Enhanced Utilization of Induced Pluripotent Stem Cell–Derived Human Intestinal Organoids Using Microengineered Chips

Abstract

Background and AimsHuman intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium’s role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell–derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli.MethodsEpithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate–dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function.ResultsThe optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability.ConclusionsWe demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.

Towards an autologous iPSC-derived patient-on-a-chip

“…We seek to obtain an intestinal epithelial cell layer that is opened at the luminal side, apically facing the top of the Transwell, while the basolateral side lies on the membrane of the Transwell to further improve our intestinal model. A similar single organ approach has recently been shown with iPSC-derived and primary small intestinal organoids in a chip system 44,45 . Moreover, a dense tight layer of endothelial cells underneath the Transwell should seal the barrier to the blood surrogate.…”

Developmentally inspired human ‘organs on chips’

“…Many adult cell types are available commercially; however, tissue-specific organoids also can be created by isolating stem cells from patient biopsies, and this approach has been leveraged as a cell source to create functional human intestine chips that form highly differentiated villus structures (Kasendra et al, 2018). Induced pluripotent stem cell (iPSC) approaches have also been leveraged to create various types of specialized cells for organ chip studies, including cardiomyocytes, kidney podocytes, brain microvascular endothelial cells, and intestinal enterocytes (Wang et al, 2014(Wang et al, , 2017Musah et al, 2017;Workman et al, 2018), but their clinical relevance is sometimes questioned because most iPSCs generated in vitro remain fetal-like or neonatal in nature. Importantly, culturing iPSC-derived motoneurons and brain microvascular endothelial cells together in an organ chip model of the neuromuscular unit significantly enhanced function and in vivo-like maturation of spinal cord neural tissue (Sances et al, 2018).…”

“…When different cell types are placed in the correct microenvironment with appropriate positioning of tissues relative to one another, they spontaneously accumulate ECM along their interface, switch on developmental programs and self-organize, resulting in formation of highly polarized and differentiated epithelial tissue structures that closely resemble those seen in the organs of our bodies, such as mucus and surfactant-producing alveolar epithelium (Huh et al, 2010), ciliated pseudostratified airway epithelium (Benam et al, 2016a), 3D finger-like intestinal villi (Kim et al, 2012;Kasendra et al, 2018;Workman et al, 2018) and reconstitution of podocyte-ECMendothelial contacts of the kidney glomerulus (Musah et al, 2017).…”

“…1A,B; Huh et al, 2010). Since this study was published, multi-channel design approaches have been used by multiple research groups to build microfluidic organ-chip models of the human lung small airway, skin, kidney, intestine, placenta, blood-retinal barrier, blood-brain barrier, neurovascular unit and neuromuscular unit, among others (Kim et al, 2012;Achyuta et al, 2013;Abaci et al, 2015;Benam et al, 2016a;Lee et al, 2016;Musah et al, 2017;Yeste et al, 2017;Wang et al, 2017;Workman et al, 2018;Kasendra et al, 2018;Sances et al, 2018; reviewed by Bhatia and Ingber, 2014).…”

Complex human gut microbiome cultured in anaerobic human intestine chips

“…We demonstrated this capability using both an established human Caco2 intestinal cell line and primary human ileal intestinal epithelium, however, the same methodology could be applied to study host-microbiota interactions in any Organ Chip (e.g., lung, skin, etc.). Furthermore, by integrating primary epithelial cells from intestinal biopsies as we did here, or patient-derived induced pluripotent stem (iPS) cells 55 , in combination with microbiomes obtained from the same patients, it should be possible to develop patient-, disease-, and location-specific, host-microbiome co-culture models. The modular nature of the Organ Chip technology also allows for the incorporation of additional cell types.…”