mRNA expression and the production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advancement of 3D culture applications and cell therapies.

A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells.

Three-dimensional (3D) cell culture platforms are increasingly utilized due to their ability to more closely mimic the in vivo microenvironment compared to traditional two-dimensional methods. Limitations of currently available 3D materials include lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. Evaluated here is a unique hydrogel comprised of polyelectrolytic complex (PEC) fibers formed by hyaluronic acid and chitosan (CT). When hydrated with fetal bovine serum containing human mesenchymal stem/stromal cells (hMSCs), a hydrogel with an elastic modulus of 264±38 Pa formed in seconds with cells distributed throughout the matrix. Scanning electron microscopy showed a lattice-like meshwork of PEC fibers forming irregular compartments. hMSCs showed 48% viability during the first 24 h, with cell populations thereafter reaching a steady state for 14 days. hMSCs in the matrix were induced to differentiate to chondrogenic, osteogenic, and adipogenic phenotypes. Emergent features, at days 56 and 70, consisted of chondrogenesis on the surface of hydrogels induced to osteogenic and adipogenic phenotypes. Results indicate that this matrix may be useful for tissue engineering and disease modeling applications.