Unraveling intestinal stem cell behavior with models of crypt dynamics

Abstract

The definition, regulation and function of intestinal stem cells (ISCs) has been hotly debated. Recent discoveries have started to clarify the nature of ISCs, but many questions remain. This review discusses the current advances and controversies of ISC biology as well as theoretical compartmental models that have been coupled with in vivo experimentation to investigate the mechanisms of ISC dynamics during homeostasis, tumorigenesis, repair and development. We conclude our review by discussing the key lingering questions in the field and proposing how many of these questions can be addressed using both compartmental models and experimental techniques.

Figure 1. Intestinal epithelial organization and markers

(a) The intestinal epithelium is organized into crypt and villus regions, with the stem and progenitor zone localized in the crypt. Current models favor the existence of two stem cell populations, the +4 stem cell and the crypt base columnar cell (CBCC), which are thought to be quiescent and active stem cells, respectively. Transit-amplifying (TA) progenitors arise from the stem cell compartment and differentiate into absorptive enterocytes or secretory goblet, enteroendocrine, tuft, or Paneth cells. Most of the differentiated cell populations migrate up the villi, but, uniquely, the Paneth cells move downward and reside between the CBCCs. (b) Molecular and functional markers that have been described for various proposed stem cell and potential stem cell populations. Of note, both TA cells and +4 cells have been shown to be Label Retaining Cells (LRCs). Sox9-EGFP has been shown to mark both CBCCs and clonogenic enteroendocrine cells, depending on the level of EGFP expression. The gene Dclk1 has been proposed to be a stem cell marker, but it has also been shown to be a specific marker of differentiated tuft cells. It is possible that there is an independent +4 cell population that is also marked with Dclk1, but this has not been verified by lineage tracing.

Figure 2. The stem cell niche is defined by several molecular signals

Activation of the Bone Morphogenetic Pathway (BMP) occurs at a gradient that is higher in the villi and lower in the crypts. Conversely, Wnt activity is highest in the crypts. The Wnt gradient is established by secretion of Wnt ligands both from the mesenchymal myofibroblasts (WNT2a) as well as from epithelial cells. WNT3, in particular, is expressed in Paneth cells. The Notch signaling pathway is also critical for niche specification. Notch ligand presentation must occur from adjacent cells, and there is evidence that Paneth cells present DLL4, and that a subset of secretory progenitors express DLL1. It is unclear if other TA cell populations can present Notch ligand to stem cells.

Figure 3. Key questions in the field of intestinal stem cell biology

(a) Schematic illustration of the base of an intestinal crypt with stem cells designated in green. The mechanisms regulating stem cell number are not known. (b) An illustration of two opposing theories regarding the role of the stem cell niche. Left: The niche (green arrows) completely specifies the stem cell. Right: the niche partially specifies a cell that possesses certain features of intrinsic stemness (yellow). Only cells acquire both extrinsic and intrinsic signals become stem cells. (c) Diagram of two possible QSC populations. Left: a single QSC that possesses a unique molecular signature (horizontal arrow) is shown. Right: QSC markers are expressed in a gradient in the crypt (vertical arrow) and any cell in this zone possesses the potential to act like a QSC. (d) Schematic of the TA cell compartment. Left: several rounds of cell divisions are shown (T1–T5). The exact number and regulation of TA cell divisions is not known. Right: a TA cell is shown to de-differentiate and replace a lost CBCC (curved arrow). Exactly which TA cells possess clonogenicity is unknown.

Figure 4. Compartmental model of homeostasis and tumorigenesis

(a) An illustration of the colonic crypt as modeled by Johnston et al.. Unlike the small intestine the colon does not have villi nor traditional Paneth cells. (b) Compartmental model of homeostasis and tumorigenesis adapted from Figure 1 of Johnston et al.. Cell populations include stem cells, semi-differentiated cells and fully differentiated cells. Cell flows into and out of the compartments are indicated by arrows and are defined by rates of death, differentiation, and renewal from the stem and semi-differentiated compartments. There is no renewal in the fully differentiated compartment and cells leave by removal.

Figure 5. Compartmental model of irradiation recovery

(a) Diagram of the cell population compartments of the crypt post-irradiation model adapted from Figure 2 of Paulus et al.. Cell populations include stem cells (A), TA cells (T1–T4), differentiated cells (D), and previously proliferative cells that stopped cycling due to irradiation injury (D′). Cells move from one compartment to the next after completing the cell cycle. Cells in A and T1 can re-enter their compartment with the probability p_A and p_T1, respectively. (b) Diagram of different cell cycle subcompartments are shown. (i) Subcompartments during steady state when the cell cycle time is 24 hours for A and 12 hours for T compartments. Cells (white circles) advance to the next subcompartments every hour of the simulation. For clarity we have included G1, S, G2 and M phases of the cell cycle, but the lengths of G2 and M that were used during the Paulus et al. simulation was not made clear in the manuscript. (ii) Normal stochasticity in the model. Cell cycle time was allowed to vary slightly for each individual cell. This variation was limited to the G1 compartment and was achieved by skipping a subcompartment. Renewal in the A and T1 compartment was accomplished by re-entering the first G1 subcompartment after completing M phase. (iii) Alteration in subcompartments after maximal irradiation injury, where cell cycle lengths are decreased to 8 hours.

Figure 6. Compartmental model of crypt development

This figure has been adapted from Figure 3 of Itzkovitz et al. (a) Definitions of types of stem and non-stem cell divisions. Stem cells can undergo two types of symmetric division, S1 and S2, or asymmetric division, A. Non-stem cells always divide symmetrically or are extruded from the crypt. (b) Depiction of the two types of “bang-bang” model outcomes. The rounds of division have been limited to 5 for clarity. The left cell lineage tree shows bang-bang division that shows a switch from S1 stem cell division to A division. The right lineage tree shows A division preceding S1 division. (c) Depiction of the “overshoot” model where stem cells undergo S1 division followed by S2 division. The final cell composition is the same as the bang-bang models, but it only takes 4 rounds of divisions instead of 5 to achieve this.