ABSTRACT
Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.
Subject(s)
Cell Dedifferentiation , Cell Transformation, Neoplastic , Colonic Neoplasms/pathology , Neoplastic Stem Cells/pathology , Animals , Colon/pathology , Epithelial Cells/pathology , Female , Humans , Male , Mice , NF-kappa B/metabolism , Wnt Signaling PathwayABSTRACT
The mammalian ovary is covered by a single-layered epithelium that undergoes rupture and remodelling following each ovulation. Although resident stem cells are presumed to be crucial for this cyclic regeneration, their identity and mode of action have been elusive. Surrogate stemness assays and in vivo fate-mapping studies using recently discovered stem cell markers have identified stem cell pools in the ovary and fimbria that ensure epithelial homeostasis. Recent findings provide insights into intrinsic mechanisms and local extrinsic cues that govern the function of ovarian and fimbrial stem cells. These discoveries have advanced our understanding of stem cell biology in the ovary and fimbria, and lay the foundations for evaluating the contribution of resident stem cells to the initiation and progression of human epithelial ovarian cancer.
Subject(s)
Ovarian Neoplasms/metabolism , Ovary/metabolism , Stem Cell Niche , Stem Cells/metabolism , Tumor Microenvironment , Animals , Female , Humans , Ovarian Neoplasms/pathology , Ovary/pathology , Ovulation/metabolism , Stem Cells/pathologyABSTRACT
The stem/progenitor cell pool is indispensable for the development, homeostasis and regeneration of the gastric epithelium, owing to its defining ability to self-renew whilst supplying the various functional epithelial lineages needed to digest food efficiently. A detailed understanding of the intricacies and complexities surrounding the behaviours and roles of these stem cells offers insights, not only into the physiology of gastric epithelial development and maintenance, but also into the pathological consequences following aberrations in stem cell regulation. Here, we provide an insightful synthesis of the existing knowledge on gastric epithelial stem cell biology, including the in vitro and in vivo experimental techniques that have advanced such studies. We highlight the contributions of stem/progenitor cells towards patterning the developing stomach, specification of the differentiated cell lineages and maintenance of the mature epithelium during homeostasis and following injury. Finally, we discuss gaps in our understanding and identify key research areas for future work.
Subject(s)
Stem Cells , Stomach , Homeostasis , Cell Differentiation , Cell LineageABSTRACT
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.
Subject(s)
Adult Stem Cells/physiology , Epithelial Cells/physiology , Intestinal Mucosa/cytology , Regeneration , Animals , Cell Lineage , Homeostasis , Humans , Intestinal Mucosa/physiology , Models, Biological , Stem Cell NicheABSTRACT
LGR5 marks resident adult epithelial stem cells at the gland base in the mouse pyloric stomach1, but the identity of the equivalent human stem cell population remains unknown owing to a lack of surface markers that facilitate its prospective isolation and validation. In mouse models of intestinal cancer, LGR5+ intestinal stem cells are major sources of cancer following hyperactivation of the WNT pathway2. However, the contribution of pyloric LGR5+ stem cells to gastric cancer following dysregulation of the WNT pathway-a frequent event in gastric cancer in humans3-is unknown. Here we use comparative profiling of LGR5+ stem cell populations along the mouse gastrointestinal tract to identify, and then functionally validate, the membrane protein AQP5 as a marker that enriches for mouse and human adult pyloric stem cells. We show that stem cells within the AQP5+ compartment are a source of WNT-driven, invasive gastric cancer in vivo, using newly generated Aqp5-creERT2 mouse models. Additionally, tumour-resident AQP5+ cells can selectively initiate organoid growth in vitro, which indicates that this population contains potential cancer stem cells. In humans, AQP5 is frequently expressed in primary intestinal and diffuse subtypes of gastric cancer (and in metastases of these subtypes), and often displays altered cellular localization compared with healthy tissue. These newly identified markers and mouse models will be an invaluable resource for deciphering the early formation of gastric cancer, and for isolating and characterizing human-stomach stem cells as a prerequisite for harnessing the regenerative-medicine potential of these cells in the clinic.
