Directed differentiation protocols for successful human intestinal organoids derived from multiple induced pluripotent stem cell lines

Abstract

Background: Relatively little has been reported comparing the ability of different induced pluripotent stem cells (iPSCs) and protocols to derive human intestinal organoids (HIO), although there is potential to supply HIO for translational research and regenerative medicine. In view of the time and effort required to differentiate HIO, protocols for differentiation were compared and five iPSC lines, produced by retroviral or non-viral reprogramming methods, were concurrently differentiated to HIO to evaluate the robustness and repeatability of using cellular markers by flow cytometry and immunohistochemistry. Methods: iPSCs were differentiated to definitive endoderm by Activin A treatment (Protocol I) or Myostatin (GDF8), GSK-3β inhibitor (CHIR99021) and B27 supplement (Protocol II) and then differentiated to hindgut using FGF4 and WNT3A (Protocol I), or KGF and Retinoic Acid (Protocol II) resulting in spheroids that were differentiated to HIO in 3-dimensional culture in matrigel containing EGF, Noggin, and R-Spondin1. Results: Definitive endoderm markers (CXCR4 and SOX17) were similar using both protocols and in all cell lines, but in their absence, spheroids to not develop. HIO derived from excised hindgut spheroids (Protocol II) showed more consistent expression of epithelial markers E-Cadherin, CDX2, villin, and chromogranin A. Morphological characteristics such as budding are predictive of robust enterocyte differentiation (villin positive) in HIO. Conclusions: Substituting different members of the growth factor families (e.g., TGFβ) is equally or more effective for producing epithelial lineages in HIO. Although there are no early quantitative predictors of level of success, all reprogrammed somatic cells could be differentiated to produce HIO. Several morphological characteristics (excisable spheroids and budding in HIO) were associated with HIO success. This could improve cost-effectiveness and ease of differentiation as HIO become more commonly available as translational 3D models of the gut.

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies

“…Recently, several researchers have reported that intestinal organoids can be differentiated from human pluripotent stem cells in vitro (Forster et al., 2014, Kauffman et al., 2015, Spence et al., 2011, Tamminen et al., 2015). Human intestinal organoids consist of all small intestinal cell types (paneth cells, goblet cells, enterocytes, and enteroendocrine cells).…”

Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids

“…Recently, 3D organoids and tissue models have been generated from iPSCs to resemble organ structure and function. Examples of complex tissues and organs modelled in 3D include Engineered Heart Tissues (EHTs) [ 5 ], Lung [ 6 ], and intestinal organoids [ 7 ], as well as so-called “mini-brains” [ 8 ]. A 3D model of the vascular system was only recently published [ 9 ].…”

Potential combination endothelial progenitor cell and antiviral therapies for reversal of impaired angiogenesis induced by SARS-CoV-2 infection

“…Furthermore, since SARS-CoV-2 infections also cause dysfunction of disparate human organs including the lungs, heart, brain, kidney, intestine, etc., disease modeling by employing the human induced pluripotent stem cells (hiPSC) derived organoidmodel systems [30][31][32][33][34][35][36][37][38][39][40], lead the path towards the mechanisms underlying organ failures due to SARS-CoV-2 infections. Such experimental investigations using hiPSC derived organoid models could well generate molecular drug therapies to treat preemptive tissue damage by SARS-CoV-2 infections.…”