Lymphatics act as a signaling hub to regulate intestinal stem cell activity

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A constant pool of Lgr5+ intestinal stem cells is required for intestinal homeostasis

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Quiescent stem cells in intestinal homeostasis and cancer

Abstract

Adult stem cell niches are characterized by a dichotomy of cycling and quiescent stem cells: while the former are responsible for tissue turnover, their quiescent counterparts are thought to become active upon tissue injury thus underlying the regenerative response. Moreover, quiescence prevents adult stem cells from accumulating mutations thus ensuring a reservoir of unaltered stem cells. In the intestine, while cycling stem cells were shown to give rise to the main differentiated lineages, the identity of their quiescent equivalents remains to date elusive. This is of relevance for conditions such as Crohn's disease and ulcerative colitis where quiescent stem cells may underlie metaplasia and the increased cancer risk associated with chronic inflammation. Tumours are thought to share a comparable hierarchical structure of adult tissues with pluripotent and self-renewing cancer stem cells (CSCs) giving rise to more differentiated cellular types. As such, neoplastic lesions may encompass both cycling and quiescent CSCs. Because of their infrequent cycling, quiescent CSCs are refractory to chemo- and radiotherapy and are likely to play a role in tumour dissemination, dormancy and recurrence.     

Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of ‘field cancerization’ describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5⁺ stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.     

Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.     

Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal

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Modulation of stem cell fate in intestinal homeostasis,injury and repair

The mammalian intestinal epithelium constitutes the largest barrier against the external environment and makes flexible responses to various types of stimuli. Epithelial cells are fast-renewed to counteract constant damage and disrupted barrier function to maintain their integrity. The homeostatic repair and regeneration of the intestinal epithelium are governed by the Lgr5⁺ intestinal stem cells (ISCs) located at the base of crypts, which fuel rapid renewal and give rise to the different epithelial cell types. Protracted biological and physicochemical stress may challenge epithelial integrity and the function of ISCs. The field of ISCs is thus of interest for complete mucosal healing, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel diseases. Here, we review the current understanding of the signals and mechanisms that control homeostasis and regeneration of the intestinal epithelium. We focus on recent insights into the intrinsic and extrinsic elements involved in the process of intestinal homeostasis, injury, and repair, which fine-tune the balance between self-renewal and cell fate specification in ISCs. Deciphering the regulatory machinery that modulates stem cell fate would aid in the development of novel therapeutics that facilitate mucosal healing and restore epithelial barrier function.     

Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67‐RFP allele

Cycling Lgr5⁺ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5⁺ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67^RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67^RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5^high stem cells are continuously in cell cycle, while a fraction of Lgr5^low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5^low Ki67⁻ cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.     

Adult mammalian stem cells: the role of Wnt, Lgr5 and R-spondins

After its discovery as oncogen and morphogen, studies on Wnt focused initially on its role in animal development. With the finding that the colorectal tumour suppressor gene APC is a negative regulator of the Wnt pathway in (colorectal) cancer, attention gradually shifted to the study of the role of Wnt signalling in the adult. The first indication that adult Wnt signalling controls stem cells came from a Tcf4 knockout experiment: mutant mice failed to build crypt stem cell compartments. This observation was followed by similar findings in multiple other tissues. Recent studies have indicated that Wnt agonists of the R-spondin family provide potent growth stimuli for crypts in vivo and in vitro. Independently, Lgr5 was found as an exquisite marker for these crypt stem cells. The story has come full circle with the finding that the stem cell marker Lgr5 constitutes the receptor for R-spondins and occurs in complex with Frizzled/Lrp.     

Hair follicle stem cells as a skin‐organizing signaling center during adult homeostasis

Stem cells are the essential source of building blocks for tissue homeostasis and regeneration. Their behavior is dictated by both cell‐intrinsic cues and extrinsic cues from the microenvironment, known as the stem cell niche. Interestingly, recent work began to demonstrate that hair follicle stem cells (HFSCs) are not only passive recipients of signals from the surroundings, but also actively send out signals to modulate the organization and function of their own niches. Here, we discuss recent findings, and briefly refer to the old, on the interaction of HFSCs and their niches with the emphasis on the outwards signals from HFSCs toward their niches. We also highlight recent technology advancements that further promote our understanding of HFSC niches. Taken together, the HFSCs emerge as a skin‐organizing center rich in signaling output for niche remodeling during various stages of adult skin homeostasis. The intricate crosstalk between HFSCs and their niches adds important insight to skin biology that will inform clinical and bioengineering fields aiming to build complete and functional 3D organotypic cultures for skin replacement therapies.     

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

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.     

Multi-therapeutic effects of human adipose-derived mesenchymal stem cells on radiation-induced intestinal injury

Radiation-induced intestinal injuries (RIII) commonly occur in patients who suffer from pelvic or abdominal cancer. However, current management of these injuries is ineffective. Recently, mesenchymal stem cells (MSCs) have been extensively used in regenerative medicine and have achieved a high level of efficacy. In the present study, we hypothesised that human adipose-derived mesenchymal stem cells (hAd-MSCs) could be used as potential tools to heal RIII. We observed that adult Sprague–Dawley rats that received whole-abdominal irradiation benefitted from hAd-MSC injection. hAd-MSCs had RIII-healing effects, including anti-inflammation, neovascularisation and maintenance of epithelium homeostasis, as indicated by elevated serum IL-10, upregulation of vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor in irradiated intestine, mobilisation of CD31-positive haematopoietic stem cells or haematopoietic progenitor cells, and the prolonged presence of Bmi1-positive cells within crypts. Consequently, after hAd-MSC treatment, irradiated rats survived longer than non-treated animals. These results suggest that hAd-MSCs have therapeutic potential for RIII management.     

Establishing a lung cancer stem cell culture using autologous intratumoral fibroblasts as feeder cells

Human LCSCs (lung cancer stem cells) were first isolated from lung cancer patients and cultured using serum-free culture methods. To recreate the intratumoural microenvironment to sustain LCSC growth, autologous intratumoral fibroblasts were used as feeder cells. In this study, we investigated the growth and maintenance of pluripotency in prolonged LCSCs culture on autologous intratumoural fibroblasts. LCSCs isolated from three clinical samples all showed vigorous growth on feeder cells for 16 weeks of continuous cultures with a doubling time of 41-47 h. The cells continued expressing stem cell marker CD133 and remained undifferentiated. Pluripotency was demonstrated by tumour formation in immunodeficient mice. In a feeder-free culture system, growth of LCSCs spheres was retarded and would cease when the diameter reached 100 μm if immediate passage was not performed. Moreover, spontaneous differentiation was more frequently seen in a serum-free culture system. In conclusion, we have successfully established a culture system using autologous intratumoural fibroblast cells as feeder cells for prolonged culture of undifferentiated LCSCs in vitro.     

Musashi 1‐positive cells derived from mouse embryonic stem cells can differentiate into neural and intestinal epithelial‐like cells in vivo

Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1‐positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1–GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1‐positive cells out of the differentiated cell population. The GFP‐positive cells (i.e. Msi1‐positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non‐obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP‐positive cells, but not in the GFP‐negative cells. The markers for neural cells (Nestin and Tubulin β III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1‐positive cells than those from Msi1‐negative cells (P<0.05). The grafts from the Msi1‐negative cells contained more mesodermal‐like tissues than those from the Msi1‐positive cells. The pMsi1–GFP vector can be used to sort Msi1‐positive cells from a cell population derived from mouse embryonic stem cells. The Msi1‐positive cells can differentiate into neural and intestinal epithelial‐like cells in vivo.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.     

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.