Ex vivo culture of the intestinal epithelium: strategies and applications

Abstract

Limited pools of resident adult stem cells are critical effectors of epithelial renewal in the intestine throughout life. Recently, significant progress has been made regarding the isolation and in vitro propagation of fetal and adult intestinal stem cells in mammals. It is now possible to generate ever-expanding, three-dimensional epithelial structures in culture that closely parallel the in vivo epithelium of the intestine. Growing such organotypic epithelium ex vivo facilitates a detailed description of endogenous niche factors or stem-cell characteristics, as they can be monitored in real time. Accordingly, this technology has already greatly contributed to our understanding of intestinal adult stem-cell renewal and differentiation. Transplanted organoids have also been proven to readily integrate into, and effect the long-term repair of, mouse colonic epithelia in vivo, establishing the organoid culture as a promising tool for adult stem cell/gene therapy. In another exciting development, novel genome-editing techniques have been successfully employed to functionally repair disease loci in cultured intestinal stem cells from human patients with a hereditary defect. It is anticipated that this technology will be instrumental in exploiting the regenerative medicine potential of human intestinal stem cells for treating human disorders in the intestinal tract and for creating near-physiological ex vivo models of human gastrointestinal disease.

Intestinal Organoids—Current and Future Applications

“…Importantly, cells that expressed low or no Lgr5 were incapable of forming these organoids. Taken together, these studies provide compelling evidence for Lgr5 being a marker of proliferating adult stem cells residing at the crypt base throughout the intestinal tract [12]. CD133 (Prominin1 in mouse) was originally reported as a novel pentaspan glycoprotein expressed on neural and hematopoietic stem cells.…”

“…Leucine-rich repeat-containing G-protein coupled receptor 5/G-protein coupled receptor 49 (Lgr5/GPR49) was originally identified as a Wingless-related integration site (Wnt) target gene encoding an orphan G-protein-coupled receptor in colorectal cancer cell-lines [12]. Investigation of its expression pattern in normal intestine revealed it to be a highly specific marker for the proliferating CBC cells [13].…”

“…Such markers are essential for driving advances in our understanding of intestinal stem cell biology, tissue homeostasis, repair, and cancer [11]. There are several cells markers to the CBC stem cells and for the +4 cells [12]. Although their specificity is controversial, some of them will be presented below and are graphically summarized in Figure 1.…”

“…More recently, it has been described as a marker of epithelial stem/progenitor populations in human kidney tubules and the prostate. However, a detailed analysis of reporter mice expressing lacZ under the control of the endogenous Prom1 promoter documented widespread expression in many adult epithelia, including the colon, casting doubt on its authenticity as a broad adult stem cell marker [12,14].…”

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“…The form of cultured stem cells became a round-shape called "spheroid'' and differed from the original shape. This spheroid was surrounded with epithelium and a basement membrane component was placed inserted [40]. When cultured stem cells were transplanted into organs, the school of thought was that contact with their basement membrane and adhesive surface was better for cell transplantation to increase its organ acceptance rate.…”