Abstract
Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.
Highlights
Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages
To create a high-resolution profile of colon crypt stem cells and their daughter cells, we developed a new flow sorting protocol using freshly dissected, wild-type C57BL/6 N mouse colons and antibodies to validated intestinal cell surface markers including Cd44 (Fig. 1a, b, Supplementary Fig. 1)
Upon discovery that Cd44 is highly sensitive to TrypLE, and other commonly used proteases[16] (Supplementary Fig. 2a), we developed a dissociation protocol that uses only ethylenediaminetetraacetic acid (EDTA) and mechanical force
Summary
Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages The kinetics of this process is rapid such that the epithelium is replaced weekly. Multiple studies have shown how absorptive and secretory cell types can respond to wounding by dedifferentiation and repopulation of the stem cell compartment[2,3,4,5] This de-differentiation process occurs promptly, it is unknown if these reverse changes in cell state and gain of stemness occur on a similar rapid timescale as loss of stemness. The transcriptome and proteome basis for loss of stemness and early commitment is unknown
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