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Isolation and in vitro expansion of human colonic stem cells

From Nature Medicine
By Stuart P. Atkinson

A paper recently published in Nature Medicine, described as a major advance for regenerative medicine, has reported for the first time the isolation and in vitro culture of stem cells from the adult human colon (CoSCs) (Jung and Sato et al). This achievement, from the group of Eduard Batlle at the Institute for Research in Biomedicine, Barcelona, achieved its success after over 10 years of intensive research into the characterization of the biology of the intestinal stem cells and its connection with cancer. The study, done in collaboration with renowned researchers Hans Clevers and María A. Blasco, represents a sub­stantial step forward toward the use of human CoSCs in regenerative medicine and toward understanding their role in pathology.

Initial analysis for potential markers for CoSCs in human intestinal crypts found a gradient of EPHB2 expression; EPHB2 was maximal in the bottom of the crypts, decreasing with the upward migration of differentiating epithelial cells. The mouse Ephb2gene encodes a receptor tyrosine kinase that is found at high levels in mouse small intestine intestinal stem cell (ISCs) as well as colorectal cancer stem cells and is also know to be involved the spatial organisation of intestinal crypt cells (Batlle et al and Merlos-Suárez et al). Further analysis demonstrated that the differentiated cells of the surface epithelium, which are positive for KRT20, stained negative for EPHB2, suggesting that EPHB2 was marking a stem cell like population. FACS segregation of different cell populations from the crypts using EpCAM+ and EPHB2 allowed the comparison of cells expressing low, medium and high levels of EPHB2. An increase in EPHB2 expression was correlated to a decrease in colonic differentiation markers and an increase in expression of genes required for proliferation and longer telomeres, again suggestive of a stem cell phenotype. Gene expression analysis also showed a correlation between EPHB2 expression and the expression of genes known to mark mouse ISCs (LGR5, ASCL2 and OLFM4) as well as EPHB3 and TERT. Overall the authors suggest that the EPHB2high cell population contained CoSCs while EPHB2medium and EPHB2low cells represent transit amplifying cells at different stages of differentiation. Following experimental analysis in the mouse model, a protocol was described for the maintenance of full human colon crypts embedded in matrigel; which mimicked the mouse protocol but with the supplementation of the vitamin nicotinamide and prostaglandin E2 (PGE2), which blocks anoikis (programmed cell death associated with a lack of cell-matrix interaction, usually affecting detached or disrupted cells) and transactivates mitogenic signaling. This allowed for the specific maintenance of the bottom two thirds of the crypt which formed closed spheroids which expanded through rapid cell proliferation and maintaining a central lumen. These microstructures could be passaged by mechanical dissociation and re-embedded within Matrigel, leading to the quick reformation of the spheroids. This system was then utilised to test for the ability of the EPHB2high cell population to from spheroids. Single EPHB2high cells grew exponentially and gave rise to macroscopic spheroids within 18–21 days at a frequency of between 6-17%. Growth as spheroids was linked intimately to the levels of EPHB2 and under this regime, EPHB2high cells could be cultured for long periods (over 4 months) in conditions of exponential growth with no notable karyotypic instability. The differentiation ability of these cells was maintained, as observed by an increase in the levels of KRT20, as well as markers of absorptive (ANPEPand CA1) and secretory lineages, MUC2 and TFF3 in response to the removal of PGE2 and Wnt3a alongside the inhibition of Notch signalling using a g-secretase inhibitor. EPHB2high cell differentiation was accompanied by marked morphological changes that included prominent cellular polarization and the presence of intermingled cells with either a well-defined apical brush border or large mucous vacuoles, characteristic of absorptive or mucus-secreting cells, respectively. This multipotent nature of the CoSCs was maintained in cells which had been propagated in culture over six weeks.

These findings will allow more detailed research into this colonic stem cell population, including the molecular basis of gastrointestinal cell proliferation and differentiation, in addition to further research on diseases in which CoSCs have been impacted; such as colorectal cancer and Crohn's disease.



Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, Meeldijk J, Robertson J, van de Wetering M, Pawson T, Clevers H.
Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB.
Cell. 2002 Oct 18;111(2):251-63.

Jung P, Sato T, Merlos-Suárez A, Barriga FM, Iglesias M, Rossell D, Gallardo MM, Blasco MA, Sancho E, Clevers H, Batlle E
Isolation and in vitro expansion of human colonic stem cells.
Nat Med. 2011 Sep 4. doi: 10.1038/nm.2470.

Merlos-Suárez A, Barriga FM, Jung P, Iglesias M, Céspedes MV, Rossell D, Sevillano M, Hernando-Momblona X, da Silva-Diz V, Muñoz P, Clevers H, Sancho E, Mangues R, Batlle E.
The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse.
Cell Stem Cell. 2011 May 6;8(5):511-24.