Cloning and variation of ground state intestinal stem cells

Abstract

SummaryStem cells of the gastrointestinal tract, pancreas, liver, and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, “ground state” stem cells of the human intestine and colon. We show that derived stem cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells likely enforces the functional specificity of the adult intestinal tract. Using clonally-derived colonic epithelia, we show that toxins A or B of the enteric pathogen C. difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modeling and regenerative medicine.

Expanding intestinal stem cells in culture

“…Stem cells grown under Wang's condition, by contrast, continue to propagate as flat 2D colonies on the feeder layer and do not differentiate [1]. Since the growth factor additions are nearly identical between the two conditions (containing Wnt agonist R-spondin, EGF, BMP antagonist and TGF-β inhibitor), the matrix as well as the focal production of Wnt3 by Paneth cells in organoids [7,8] might be a critical factor for the difference between the two systems.…”

“…Wang et al [1] showed that clonal stem cells yield all types of differentiated cells upon induction differentiation. However, it remains unknown whether the dynamic turnover of epithelia oc- curs in these differentiated conditions and, even so, how closely this turnover rate might resemble the in vivo situation.…”

“…Wang et al [1] applied this culture model to probe molecular properties of intestinal stem cells. It was previously reported that adult intestinal stem cells carry information of commitment to region-specific differentiation along intestinal tract by analyzing gene expression of organoid system [9].…”

“…Over the past decade, significant progress has been made for establishing culture methods for tissue stem cells directly from healthy or diseased mouse and human organs. Now Wang et al [1] developed a new method for the specific expansion of intestinal stem cells.…”

“…In a paper recently published in Nature, Wang and colleagues describe a condition to support long-term and highly homogeneous preparations of stem cells from human fetal and adult small intestine and colon [1]. Single cell suspensions were seeded on a feeder layer of mouse fibroblasts.…”