A Coculture System for Modeling Intestinal Epithelial-Fibroblast Crosstalk.

Epithelial organoid monoculture is a powerful tool to model stem cell dynamics in vitro. However, extensive efforts have recently revealed various niche players and their significant roles in regulating epithelial stem cells. Among these niche components, fibroblasts have been heavily recognized in the field as a critical niche signal secretor. Thus, understanding the roles of fibroblasts in epithelial dynamics has become increasingly relevant and crucial. This propels the development of approaches to coculture epithelial 3D organoids with fibroblasts to model epithelial-fibroblast crosstalk in vitro. Here, we describe a stepwise coculture method to isolate and culture primary intestinal fibroblasts and epithelial organoids together. Aligned with the recent literature, our coculture protocol allows for primary intestinal fibroblast support of epithelial organoid growth.

A therapeutic vascular conduit to support in vivo cell-secreted therapy.

A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro.

Formation of a perfusable microvascular network (µVN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a µVN, but distinct stromal cell populations may play different roles in this process. Here we describe the differential effects that two widely used stromal cell populations, fibroblasts (FBs) and pericytes (PCs), have on µVN formation. We examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable µVNs in fibrin gels cast within a microfluidic chamber. ECs alone failed to fully assemble a perfusable µVN. Human lung FBs stimulated the formation of EC-lined µVNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF). Addition of recombinant HGF improved while the c-MET inhibitor, Capmatinib (INCB28060), reduced µVN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. Different stromal cell types provide different functions in microvessel assembly by ECs. FBs support µVN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology.