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AimsEndothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution.Methods and resultsWe carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature.ConclusionThis study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover from a range of cardiac insults. Over the past two decades, our understanding of the complexities of both the independent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has increased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularization and lymphangiogenesis, which in turn require epicardial, endocardial, and inflammatory cell signalling within an extracellular milieu that is optimized for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury. Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are re-activated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model organism to explore this concept. In this review, we summarize recent advances from the zebrafish cardiovascular research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.

Cardiac injury leads to the loss of cardiomyocytes which are rapidly replaced by proliferation of surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronised live imaging, RNA sequencing and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterise macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche triggering the expansion of the epicardium which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signalling. Identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

The human transcriptome comprises a complex network of coding and non-coding RNAs implicated in a myriad of biological functions. Non-coding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathological states, including in the cardiovascular system. This review defines the current knowledge gaps, unmet methodological needs, and describes the challenges in dissecting and understanding the role and regulation of the non-coding transcriptome in cardiovascular disease. These challenges include poor annotation of the non-coding genome, determination of the cellular distribution of transcripts, assessment of the role of RNA processing and identification of cell-type specific changes in cardiovascular physiology and disease. We highlight similarities and differences in the hurdles associated with the analysis of the non-coding and protein-coding transcriptomes. In addition, we discuss how the lack of consensus and absence of standardized methods affect reproducibility of data. These shortcomings should be defeated in order to make significant scientific progress and foster the development of clinically applicable non-coding RNA-based therapeutic strategies to lessen the burden of cardiovascular disease. ispartof: CARDIOVASCULAR RESEARCH vol:118 issue:4 pages:1004-1019 ispartof: location:England status: published

Aims Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide SPAAR in EC function. Methods and results Deep sequencing of human embryonic stem cell differentiation to ECs was combined with ENCODE RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as a lncRNA but reassigned as a protein coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC-enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation, however overexpression of the full length transcript did not, despite production of SPAAR. Further, overexpression of an ATG mutant of the full length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated a LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterise LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin binding protein, SYNE1. Conclusion The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961. Translational perspective Treatment of ischemic conditions remains a major cardiovascular health burden. Identification of genes and non-coding RNAs that regulate the function of the vascular endothelium is important to understand and evolve potential new strategies that might enhance vascular regeneration. Here, we describe and dissect the functional importance of a micropeptide-encoding RNA transcript in the vascular endothelium, and demonstrate that both the RNA and the peptide regulate endothelial biology. Modulation of this axis may be a novel approach to regulate angiogenesis.

Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.

Long non-coding RNAs (lncRNAs) have structural and functional roles in development and disease. We have previously shown that the LINC00961/SPAAR (small regulatory polypeptide of amino acid response) locus regulates endothelial cell function, and that both the lncRNA and micropeptide counter-regulate angiogenesis. To assess human cardiac cell SPAAR expression, we mined a publicly available scRNSeq dataset and confirmed LINC00961 locus expression and hypoxic response in a murine endothelial cell line. We investigated post-natal growth and development, basal cardiac function, the cardiac functional response, and tissue-specific response to myocardial infarction. To investigate the influence of the LINC00961/SPAAR locus on longitudinal growth, cardiac function, and response to myocardial infarction, we used a novel CRISPR/Cas9 locus knockout mouse line. Data mining suggested that SPAAR is predominantly expressed in human cardiac endothelial cells and fibroblasts, while murine LINC00961 expression is hypoxia-responsive in mouse endothelial cells. LINC00961–/– mice displayed a sex-specific delay in longitudinal growth and development, smaller left ventricular systolic and diastolic areas and volumes, and greater risk area following myocardial infarction compared with wildtype littermates. These data suggest the LINC00961/SPAAR locus contributes to cardiac endothelial cell and fibroblast function and hypoxic response, growth and development, and basal cardiovascular function in adulthood.

