Hugo J. Snippert

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt–villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24+ Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

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.

Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas

Cancer Stem Cells in Color One of the liveliest debates in contemporary cancer research centers on whether cancer stem cells (CSCs) exist and, if so, how these cells are defined phenotypically. CSCs are hypothesized to be a small population of cells within a tumor that are endowed with the unique capacity to drive tumor growth—a scenario that in principle would offer important therapeutic opportunities. By studying mice expressing multicolor reporter genes, Schepers et al. (p. 730, published online 1 August) were able to visualize and monitor the fate of a candidate stem cell for intestinal adenomas, an early stage of cancer. This “lineage tracing” analysis suggests that tumor cells expressing the intestinal crypt stem cell marker Lgr5 (leucine-rich repeat containing G protein–coupled receptor 5) are the cells that fuel the growth of intestinal adenomas. Multicolor reporter genes signal the fate of stem cells that fuel the growth of intestinal tumors in mice. The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By “lineage retracing” using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat–containing heterotrimeric guanine nucleotide–binding protein–coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5+ cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5+ cells as well as all other adenoma cell types. The Lgr5+ cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.

Lgr5 marks cycling, yet long-lived, hair follicle stem cells

In mouse hair follicles, a group of quiescent cells in the bulge is believed to have stem cell activity. Lgr5, a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Here we show that Lgr5+ cells comprise an actively proliferating and multipotent stem cell population able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time. Lgr5+ progeny repopulate other stem cell compartments in the hair follicle, supporting the existence of a stem or progenitor cell hierarchy. By marking Lgr5+ cells during trafficking through the lower outer root sheath, we show that these cells retain stem cell properties and contribute to hair follicle growth during the next anagen. Expression analysis suggests involvement of autocrine Hedgehog signaling in maintaining the Lgr5+ stem cell population.

Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.

Hair Today, Skin Tomorrow The epidermis of mammals contains hair follicles, sebaceous glands, and interfollicular epidermis, but it has not been clear how the development and repair of these structures is regulated. Snippert et al. (p. 1385) show that a stem-cell cluster in the hair follicle, characterized by the expression of Lgr6, a close homolog of the Lgr5 marker for stem cells in the small intestine and colon, resides directly above the hair bulge and gives rise to all cell lineages of the skin. Skin wounds in adult mice are repaired by Lgr6 stem cells in the hair follicles that flank the damage. After hair morphogenesis, Lgr6 stem cells give rise to epidermal and sebaceous gland lineages to generate fully differentiated new skin. Skin wounds can be repaired by primitive stem cells into fully differentiated tissue, complete with hairs and sebaceous glands. Mammalian epidermis consists of three self-renewing compartments: the hair follicle, the sebaceous gland, and the interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative of the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland, and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, whereas contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell.

Sequential cancer mutations in cultured human intestinal stem cells

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation

Astrocytes are thought to have important roles after brain injury, but their behavior has largely been inferred from postmortem analysis. To examine the mechanisms that recruit astrocytes to sites of injury, we used in vivo two-photon laser-scanning microscopy to follow the response of GFP-labeled astrocytes in the adult mouse cerebral cortex over several weeks after acute injury. Live imaging revealed a marked heterogeneity in the reaction of individual astrocytes, with one subset retaining their initial morphology, another directing their processes toward the lesion, and a distinct subset located at juxtavascular sites proliferating. Although no astrocytes actively migrated toward the injury site, selective proliferation of juxtavascular astrocytes was observed after the introduction of a lesion and was still the case, even though the extent was reduced, after astrocyte-specific deletion of the RhoGTPase Cdc42. Thus, astrocyte recruitment after injury relies solely on proliferation in a specific niche.

Prominin-1/CD133 Marks Stem Cells and Early Progenitors in Mouse Small Intestine

BACKGROUND & AIMS Prominin-1(Prom1)/CD133 is used, alone or in combination with other cell surface markers, to identify and isolate stem cells from various adult tissues. We recently identified leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) as a marker of the intestinal stem cells from which all cellular lineages of the gastrointestinal epithelium are derived. To determine whether there is a relationship between these markers, we investigated the intestinal expression pattern of Prom1/CD133 and created knock-in mice to visualize and trace Prom1(+) cells. METHODS We analyzed Prom1 mRNA and protein expression among stem cells within intestinal crypts. Prom1/CD133 knock-in mice (Prom1(-mCherry-IRES-CreERT2) KI) were generated that express a fusion of red fluorescent protein mCherry with the C-terminus of Prom1. The knock-in allele also contains the tamoxifen-inducible CreERT2 recombinase, allowing for genetic tracing of progeny derived from Prom1-positive cells. RESULTS In the small intestine, Prom1 mRNA was detected throughout the lower half of crypts and was not restricted to the rare stem cells that are sandwiched between Paneth cells. Prom1 protein was detected at the apical membranes of Lgr5(+) intestinal stem cells, but also on the transit-amplifying progenitors located above the Paneth cells. Analyses of the Prom1(-mCherry-IRES-CreERT2) KI mice showed that Prom1 is not exclusively expressed in Lgr5(+) intestinal stem cells but marks a much larger stem cell/transit-amplifying progenitor compartment. CONCLUSIONS Prom-1 marks intestinal stem cells, as well as transit-amplifying progenitors, so it is not a specific marker for Lgr5(+) intestinal stem cells.