Crypt region localization of intestinal stem cells in adults

Abstract

The intestinal epithelial lining plays a central role in the digestion and absorption of nutrients, but exists in a harsh luminal environment that necessitates continual renewal. This renewal process involves epithelial cell proliferation in the crypt base and later cell migration from the crypt base to the luminal surface. This process is dependent on multi-potent progenitor cells, or stem cells, located in each crypt. There are about 4 to 6 stem cells per crypt, and these stem cells are believed to generate distinct end-differentiated epithelial cell types, including absorptive cells, goblet cells, enteroendocrine cells and Paneth cells, while also maintaining their own progenitor cell state. Earlier studies suggested that intestinal stem cells were located either in the crypt base interspersed between the Paneth cells [i.e. crypt base columnar (CBC) cell model] or at an average position of 4 cells from the crypt base [i.e. label-retaining cells (LRC +4) model]. Recent studies have employed biomarkers in the in vivo mammalian state to more precisely evaluate the location of these progenitor cells in the intestinal crypt. Most notable of these novel markers are Lgr5, a gene that encodes a G-protein-coupled receptor with expression restricted to CBC cells, and Bmi 1, which encodes a chromatin remodeling protein expressed by LRC. These studies raise the possibility that there may be separate stem cell lines or different states of stem cell activation involved in the renewal of normal mammalian intestinal tract.

Key Words: Crypt base columnar cells, Intestinal epithelial cell renewal, Lgr5 gene, Polycomb bmi1 protein, Progenitor cells, Stem cells, +4 stem cell model

INTRODUCTION

An improved understanding of stem cell biology and its possible application in the treatment of diseases has emerged as an important fundamental area in clinical medicine. The intestinal epithelial lining is relatively unique in its ability to rapidly regenerate. This has led to an ever increased focus on the crypt cell as a potential model of adult stem cell biology. The mammalian intestinal epithelial lining plays a critical role in digestion and in the absorption of nutrients and requires constant renewal due to the very harsh luminal environment. This renewal process involves rapid and continuous proliferation of epithelial cells in the crypt base with subsequent migration of these cells to the luminal surface. This process of epithelial cell renewal within the intestine appears to be entirely dependent upon a limited number of long-lived multi-potent intestinal progenitor cells or stem cells.

An intestinal epithelial stem cell may be broadly defined as a cell having at least 2 important properties: firstly, an ability to generate distinct differentiated epithelial cell types found in the intestine; and secondly, an ability to maintain itself as a progenitor cell over prolonged periods. It appears that each intestinal crypt contains approximately 4 to 6 stem cells. Each of these cells is believed to be capable of essential regenerative activity which is required to produce all the distinctive end-differentiated epithelial cell types found in the intestine. However, recent studies employing specific biomarkers for identification of stem cells have raised new and intriguing issues which have significant implications for the regenerative processes in the digestive tract involved in both health and disease.

INTESTINAL STEM CELL IDENTIFICATION

Over 30 years ago, Cheng and Leblond identified small cycling epithelial cells interspersed between the Paneth cells, or the so-called crypt base columnar (CBC) cells, using morphological methods in mammalian intestine[1,2]. Later, Bjerkness and Cheng provided additional information on these specialized cells using elegant clonal marking techniques[3].

These investigators theorized that the CBC cells located within the stem cell zone of the crypt base might represent the actual intestinal epithelial stem cells[3,4]. All of the end-differentiated intestinal epithelial cells were hypothesized to develop from these CBC cells including intestinal columnar cells, intestinal goblet cells, enteroendocrine cells and Paneth cells.

An alternative hypothesis has also suggested that intestinal stem cells were actually located elsewhere at a position that averaged +4 from the bottom of the crypts with the lowest three positions generally relegated to the terminally differentiated Paneth cells. Evidence supporting this hypothesis of the +4 stem cell model was provided by Potten et al[5,6]. These investigators, using the DNA-labeling reagents, bromo-deoxyuridine or (³H)-thymidine, on radiation-sensitive, label-retaining cells (LRC) showed that the LRC were located specifically at the +4 position in the intestinal crypt region, precisely at the origin of the migratory epithelial cell column.

INTESTINAL STEM CELL SIGNALING

Intestinal stem cells, although compartmentalized into the crypt region, do not function in isolation independent of specific regulatory controls. Clearly, these cells play critical roles in the proliferation and differentiation of normal and neoplastic epithelial cells, However, the complex regulation of these cells involves a wide array of critical signaling pathways, recently well reviewed elsewhere[7]. These include Wnt, BMP, PTEN-controlled PI3K/Akt and Notch pathways. While studies are needed to further elucidate all of the specific mechanisms involved in each signaling pathway, recent reports focused on the elusive position of these progenitor cells have served to add a new dimension to the intricate biology of these cell processes.

INTESTINAL STEM CELL LOCALIZATION

In 2007, Barker and Clevers reported a highly restricted molecular marker for intestinal stem cells[8]. These investigators identified Lgr5, a gene encoding a specific G-protein-coupled receptor, which was expressed specifically in CBC cells. Later, using engineered mice, a DNA marker on Lgr5-positive cells was created which permitted subsequent tracing of their lineage into long-lived epithelial clones with all of the cell types in normal ratios. Interestingly, CBC cells were not quiescent (as might have been expected) but completed a cell cycle in about one day suggesting that these CBC cells would have to undergo many hundreds or thousands of cell divisions during the lifetime of the animal without loss of genetic information or malignant transformation. The Lgr5 gene was also found to be expressed in the stomach and throughout the intestinal tract as well as in colon cancer cells leading to speculation that Lgr5 might be a cancer stem cell marker. Moreover, as the Lgr5 gene encoded a receptor on the cell surface, it was suggested that this might permit recognition with a monoclonal antibody and a means of eventually eradicating Lgr5-positive cancer stem cells[9].

In a recent 2008 report, another new stem cell marker was reported by Sangiorgi et al[10]. In their studies, Bmi 1, a Polycomb group protein known to play an important role in the renewal of hematopoietic and neural stem cells, characterized the progeny derived from Bmi 1-positive cells using a similar lineage tracing approach employed for Lgr5-positive cells. The Bmi 1 locus marked long-lived cell clones by all intestinal cell types. Ablation of Bmi 1-positive cells resulted in depletion from the epithelium in entire intestinal crypts. Of note, Bmi 1 was expressed in cells in the +4 position region, not at the location of the CBC cells, and primarily in the proximal small intestine, suggesting that a different stem cell population may have been characterized. Moreover, crypt Bmi 1-positive cells appeared to have slow turnover with relatively slow kinetics, another difference from Lgr5-positive cells which rapidly turnover[11].

FUTURE STUDIES

The possible relationship of these two apparently distinctive intestinal stem cell populations requires further definition. In addition, studies for other new stem cell markers are likely to already be in progress. Are these studies likely to indicate that there are multiple stem cell populations in the intestine similar to other stem cell organs, such as the epidermis, with distinctive stem cell populations? Or, alternatively, is this simply the expression of a single stem cell population in different proliferative states? Further definition of this issue is essential. The regenerative processes in the intestinal epithelial lining in health and disease are both fascinating and complex. More importantly, the possible implications for clinical medicine may be enormous. As clinicians caring for patients, close attention to this rapidly progressing fundamental investigative endeavour is needed.