CD133 (prominin-1) was the first in a class of novel pentaspan membrane proteins to be identified in both humans and mice, and was originally classified as a marker of primitive haematopoietic and neural stem cells. Due to the highly restricted expression of CD133 family molecules on plasma membrane protrusions of epithelial and other cell types, in association with membrane cholesterol, a role in the organization of plasma membrane topology has also recently been assigned to this family. Studies have now confirmed the utility of CD133 as a marker of haematopoietic stem cells for human allogeneic transplantation. In addition, CD133 represents a marker of tumour-initiating cells in a number of human cancers, and therefore it may be possible to develop future therapies towards targeting cancer stem cells via this marker. The development of such therapies will be aided by a clearer understanding of the molecular mechanisms and signalling pathways that regulate the behaviour of CD133-expressing cells, and new data outlining the role of Wnt, Notch, and bone morphogenetic protein (BMP) signalling in CD133(+) cancer stem cell regulation are discussed within.

The authors have previously reported the derivation of colonic subepithelial myofibroblasts (SEMFs) in both humans and mice from bone marrow (BM). In the pathogenesis of inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, colonic SEMFs mediate several types of inflammatory response. In the present study, interleukin (IL)-10−/− mice were used as a model of IBD to investigate the involvement of BM-derived cells in the inflamed mucosa. Male whole BM [either C57/BL10 (wild type: WT) or IL-10−/− donor mice] was used to perform bone marrow transplantation (BMT) into both WT and IL-10−/− female mice. Tissue samples were evaluated by immunohistochemistry for α-smooth muscle actin expression and by in situ hybridization using a Y-chromosome-specific probe to track the donor-derived colonic SEMFs. The mucosal expression of mRNA for pro-inflammatory cytokines was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, mRNA expression of matrix metalloproteinase (MMP)-7 and osteopontin in the inflamed mucosa was assessed using in situ hybridization. Body weights and histological scores showed that IL-10−/− mice that received WT BM had an improved course of colitis, decreased mucosal pro-inflammatory mRNA expression, and up to 30% of their SEMFs were of BM origin. Conversely, IL-10−/− mice receiving IL-10−/− BM progressed to extensive colitis, and Y probe analysis revealed that up to 45% of colonic SEMFs were of BM origin. WT mice receiving IL-10−/− or WT BM had no signs of colonic inflammation. The expression of MMP-7 and osteopontin was up-regulated in the inflamed mucosa. In conclusion, IL-10−/− mice displayed ameliorated disease activity after WT BMT, whilst colitis was not induced in WT mice by IL-10−/− BMT. The contribution of BM-derived cells to colonic SEMFs was significantly increased in the inflamed mucosa compared with non-inflamed mucosa. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stem cells are primitive cells located in a specialised mesenchymal “niche” that lack expression of any definitive markers of lineage commitment and are therefore difficult to define and identify. Stem cells maintain their capacity for limitless self replication throughout the lifetime of their host, and can also divide to produce daughter cells, committed to the formation of every adult cell lineage within their tissue of origin. The stem cells of the gastrointestinal tract remain unidentified which has led to many conflicting hypotheses as to their precise nature and function. For example, the numbers and location of stem cells in the intestinal crypts and gastric glands have never been conclusively proven and, consequently, the clonal origins of these structures under normal circumstances and in neoplasia are clouded issues. The morphological events of gastrointestinal carcinoma formation are hotly debated, with two main conflicting hypotheses of the mechanisms of expansion of a mutated stem cell clone. However, with the emergence of the molecular pathways governing gastrointestinal stem cell function, and the identification of putative intestinal molecular stem cell markers, such as Musashi-1, comes a clearer insight into the properties of the gastrointestinal stem cell. Adult stem cells from several tissues can leave their niche and engraft into extraneous tissues, including the gastrointestinal mucosa and underlying mesenchyme, and transform to produce adult cell lineages common to these foreign environments. This process is optimal when the requirement for regeneration is enhanced (that is, in diseased or damaged tissue) and indeed, the contribution of transplanted bone marrow stem cells to intestinal myofibroblasts is significantly upregulated in colitis. However, adult stem cell plasticity has recently been disparaged by reports suggesting that stem cells spontaneously fuse with indigenous adult cells to form a diploid cell with an aberrant karyotype, and it is important to investigate if bone marrow …

