Scientists say they have succeeded in isolating, characterizing, and expanding human colon stem cells (CoSCs) from fresh biopsy samples. The international team, led by Professor Eduard Battle, M.D., at Barcelona’s Institute for Research in Biomedicine (IRB), and Toshiro Sato, Ph.D., at University Medical Center Utrecht in The Netherlands, suggest the achievement is, to their knowledge, a world first. The work is reported in Nature Medicine in a paper titled “Isolation and in vitro expansion of human colonic stem cells,”
CoSCs renew the epithelium lining of the large intestine, and changes in these cells are believed to account for the pathophysiology of large-bowel disorders including colorectal cancer, the authors report. Unfortunately, scientists have to date been unable to isolate CoSCs. Drs. Battle et al.’s search for CoSCs in humans was founded on previous work indicating that mouse small intestinal stem cells (ICSs) and cancer stem cells express high amounts of EPHB2.
Building on this, the IRB-led team used an antibody against the EPHB2 extracellular domain to purify EPHB2-positive intestinal epithelial cells from histologically normal fresh tissue biopsied from patients with colorectal cancer or diverticulitis. From these sampless they isolated EpCAM+ crypt epithelial cell populations expressing various levels of surface EPHB2.
Further analysis of these cells showed that those with the lowest levels of surface EPHB2 expressed the highest levels of colonic differentiation markers. In addition, EPHB2-negative cells showed up to a 2,500% reduction in the expression of proliferation genes, “implying that this population was composed of cell cycle-arrested, terminally differentiated cells,” they note.
The researcher then subdivided EPHB2-expressing cells on the basis of expression levels of the marker. They found that EPHB2high cells had the longest average telomere length and expressed the highest levels of mouse ISC markers. Expression profiling studies showed that in EPHB2high cells there was a set of 90 genes including LGR5, ASCL2, and EPHB3 that was consistently expressed at higher levels in comparison with EPHB2medium cells.
“From these data, we tentatively concluded that the EPHB2high cell population is largely enriched in CoSCs, whereas EPHB2medium and EPHB2low cells represent transient amplifying cells at different stages of differentiation,” the authors state.
Development of a suitable culturing system for the cells was based on recent protocols for maintaining mouse gastrointestinal organoids, which involves reproducing ISC niche signals such as activating the Wnt and Notch pathways and inhibiting bone morphogenetic protein signals. However, Dr. Battle et al. found that this mouse protocol required critical changes.
In particular, the maintenance of human colon crypts was dependent on supplementation with nicotinamide and was significantly boosted by the addition of prostaglandin E2. When human colon crypts were maintained under the new culture conditions, they lost their upper third compartment during the first days of culture, while the bottom regions formed closed spheroids that expanded rapidly through cell proliferation but maintained a central lumen. Spheroids that were dissected and re-embedded in matrigel quickly reformed closed structures that kept growing.
Importantly, when cultured under the same conditions, EPHB2high cells sorted from human colon biospies grew exponentially and gave rise to macroscopic spheroids within 18–21 days. The spheroids could be passaged by disaggregation every week, a regime that allowed EPHB2high to be cultured for long periods (over 4 months) in conditions of exponential growth, the authors write. Significantly, individual cells from these spheroids could be separated and recultured, again generating macroscopic spheroids with no evidence of genomic instability.
Under appropriate culture conditions EPHB2high-derived spheroids could also be triggered to differentiate, a process that was accompanied by upregulation of both the pan-differentiation marker KRT20 and differentiation markers of absorptive and secretory cell lineages as well as silencing of ISC genes. Differentiation was accompanied by marked morphological changes characteristic of absorptive or mucus-secreting cells.
“Notably, upon switching to differentiation medium, all single-cell-derived spheroids analyzed generated a mixed population of absorptive and mucosecreting cells, implying that a substantial proportion of CoSCs stay multipotent,” the researchers remark.
“Collectively, these data represent what is to our knowledge the first isolation, characterization, and in vitro expansion of a cell population largely enriched in human CoSCs,” they conclude. “The cell culturing protocol described here allows the maintenance of an undifferentiated and multipotent ISC-like phenotype in vitro. Our work represents a substantial step forward toward the use of human CoSCs in regenerative medicine and toward understanding their role in pathology.”