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author:("Li, jisheng")
1.  Developmental Programming of Long Non-Coding RNAs during Postnatal Liver Maturation in Mice 
PLoS ONE  2014;9(12):e114917.
The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day −2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.
doi:10.1371/journal.pone.0114917
PMCID: PMC4263710  PMID: 25503252
2.  CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells 
Stem cells (Dayton, Ohio)  2013;31(9):2024-2030.
Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive “active” stem cells as well as HOPX positive “facultative” stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 cells (CD24−/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease.
doi:10.1002/stem.1391
PMCID: PMC3783577  PMID: 23553902
3.  Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay 
Gastroenterology  2013;145(2):383-95.e1-21.
BACKGROUND & AIMS
Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
METHODS
We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.
RESULTS
CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.
CONCLUSIONS
We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
doi:10.1053/j.gastro.2013.04.050
PMCID: PMC3781924  PMID: 23644405
Stemness; Differentiation; Single-Cell Sorting; Flow Cytometry Analysis
4.  Coexistence of Quiescent and Active Adult Stem Cells in Mammals 
Science (New York, N.Y.)  2010;327(5965):542-545.
Adult stem cells are crucial for physiological tissue renewal and regeneration after injury. Prevailing models assume the existence of a single quiescent population of stem cells residing in a specialized niche of a given tissue. Emerging evidence indicates that both quiescent (out of cell cycle and in a lower metabolic state) and active (in cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in several tissues, in separate yet adjoining locations. Here, we summarize these findings and propose that quiescent and active stem cell populations have separate but cooperative functional roles.
doi:10.1126/science.1180794
PMCID: PMC4105182  PMID: 20110496
5.  Maternal-imprinting at H19-Igf2 locus maintains adult hematopoietic stem cell quiescence 
Nature  2013;500(7462):345-349.
The epigenetic regulation of imprinted genes via monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development1. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells2, 3,4; however, a role for imprinting per se in adult stem cells remains elusive. Here we show up-regulation of growth-restricting imprinted genes, including within the H19-Igf2 locus5, in long-term hematopoietic stem cells (LT-HSCs) and their down-regulation upon HSC activation and proliferation. A differentially methylated region (DMR) upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele1. In addition, H19 also serves as a source of miR-675, which restricts Igf1r expression6. We demonstrated that conditional deletion of the maternal but not the paternal H19-DMR reduced adult HSC quiescence, a state required for long-term maintenance of HSCs, and compromised HSC function. Maternal-specific H19-DMR deletion resulted in activation of the Igf2-Igfr1 pathway as revealed by the translocation of phosphorylated Foxo3 (an inactive form) from nucleus to cytoplasm and the release of Foxo3-mediated cell-cycle arrest, thus leading to increased activation, proliferation, and eventual exhaustion of HSCs. Mechanistically, maternal-specific H19-DMR deletion led to Igf2 up-regulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partially rescued the H19-DMR deletion phenotype. Our work establishes a novel role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.
doi:10.1038/nature12303
PMCID: PMC3896866  PMID: 23863936
6.  Intestinal Subepithelial Myofibroblasts Support the Growth of Intestinal Epithelial Stem Cells 
PLoS ONE  2014;9(1):e84651.
Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.
doi:10.1371/journal.pone.0084651
PMCID: PMC3882257  PMID: 24400106
7.  The Wnt Antagonist Dkk1 Regulates Intestinal Epithelial Homeostasis and Wound Repair 
Gastroenterology  2011;141(1):259-268.e8.
BACKGROUND & AIMS
Dkk1 is a secreted antagonist of the Wnt/β-catenin signaling pathway. It is induced by inflammatory cytokines during colitis and exacerbates tissue damage by promoting apoptosis of epithelial cells. However, little is known about the physiologic role of Dkk1 in normal intestinal homeostasis and during wound repair following mucosal injury. We investigated whether inhibition of Dkk1 affects the morphology and function of the adult intestine.
METHODS
We used doubleridge mice (Dkk1d/d), which have reduced expression of Dkk1, and an inhibitory Dkk1 antibody to modulate Wnt/β-catenin signaling in the intestine. Intestinal inflammation was induced with dextran sulfate sodium (DSS), followed by a recovery period in which mice were given regular drinking water. Animals were killed before, during, or after DSS administration; epithelial homeostasis and the activity of major signaling pathways were investigated by morphometric analysis, bromo-2′-deoxyuridine incorporation, and immunostaining.
RESULTS
Reduced expression of Dkk1 increased proliferation of epithelial cells and lengthened crypts in the large intestine, which was associated with increased transcriptional activity of β-catenin. Crypt extension was particularly striking when Dkk1 was inhibited during acute colitis. Dkk1d/d mice recovered significantly faster from intestinal inflammation but exhibited crypt architectural irregularities and epithelial hyperproliferation compared with wild-type mice. Survival signaling pathways were concurrently up-regulated in Dkk1d/d mice, including the AKT/β-catenin, ERK/Elk-1, and c-Jun pathways.
CONCLUSIONS
Dkk1, an antagonist of Wnt/β-catenin signaling, regulates intestinal epithelial homeostasis under physiologic conditions and during inflammation. Depletion of Dkk1 induces a strong proliferative response that promotes wound repair after colitis.
doi:10.1053/j.gastro.2011.03.043
PMCID: PMC3551610  PMID: 21440550
IBD; Crohn’s Disease; Mucosa; Intestinal Cell Signaling
9.  CD133, Stem Cells, and Cancer Stem Cells: Myth or Reality? 
CD133, a member of the prominin family, is found in a variety of tissues with at least three variants. The function of CD133 is not well understood, but its expression is subject to changes in the microenvironment cues including bioenergetic stress. Knockout of CD133 does not affect renewal, but mammary gland branching. A point mutation of CD133 (R733C) leads to retinal disorder. CD133 is found in embryonic stem cells, normal tissue stem cells, stem cell niches, and circulating endothelial progenitors as well as cancer stem cells. Maintenance of stemness in cancer may be attributable to asymmetric cell division in association with a set of embryonic expression signatures in CD133+ tumor cells. CD133 could enrich cancer stem cells, which are associated with chemo- and radiation resistance phenotype. High CD133 is associated with poor survival in a variety of solid tumors, including lung, colon, prostate, etc. Monitoring CD133+ cells in peripheral blood, and targeting CD133 in cancer, may further predict and improve the clinical outcomes.
doi:10.1007/s11888-011-0106-1
PMCID: PMC3207123  PMID: 22131911
CD133; Cancer stem cells; Endothelial progenitors; Colorectal cancer
10.  Phosphoinositide 3-Kinase Signaling Mediates □-Catenin Activation in Intestinal Epithelial Stem and Progenitor Cells in Colitis 
Gastroenterology  2010;139(3):869-881.e9.
Background & Aims
Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin552). We investigated whether phosphoinositide 3- kinase (PI3K) is required for Akt-mediated activation of β-catenin during intestinal inflammation.
Methods
The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice. Acute inflammation was induced in these mice (I-pik3r1KO) and their intestines were analyzed by biochemical and histological methods. The effects of chemically blocking PI3K in colitic IL-10−/− mice were examined. Biopsy samples from patients were examined.
Results
Compared to wild type mice, I-pik3r1KO mice had reduced T-cell–mediated Akt and β-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K–Akt signaling increased nuclear total β-catenin and P-β-catenin552 levels and reduced phosphorylation of N-terminal β-catenin, which is associated with degradation. PI3K inhibition in IL-10−/− mice impaired colitis-induced epithelial Akt and β-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-β-catenin552 throughout expanded crypts and increased mRNA expression of β-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples.
