A system is needed to isolate, clone, and examine the mucosal stem cells away from confounding effects of immune, stromal, and microbe cells. Markers of stem cells such as LGR5 and BMI1 are used as stable lineage tracers in mice, and methods have been developed to isolate and analyze epithelial cells of the gastrointestinal tract—either by differentiating induced pluripotent stem cells to intestinal lineages or with organoids or miniguts.
However, researchers have not been able to maintain patient-specific human intestinal stem cells for analysis, or expand clones to analyze their function, response to therapeutic agents, or regenerative properties.
Marcin Duleba et al developed robust methods to generate libraries of 100–300 independent stem cell clones from standard 1-mm biopsies of human intestinal mucosa (see figure).
The authors enzymatically digested the biopsy and placed the tissue on irradiated 3T3-J2 feeder cells in the presence of specialized growth medium. To induce differentiation, the stem cells were seeded on transwell inserts until they reached confluency. The apical media was removed and the cultures were continued for 6–12 more days.
Duleba et al reported that the method allowed them to expand the clones as stem cells to practically unlimited numbers in vitro and achieve approximately 1 billion cells in <60 days. The clones met all the key criteria for stem cells, including long-term self-renewal and multipotency.
The authors showed that a single stem cell from this system can proliferate to 1 billion cells in fewer than 60 days, sufficient to establish 10,000 3-dimensional intestinal cultures in the air–liquid interface system. These stem cells are able to autonomously form complex 3-dimensional epithelia of the native mucosa from which they were derived.
Advantages of this system over previous systems include the rapid production of a homogeneous population of intestinal stem cells. The system allows researchers to generate topologically precise, region-specific and region-committed stem cells and also to easily generate clones with uniform somatic genotypes for comparitive studies. The cells can also be used to identify factors that control their differention or proliferation, and for analyses of gene expression patterns associated with specific diseases, such as inflammatory bowel diseases (IBD).
Duleba et al state that this is a remarkably simple process for generating unlimited numbers of genetically stable and regionally committed stem cells from any patient for analysis by multiple technologies, in any laboratory.