BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt protein variant can fulfill most functions of a normal Wnt protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.

An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.

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A reserve stem cell population in small intestine renders Lgr5 -positive cells dispensable

Small populations of adult stem cells are responsible for the remarkable ability of the epithelial lining of the intestine to be efficiently renewed and repaired throughout life. The recent discovery of specific markers for these stem cells, together with the development of new technologies to track endogenous stem cell activity in vivo and to exploit their ability to generate new epithelia ex vivo, has greatly improved our understanding of stem cell-driven homeostasis, regeneration and cancer in the intestine. These exciting new insights into the biology of intestinal stem cells have the potential to accelerate the development of stem cell-based therapies and ameliorate cancer treatments.

Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration

Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells in multiple adult organs of mice and humans. In R-spondin-based three-dimensional cultures, these Lgr5 stem cells can grow into ever-expanding epithelial organoids that retain their original organ identity. Single Lgr5 stem cells derived from the intestine can be cultured to build epithelial structures that retain hallmarks of the in vivo epithelium. Here, we review the mechanisms that support this notable example of self-organization and discuss applications of this technology for stem cell research, disease modeling (e.g., for colorectal cancer and cystic fibrosis), and regenerative medicine.

http://science.sciencemag.org/content/sci/340/6137/1190.full.pdf

Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications

http://crcooper01.people.ysu.edu/Clevers%202013%20Review%20ISCs%5B1%5D.pdf

The Intestinal Crypt, A Prototype Stem Cell Compartment

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

https://www.pnas.org/content/pnas/108/1/179.full.pdf

Mariette E. G. Kranendonk

Papers

Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells