Growth Of Epithelial Stem Cells Research Articles

Mesenchymal stem cells and the embryonic reawakening theory of BPH.

The prostate is the only organ in a man that continues to grow with age. John McNeal proposed, 40 years ago, that this BPH is characterized by an age-related reinitiation of benign neoplastic growth selectively in developmentally abortive distal ducts within the prostate transition-periurethral zone (TPZ), owing to a reawakening of inductive stroma selectively within these zones. An innovative variant of this hypothesis is that, owing to its location, the TPZ is continuously exposed to urinary components and/or autoantigens, which produces an inflammatory TPZ microenvironment that promotes recruitment of bone marrow-derived mesenchymal stem cells (MSCs) and generates a paracrine-inductive stroma that reinitiates benign neoplastic nodular growth. In support of this hypothesis, MSCs infiltrate human BPH tissue and have the ability to stimulate epithelial stem cell growth. These results provide a framework for defining both the aetiology of BPH in ageing men and insights into new therapeutic approaches.

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

Effects of Flow-Induced Shear Stress on Limbal Epithelial Stem Cell Growth and Enrichment

The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.

TGF-β signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung

Tissue resident mesenchymal stromal cells (MSCs) contribute to tissue regeneration through various mechanisms, including the secretion of trophic factors that act directly on epithelial stem cells to promote epithelialization. However, MSCs in tissues constitute a heterogeneous population of stromal cells and different subtypes may have different functions. In this study we show that CD166neg and CD166pos lung stromal cells have different proliferative and differentiative potential. CD166neg lung stromal cells exhibit high proliferative potential with the capacity to differentiate along the lipofibroblastic and myofibroblastic lineages, whereas CD166pos lung stromal cells have limited proliferative potential and are committed to the myofibroblastic lineage. Moreover, we show that CD166pos lung stromal cells do not share the same epithelial-supportive capacity as their CD166neg counterparts, which support the growth of lung epithelial stem cell (EpiSPC) colonies in vitro. In addition, ex vivo expansion of lung stromal cells also resulted in the loss of epithelial-supportive capacity, which could be reinstated by inhibition of the TGF-β signaling pathway. We show that epithelial-supportive capacity correlated with the level of FGF-10 expression and the reactivation of several lung development-associated genes. In summary, these studies suggest that TGF-β signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung.

281 Mutant K-RAS in the Epithelial Stem Cell Compartment Promotes Symmetric Stem Cell Division and Crypt Fission but Not Tumorigenesis in the Mouse Intestine and Colon

cell sorting and 3) significantly expand organoids as either enriched for stem cells or as differentiated cells that retain a site-specific program of differentiation and are free from mesenchymal contamination. Here we present an improved isolation and culture method that addresses these needs. METHOD: Human gastrointestinal epithelial cells were cultured using an adaptation of our published mouse epithelium culturing system. This system utilizes conditioned media collected from an L cell line (L-WRN) engineered to secrete the factors required for epithelial stem cell growth (Wnt3a, R-spondin3 and Noggin). Importantly, cultures were established from biopsy tissue (2-3 biopsies with area of 6-24 mm2) collected during routine endoscopy. Cell sorting enrichment was not required. Epithelial cells were maintained as 3-dimensional organoids in basement membrane matrix (Matrigel) with 50% conditioned media (1:1 dilution with fresh crypt culture media). Analysis of cellular markers of proliferative and differentiated cells was performed using qRT-PCR, immunofluorescence and flow assisted cell sorting. RESULTS: Human epithelial cultures were established from multiple regions of the gastrointestinal tract, including small intestine (n=9), colon (n=23), stomach (n=2) and Barrett's esophagus (n=2). Biopsy donors included healthy and IBD patients. Mesenchymal cells carried over from the isolation process were eliminated during initial passages. Subsequent studies focused on intestinal organoids, which were maintained for.40 passages (.120 days) and were able to be stored as frozen stocks. In 50% conditioned media, the majority of cells were proliferative and expressed high levels of the stem cell markers, LGR5 and OLFM4. Accordingly, organoids expanded at a rapid rate and were split 1:3 every 3 days. An intestine-specific program of differentiation was achieved using lower percentage conditioned media plus additional factors, including the Notch inhibitor DAPT. CONCLUSIONS:We have generated a culture system that will allow for large-scale, functional experiments of human intestinal epithelial cells. This system will enable us to create a biobank of human epithelial cell lines. Moreover, because cultures can be established from endoscopic biopsies, tissue donors could potentially be pre-selected based on genotype to facilitate studies of the interactions between genotype and environmental factors.

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).

Transforming Growth Factor-beta2 protects the small intestine during methotrexate treatment in rats possibly by reducing stem cell cycling.

During chemo- and radiation therapy, the balance between epithelial cell proliferation, differentiation, and cell death at the villus tip is disrupted by premature death of dividing epithelial cells. This will subsequently lead to the onset of mucosal barrier injury in the whole gastrointestinal tract. Up till now there is no validated method to treat side effects occurring due to therapy. An approach to manage this side effect might be to reversibly arrest growth of epithelial stem cells during therapy using Transforming Growth Factor-β2. A Transforming Growth Factor-β2 enriched fraction prepared from bovine milk was shown to protect small intestinal epithelial cells against cell cycle specific chemotherapeutic agents by arresting the cells in G1-phase. Secondly, in a rat model for induced small intestinal damage, oral supplementation of rats exposed to methotrexate with the Transforming Growth Factor-β2 enriched fraction significantly reduced the chemotherapy-associated weight loss and ileal villus atrophy by reducing cell proliferation in the normal stem cell population. Thus oral supplementation with a bovine milk fraction enriched for Transforming Growth Factor-β2 attenuated the side effects of chemotherapy in the small intestine in rats.British Journal of Cancer (2002) 87, 113–118. doi:10.1038/sj.bjc.6600342 www.bjcancer.com© 2002 Cancer Research UK