Subject(s)
Aquaporin 5/metabolism , Carcinogenesis/pathology , Neoplastic Stem Cells/pathology , Stomach Neoplasms/pathology , Stomach/pathology , Animals , Biomarkers/metabolism , Humans , Mice , Neoplastic Stem Cells/metabolism , Pylorus/pathology , Receptors, G-Protein-Coupled/metabolism , Wnt Signaling PathwayABSTRACT
We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.
Subject(s)
Proteasome Inhibitors , Tumor Suppressor Protein p53 , Mice , Animals , Bortezomib/pharmacology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Proteasome Inhibitors/pharmacology , Proteasome Inhibitors/metabolism , Hematopoietic Stem Cells/metabolism , Myeloid Progenitor Cells/metabolism , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Cell Lineage , Intestine, Small/cytology , Stem Cells/cytology , Animals , Clone Cells , Mice , Models, Biological , Receptors, G-Protein-Coupled/metabolismABSTRACT
The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.
Subject(s)
Adult Stem Cells/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Intestine, Small/cytology , Animals , Cell Separation , Gene Deletion , Gene Expression Profiling , Mice , Mice, TransgenicABSTRACT
In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.
ABSTRACT
The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Extremities/embryology , Intercellular Signaling Peptides and Proteins/metabolism , Limb Deformities, Congenital/genetics , Receptors, G-Protein-Coupled/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , DNA-Binding Proteins/metabolism , Female , Fibroblasts , Gene Knockout Techniques , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/metabolism , Phenotype , Receptors, G-Protein-Coupled/deficiency , Ubiquitin-Protein Ligases/metabolism , Xenopus/geneticsABSTRACT
An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.
Subject(s)
Adult Stem Cells/metabolism , Anaplastic Lymphoma Kinase/genetics , Epithelial Cells/metabolism , Gene Editing/methods , Organoids/metabolism , RNA-Binding Proteins/genetics , Wnt Signaling Pathway/genetics , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/metabolism , Adult Stem Cells/cytology , Anaplastic Lymphoma Kinase/metabolism , Animals , CRISPR-Cas Systems , Cell Proliferation , Epithelial Cells/cytology , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Gene Expression Regulation , Gene Library , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , HEK293 Cells , Humans , Interleukins/genetics , Interleukins/metabolism , Mice , Organoids/cytology , RNA-Binding Proteins/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Stomach/cytologyABSTRACT
Glioblastoma (GBM) is the most common primary malignant brain cancer in adults with a dismal prognosis. Temozolomide (TMZ) is the first-in-line chemotherapeutic; however, resistance is frequent and multifactorial. While many molecular and genetic factors have been linked to TMZ resistance, the role of the solid tumor morphology and the tumor microenvironment, particularly the blood-brain barrier (BBB), is unknown. Here, the authors investigate these using a complex in vitro model for GBM and its surrounding BBB. The model recapitulates important clinical features such as a dense tumor core with tumor cells that invade along the perivascular space; and a perfusable BBB with a physiological permeability and morphology that is altered in the presence of a tumor spheroid. It is demonstrated that TMZ sensitivity decreases with increasing cancer cell spatial organization, and that the BBB can contribute to TMZ resistance. Proteomic analysis with next-generation low volume sample workflows of these cultured microtissues revealed potential clinically relevant proteins involved in tumor aggressiveness and TMZ resistance, demonstrating the utility of complex in vitro models for interrogating the tumor microenvironment and therapy validation.