Conclusions
PI3K–Akt signaling cooperates with Wnt to increase β-catenin signaling during inflammation. PI3K-induced and Akt-mediated β-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.
doi:10.1053/j.gastro.2010.05.037
PMCID: PMC2930080  PMID: 20580720
PI3K; pik3r1; intestinal stem cells; intestinal progenitor cells; ulcerative colitis; inflammatory bowel disease; colon cancer; β-catenin; T-cell activation
11.  Interferon-γ regulates intestinal epithelial homeostasis through converging β-catenin signaling pathways 
Immunity  2010;32(3):392-402.
SUMMARY
Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-γ (IFN-γ) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-β-catenin and Wingless-Int (Wnt)-β-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-γ resulted in activation of β-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-γ treatment, and reduced proliferation through depletion of the Wnt co-receptor LRP6. These effects were enhanced by tumor necrosis factor-α (TNF)-α, suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased β-catenin-T-cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-γ - deficient mice. Thus, we propose that IFN-γ regulates intestinal epithelial homeostasis by sequential regulation of converging β-catenin signaling pathways.
doi:10.1016/j.immuni.2010.03.001
PMCID: PMC2859189  PMID: 20303298
12.  5-ASA inhibits epithelial β-catenin activation in chronic ulcerative colitis 
Gastroenterology  2009;138(2):595.
Background & aims
5-aminosalicylic acid (5-ASA) is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) where it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that 5-ASA reduces β-catenin-associated progenitor cell activation, Akt-phosphorylated β-cateninSer552 (P-β-catenin), and colitis-induced dysplasia (CID).
Methods
Effects of 5-ASA on P-β-catenin staining and function were assessed by IHC and qRT-PCR in biopsies of CUC in mild or “refractory” severe mucosal inflammation. Effects of 5-ASA on epithelial proliferation, and activation of Akt and β-catenin were assessed in IL-10−/− colitis and CID by IHC and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon.
Results
Data from IL-10−/− and human colitis samples show 5-ASA reduced Akt activation and P-β-catenin levels in the mid and upper crypt. Reductions in P-β-catenin in CUC biopsies with severe inflammation suggested that 5-ASA reduced P-β-catenin levels in tissue refractory to 5-ASA’s anti-inflammatory effects. In IL-10−/− mice, 5-ASA reduced CID concordant with inhibition of crypt Akt and β-catenin signaling.
Conclusions
The results are consistent with the model that 5-ASA contributes to chemoprevention in CAC by reducing β-catenin signaling within intestinal progenitors.
doi:10.1053/j.gastro.2009.10.038
PMCID: PMC2819654  PMID: 19879273
13.  The stem cell niches in bone 
Journal of Clinical Investigation  2006;116(5):1195-1201.
The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for osteogenesis, and hematopoietic cells, responsible for hematopoiesis, are closely associated in the bone marrow, suggesting a reciprocal relationship between the two. It was recently discovered that a subset of osteoblasts functions as a key component of the HSC niche (namely, the osteoblastic niche), controlling HSC numbers. HSCs interact not only with osteoblasts but also with other stromal cells, including endothelial cells. Sinusoidal endothelial cells in bone marrow have been revealed as an alternative HSC niche called the vascular niche. In this Review we compare the architecture of these 2 HSC niches in bone marrow. We also highlight the function of osteoblasts in maintaining a quiescent HSC microenvironment and the likely role of the vascular niche in regulating stem cell proliferation, differentiation, and mobilization. In addition, we focus on studies of animal models and in vitro assays that have provided direct insights into the actions of these osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules. Many of the discoveries described herein may contribute to future clinical treatments for hematopoietic and bone-related disorders, including cancer.
doi:10.1172/JCI28568
PMCID: PMC1451221  PMID: 16670760
14.  Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation 
Molecular and Cellular Biology  2002;22(8):2830-2841.
Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.
doi:10.1128/MCB.22.8.2830-2841.2002
PMCID: PMC133705  PMID: 11909975

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