Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2 eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.

Towards the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ~95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD and displayed cortical organization of F-actin. Compared to the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD is a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs.

There is a critical need for the establishment of an engineered model of the vocal fold epithelium that can be used to gain understanding of its role in vocal fold health, disease, and facilitate the development of new treatment options. Toward this goal, we isolated primary vocal fold epithelial cells (VFECs) from healthy porcine larynxes and used them within passage 3. Culture-expanded VFECs expressed the suprabasal epithelial marker cytokeratin 13 and intercellular junctional proteins occludin, E-cadherin, and zonula occludens-1. To establish the engineered model, we cultured VFECs on a hyaluronic acid-derived synthetic basement membrane displaying fibronectin-derived integrin-binding peptide (RGDSP) and/or laminin 111-derived syndecan-binding peptide AG73 (RKRLQVQLSIRT). Our results show that matrix stiffness and composition cooperatively regulate the adhesion, proliferation, and stratification of VFECs. Cells cultured on hydrogels with physiological stiffness (elastic shear modulus, G′ = 1828 Pa) adopted a cobblestone morphology with close cell–cell contacts, whereas those on softer matrices (G′ = 41 Pa) were spindle shaped with extensive intracellular stress fibers. The development of stratified epithelium with proliferating basal cells and additional (1–2) suprabasal layers requires the presence of both RGDSP and AG73 peptide signals. Supplementation of cytokines produced by vimentin positive primary porcine vocal fold fibroblasts in the VFEC culture led to the establishment of 4–5 distinct cell layers. The engineered vocal fold epithelium resembled native tissue morphologically; expressed cytokeratin 13, mucin 1, and tight/adherens junction markers; and secreted basement membrane proteins collagen IV and laminin 5. Collectively, our results demonstrate that stiffness matching, cell–matrix engagement, and paracrine signaling cooperatively contribute to the stratification of VFECs. The engineered epithelium can be used as a versatile tool for investigations of genetic and molecular mechanisms in vocal fold health and disease.

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