Allon M. Klein

Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

A single type of progenitor cell maintains normal epidermis

According to the current model of adult epidermal homeostasis, skin tissue is maintained by two discrete populations of progenitor cells: self-renewing stem cells; and their progeny, known as transit amplifying cells, which differentiate after several rounds of cell division. By making use of inducible genetic labelling, we have tracked the fate of a representative sample of progenitor cells in mouse tail epidermis at single-cell resolution in vivo at time intervals up to one year. Here we show that clone-size distributions are consistent with a new model of homeostasis involving only one type of progenitor cell. These cells are found to undergo both symmetric and asymmetric division at rates that ensure epidermal homeostasis. The results raise important questions about the potential role of stem cells on tissue maintenance in vivo.

Intestinal stem cell replacement follows a pattern of neutral drift.

Gut Stem Cell Replacement Gut cell turnover is characteristically rapid and relies on stem cells in the crypts that lie between the intestinal villi. The prevailing view is that stem cell division is asymmetric with one daughter retaining a stem cell character; however, this pattern of stem cell turnover does not always apply. Using long-term lineage tracing, Lopez-Garcia et al. (p. 822, published online 23 September) showed that the loss of a stem cell was compensated for by the multiplication of a neighboring cell. The rate of stem-cell loss was found to be equivalent to the rate of cell division, indicating that symmetric cell division was the rule for gut stem cells and implying stochastic expansion, contraction, and extinction of clones occurs. Intestinal stem cells form an equipotent population where loss of a stem cell is compensated for by multiplication of a neighbor. With the capacity for rapid self-renewal and regeneration, the intestinal epithelium is stereotypical of stem cell–supported tissues. Yet the pattern of stem cell turnover remains in question. Applying analytical methods from population dynamics and statistical physics to an inducible genetic labeling system, we showed that clone size distributions conform to a distinctive scaling behavior at short times. This result demonstrates that intestinal stem cells form an equipotent population in which the loss of a stem cell is compensated by the multiplication of a neighbor, leading to neutral drift dynamics in which clones expand and contract at random until they either take over the crypt or they are lost. Combined with long-term clonal fate data, we show that the rate of stem cell replacement is comparable to the cell division rate, implying that neutral drift and symmetrical cell divisions are central to stem cell homeostasis.

Clonal dynamics of native haematopoiesis.

It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease.

Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling

Epithelial Stem Cells Much remains to be known about how epithelial stem cells are generated and maintained. Lim et al. (p. 1226; see the Perspective by Frede and Jones) describe a mechanism of stem cell maintenance where epidermal stem cells generate their own self-renewing Wnt signals rather than being controlled by adjacent “niche” signals. These stem cells also express secreted Wnt inhibitors that become localized to more differentiated progeny cells. These autocrine Wnt signals and paracrine long-range Wnt inhibitors may balance stem cell self-renewal and differentiation. Stem cells produce short-range signals to support self-renewal and long-range signal inhibitors to allow differentiation. The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

Mouse Germ Line Stem Cells Undergo Rapid and Stochastic Turnover

In cycling tissues, adult stem cells may be lost and subsequently replaced to ensure homeostasis. To examine the frequency of stem cell replacement, we analyzed the population dynamics of labeled stem cells in steady-state mouse spermatogenesis. Our results show that spermatogenic stem cells are continuously replaced, on average within 2 weeks. The analysis exposes a simple and robust scaling behavior of clone size distributions that shows stem cell replacement to be stochastic, meaning that stem cells are equipotent and equally likely to be lost or to multiply to replace their neighbors, irrespective of their clonal history. The surprisingly fast rate of stem cell replacement is supported experimentally by 3D clone morphology and by live-imaging of spermatogonial migration. These results suggest that short-lived stem cells may be a common feature of mammalian stem cell systems and reveal a natural mechanism for matching the rates of cell proliferation and loss in tissue.