Myofibroblasts are ubiquitous cells with features of both fibroblasts and smooth muscle cells. We suggest that the bone marrow can contribute to myofibroblast populations in a variety of tissues and that this is exacerbated by injury. To assess this, female mice were transplanted with male bone marrow and the male cells were tracked throughout the body and identified as myofibroblasts. Skin wounding and paracetamol administration were used to assess whether myofibroblast engraftment was modulated by damage. Following radiation injury, a proportion of myofibroblasts in the lung, stomach, esophagus, skin, kidney, and adrenal capsule were bone-marrow derived. In the lung, there was significantly greater engraftment following paracetamol administration (17% versus 41% p < 0.005). Bone-marrow-derived fibroblasts were also found. We suggest that bone marrow contributes to a circulating population of cells and, in the context of injury, these cells are recruited and contribute to tissue repair.

Turnover of the epithelial cell lineages within the gastrointestinal tract is a constant process, occurring every two to seven days under normal homeostasis and at increased rates after damage. This process is regulated by multipotent stem cells, which generate all gastrointestinal epithelial cell lineages and can regenerate whole intestinal crypts and gastric glands. The stem cells of the gastrointestinal tract are as yet undefined, although it is generally agreed that they are located within a ’niche’ in the intestinal crypts and gastric glands. Studies of allophenic, tetraparental chimeric mice and targeted stem cell mutations suggest that a single stem cell undergoes an asymmetrical division to produce an identical daughter cell, thus replicating itself, and a committed progenitor cell, which further differentiates into an adult epithelial cell type. The discovery of stem cell plasticity in many tissues, including the ability of transplanted bone marrow to transdifferentiate into intestinal subepithelial myofibroblasts, provides a potential use of bone marrow cells to deliver therapeutic genes to damaged tissues, for example, in the treatment of mesenchymal diseases in the gastrointestinal tract, such as fibrosis and Crohn’s disease. Studies are beginning to identify the molecular pathways that regulate stem cell proliferation and differentiation into adult gastrointestinal cell lineages, such as the Wnt and Notch–Delta signaling pathways, and to discover the importance of mesenchymal–epithelial interactions in normal gastrointestinal epithelium and in development and disease. Finally, despite some dispute, a strong case can be made for intestinal neoplasia arising as a result of a series of mutations in stem cells. The mechanism and direction of the spread of this mutated clone in the gastrointestinal mucosa is hotly disputed, and central to this argument is the position and nature of the gastrointestinal stem cell.

Expression of the cell adhesion molecule E-cadherin in the developing mouse has been investigated by immunocytochemistry and disaggregated organ culture. The principal aims were firstly, to determine whether E-cadherin is expressed in the indifferent gonad and if so with which cell population(s) it is associated. Secondly, to investigate the pattern of expression in the mesonephros, especially in relation to ventral mesonephric tubule cells, which contribute to the somatic cell population of the gonad. Thirdly, to discover whether there are any sex differences in expression of E-cadherin in differentiating gonads. Germ cells in the indifferent gonad showed strong immunoreactivity whereas somatic cells were unstained unless their membranes were in contact with those of germ cells. Positive staining for E-cadherin was found in epithelial cells of the mesonephric duct and tubules. Staining was weak at the ventral margins of the ventral mesonephric tubules. At later stages, germ cell immunoreactivity could be correlated with stages of ovarian differentiation, being reduced or absent between germ cells at 16 days post coitum, when ovigerous cords become dissociated as a prelude to follicle formation. Stronger staining reappeared briefly at 17 days post coitum, the time of follicular cell attachment to oocytes, before waning again 2 days later. Similarly, immunoreactivity in the differentiating testis was initially restricted to the germ cell population but pre-Sertoli cells were strongly positive between 16 and 19 days post coitum. The most striking sex difference was seen in the somatic cell population, where Leydig cells of the testis became strongly positive for E-cadherin from 17 days post coitum onwards. At this time, unlike controls, dissociated cells from gonads of either sex were unable to reform their initial contacts when cultured in the presence of the antibody to E-cadherin, confirming its functional importance.

