Generation of biologically responsive colon-like intestinal tissue patches from human induced pluripotent stem cells using a rapid co-differentiation platform

Abstract

The intestinal mucosa is a complex functional layer formed from diverse cell types including epithelial cells within crypts and villi and an array of mesenchymal cells. Many intestinal diseases involve loss of the surface mucosa, which is difficult to restore and delays healing. We hypothesized that a transplantable intestinal mucosal tissue graft could aid healing. To create such a graft, we developed a novel early-stage human induced pluripotent stem cell (hiPSC) co-differentiation platform capable of generating multiple intestinal cell lineages (epithelial, mesenchymal, and endothelial) in 8 days. This protocol is simple, serum-free, and reduces animal product use. We confirmed cell identity via RNA and protein expression profiles typical of intestinal lineages. Using bulk and single-cell RNA sequencing, we characterized global transcriptional profiles, showing intestinal identity with early colonic polarization. Results were replicated across multiple hiPSC lines and an independent center. Culturing derived cells on collagen hydrogels formed colon-like intestinal patches (CL-IPs). Upon transplantation into mouse subcutis, CL-IPs developed into colon-like tissue structures including crypts, stromal and muscle layers, and human-origin vasculature that anastomosed with murine vasculature. Teratoma assays and molecular analyses showed no residual pluripotency. This platform shows potential for intestinal mucosal regeneration therapy and as a physiologically relevant in vitro model of intestinal pathobiology.

1. Introduction

Intestinal diseases are common and constitute a major health burden [1]. Many such diseases involve loss of the surface mucosa. As the mucosa is the main functional layer of the intestine – involved in absorption of nutrients and water – dysfunction results in many of the symptoms of bowel disease, such as diarrhoea, dehydration, nutrient deficiencies and haemorrhage. Loss of mucosal integrity is a prominent feature in inflammatory bowel disease (IBD) and thus restoring healthy mucosa may aid in breaking the cycle of chronic inflammation and microbial colonisation, to aid healing [2–4]. The mucosa is thus a focus for regeneration therapies [3, 5].

The intestinal mucosa is characterised by epithelial crypts supported by a range of mesenchymal cells including fibroblasts and endothelial cells and a deep band of smooth muscle, called muscularis mucosae [6–8]. These cell populations derive from endoderm and splanchnic mesoderm during embryogenesis and arise in tandem from the developing gut tube. Other cell populations, such as haemopoietic and neural cells migrate into the intestinal mucosa during embryonic life from other germ layers [8]. Interactions between epithelial and neighbouring stromal cells are particularly important in maintaining normal intestine mucosal tissue homeostasis, through the establishment of signalling morphogen gradients. These factors promote appropriate cell differentiation to allow normal gut function (for example, cells such as goblet cells, absorptive cells and endocrine cells) [9]. They also create a stem cell niche that facilitates replacement of differentiated cells that are shed from the crypt apex, while shielding stem cells from toxicity inherent to the gut luminal contents [10].