Subject(s)
Brain Neoplasms , Glioblastoma , Humans , Temozolomide/pharmacology , Temozolomide/therapeutic use , Glioblastoma/drug therapy , Glioblastoma/metabolism , Blood-Brain Barrier/metabolism , Tumor Microenvironment , Proteomics , Drug Resistance, Neoplasm/genetics , Cell Line, Tumor , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Intestinal stem cells (ISCs) marked by Lgr5 are located at the bottom of the epithelial crypt compartment. Canonical Wnt signaling, activated by Wnt/Rspo ligands, determines the stem cell identity of Lgr5â¯+â¯ISCs and is strictly regulated by the ISC niche. Emerging evidence indicates that both epithelial and stromal compartments provide the requisite Wnt/Rspo ligands, confining the ISC niche to the lower crypt regions of the intestine. Recent studies have also shown that the ISC niche can reprogram differentiated cells to replenish lost ISCs following tissue injury, accounting for the epithelial cell plasticity within the crypt compartment. Here we review these recent advances and discuss the role of canonical Wnt signaling in maintaining homeostasis and effecting cell plasticity following tissue injury in the intestine, which could reveal potential novel therapeutic opportunities in the clinic.
Subject(s)
Intestinal Mucosa/metabolism , Stem Cell Niche , Stem Cells/metabolism , Wnt Signaling Pathway , Animals , Cell Self Renewal , Homeostasis , Humans , Intestinal Mucosa/cytology , Stem Cells/cytologyABSTRACT
Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.
Subject(s)
Intestinal Mucosa/metabolism , Intestines/cytology , Transcription Factors/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Lineage , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Signal Transduction/genetics , Signal Transduction/physiology , Snail Family Transcription Factors , Transcription Factors/geneticsABSTRACT
Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in ß-catenin (CTNNB1). We have compared the dynamics and the potency of ß-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of ß-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of ß-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of ß-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:ß-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of ß-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of ß-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated ß-catenin.
Subject(s)
Cadherins/metabolism , Cell Transformation, Neoplastic , Colonic Neoplasms , Mutation , Neoplasm Proteins , Wnt Signaling Pathway , beta Catenin , Animals , Cadherins/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Mice , Mice, Transgenic , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolismSubject(s)
Neoplasms , Stomach Neoplasms , Humans , Stem Cells , Cognition , SOX9 Transcription Factor/genetics , Neoplastic Stem Cells , Cell Line, TumorABSTRACT
Hepatocyte growth factor (HGF) is secreted as an inactive single-chain HGF (scHGF); however, only proteolytically processed two-chain HGF (tcHGF) can activate the MET receptor. We investigated the localization of tcHGF and activated/phosphorylated MET (pMET) using a tcHGF-specific antibody. In day 16.5 mouse embryos, total HGF (scHGF + tcHGF) was mainly localized in smooth muscle cells close to, but separate from, MET-positive epithelial cells in endodermal organs, including the stomach. In the adult stomach, total HGF was localized in smooth muscle cells, and tcHGF was mainly localized in the glandular base region. Immunostaining for pMET and Lgr5-driven green fluorescent protein (GFP) indicated that pMET localization overlapped with Lgr5+ gastric stem cells. HGF promoted organoid formation similar to EGF, indicating the potential for HGF to promote the survival and growth of gastric stem cells. pMET and tcHGF localizations changed during regeneration following gastric injury. These results indicate that MET is constantly activated in gastric stem cells and that the localization of pMET differs from the primary localization of precursor HGF but has a close relationship to tcHGF. Our results suggest the importance of the microenvironmental generation of tcHGF in the regulation of development, regeneration, and stem cell behavior.
Subject(s)
Hepatocyte Growth Factor/metabolism , Organogenesis , Wound Healing , Animals , Biomarkers , Hepatocyte Growth Factor/genetics , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Organogenesis/genetics , Phosphorylation , Protein Transport , Proto-Oncogene Proteins c-met/metabolism , Regeneration , Stem Cells/cytology , Stem Cells/metabolism , Wound Healing/geneticsABSTRACT
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.
Subject(s)
Intestines/cytology , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Paneth Cells/cytology , Receptors, G-Protein-Coupled/metabolism , Stem Cell Niche/cytology , Animals , CD24 Antigen/metabolism , Cell Count , Cell Proliferation , Coculture Techniques , Humans , Mice , Paneth Cells/metabolism , Stem Cell Niche/metabolism , Wnt Proteins/metabolism , Wnt3 ProteinABSTRACT
The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.