The Ordered Architecture of Murine Ear Epidermis Is Maintained by Progenitor Cells with Random Fate

Typical murine epidermis has a patterned structure, seen clearly in ear skin, with regular columns of differentiated cells overlying the proliferative basal layer. It has been proposed that each column is a clonal epidermal proliferative unit maintained by a central stem cell and its transit amplifying cell progeny. An alternative hypothesis is that proliferating basal cells have random fate, the probability of generating cycling or differentiated cells being balanced so homeostasis is achieved. The stochastic model seems irreconcilable with an ordered tissue. Here we use lineage tracing to reveal that basal cells generate clones with highly irregular shapes that contribute progeny to multiple columns. Basal cell fate and cell cycle time is random. Cell columns form due to the properties of postmitotic cells. We conclude that the ordered architecture of the epidermis is maintained by a stochastic progenitor cell population, providing a simple and robust mechanism of homeostasis.

A Single Progenitor Population Switches Behavior to Maintain and Repair Esophageal Epithelium

Epithelial Defense Force The nature of the cells that maintain and heal the epithelium lining the esophagus has been controversial. Doupé et al. (p. 1091, published online 19 July; see the Perspective by Kushner) show that, unlike many other tissues, mouse esophagus is devoid of slow cycling stem cells. Instead, the epithelium is maintained and repaired by a single population of proliferating cells that can switch rapidly from homeostatic behavior into “repair mode” in the vicinity of a wound. Dividing cells in the mouse esophagus contribute to wound healing without the need for quiescent stem cells. Diseases of the esophageal epithelium (EE), such as reflux esophagitis and cancer, are rising in incidence. Despite this, the cellular behaviors underlying EE homeostasis and repair remain controversial. Here, we show that in mice, EE is maintained by a single population of cells that divide stochastically to generate proliferating and differentiating daughters with equal probability. In response to challenge with all-trans retinoic acid (atRA), the balance of daughter cell fate is unaltered, but the rate of cell division increases. However, after wounding, cells reversibly switch to producing an excess of proliferating daughters until the wound has closed. Such fate-switching enables a single progenitor population to both maintain and repair tissue without the need for a “reserve” slow-cycling stem cell pool.

Universal patterns of stem cell fate in cycling adult tissues

In cycling tissues that exhibit high turnover, tissue maintenance and repair are coordinated by stem cells. But, how frequently stem cells are replaced following differentiation, aging or injury remains unclear. By drawing together the results of recent lineage-tracing studies, we propose that tissue stem cells are routinely lost and replaced in a stochastic manner. We show that stem cell replacement leads to neutral competition between clones, resulting in two characteristic and recurring patterns of clone fate dynamics, which provide a unifying framework for interpreting clone fate data and for measuring rates of stem cell loss and replacement in vivo. Thus, we challenge the concept of the stem cell as an immortal, slow-cycling, asymmetrically dividing cell.

Kinetic Responses of β-Catenin Specify the Sites of Wnt Control

Dissecting Wnt Signaling The Wnt signaling pathway plays a key role in regulating a broad range of functions from development to cancer. But a precise understanding of how Wnt proteins act through their receptors (Frizzled proteins) has been elusive. By paring the system down to its core reactions and performing kinetic analysis in cultured cells, Hernández et al. (p. 1337, published online 8 November) were able to deduce the mechanism of Wnt action without invoking any previous assumptions. Such quantitative analysis of mass balance may offer a way to identify essential control points in other complicated signaling systems, and thus help define targets for therapeutic intervention. Reducing the rate of phosphorylation of β-catenin leads to an increase in the steady-state level of the unmodified form. Despite more than 30 years of work on the Wnt signaling pathway, the basic mechanism of how the extracellular Wnt signal increases the intracellular concentration of β-catenin is still contentious. Circumventing much of the detailed biochemistry, we used basic principles of chemical kinetics coupled with quantitative measurements to define the reactions on β-catenin directly affected by the Wnt signal. We conclude that the core signal transduction mechanism is relatively simple, with only two regulated phosphorylation steps. Their partial inhibition gives rise to the full dynamics of the response and subsequently maintains a steady state in which the concentration of β-catenin is increased.