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte-colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real-time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow-derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow-derived circulating precursor for endothelial cells.

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kδ. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

Publisher Summary The cells of the gastrointestinal tract undergo constant renewal and respond to damages by regeneration and repopulation. Although the stem cells are the most important cells of the gastrointestinal tract, responsible for the production of every other cell type in the gastrointestinal mucosa, they have not yet been closely characterized. This chapter reviews evidence that the multipotential stem cells generate all gastrointestinal epithelial cell lineages through committed precursor cells housed in the proliferative compartments of intestinal crypts and gastric glands. The gastrointestinal stem cells remain elusive and unidentified, mainly because of a lack of accepted morphological and functional markers at the single-cell level. Concepts of stem cell number, location, and fate, and the ability of gastrointestinal stem cells to regenerate cell lineages of whole intestinal crypts and villi after damage is also explored. There is a single stem cell in every intestinal crypt or gastric gland that indirectly generates a clone containing further stem cells, transit amplifying, and differentiated cells, through the production of committed progenitor cells. This cell also produces new crypts by crypt fission, repairs entire crypts and villi when damaged, and generates gastrointestinal tumors. The stem cell or cells occupy a niche, formed by mesenchymal cells, such as the intestinal subepithelial myofibroblasts, and extracellular matrix molecules, which regulate epithelial stem cells through mesenchymalepithelial crosstalk. Finally, it is concluded that the colonic stem cell is pivotal in understanding mechanisms of tumorigenesis in the colon. The isolation and characterization of gastrointestinal stem cells are a priority in gut biology.

Complex biomolecules absorb in the mid-infrared (λ = 2–20 μm), giving vibrational spectra associated with structure and function. We used Fourier transform infrared (FTIR) microspectroscopy to “fingerprint” locations along the length of human small and large intestinal crypts. Paraffin-embedded slices of normal human gut were sectioned (10 μm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 μm × 15 μm aperture) FTIR microspectroscopy in reflection mode, synchrotron (≤10 μm × 10 μm aperture) FTIR microspectroscopy in transmission mode or near-field photothermal microspectroscopy. Dependent on the location of crypt interrogation, clear differences in spectral characteristics were noted. Epithelial-cell IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in “hyperspace” might on the basis of multivariate distance reveal biophysical differences along the length of gut crypts. Following spectroscopic analysis, plotted clusters and their loadings plots pointed toward symmetric (νs)PO2− (1,080 cm−1) vibrations as a discriminating factor for the putative stem cell region; this proved to be a more robust marker than other phenotypic markers, such as β-catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of nondestructively identifying a tissue's stem cell location. νsPO2−, probably associated with DNA conformational alterations, might facilitate a means of identifying stem cells, which may have utility in other tissues where the location of stem cells is unclear. Disclosure of potential conflicts of interest is found at the end of this article.

Inflammatory bowel disease (IBD) is characterized by an ongoing mucosal inflammation caused by a dysfunctional host immune response to commensal microbiota and dietary factors. In the pathophysiology of IBD, mesenchymal cells such as intestinal subepithelial myofibroblasts (ISEMF) affect the recruitment, retention and activation of immune cells. Mesenchymal cells also promote resolution of inflammatory activity accompanied with balanced repair processes. The transient appearance of mesenchymal cells is a feature of normal wound healing, but the persistence of these cells is associated with tissue fibrosis. Recent studies suggest that mesenchymal cells derived from bone marrow (BM) stem cells play a crucial role in intestinal repair and fibrosis. This article focuses on recent knowledge about ISEMF in the field of immune response inflammation and repair. Two major topics were documented: interaction between interleukin (IL)-17-secreting CD4+ cells (Th-17 cells) and about role of BM-derived stem cells in mucosal regenerative response via differentiation to ISEMF. Recent therapeutic strategies targeting BM stem cells for IBD patients were also documented.

In the twenty-first century, diabetic patients are likely to be one of the major beneficiaries from the advancement of regenerative medicine through cellular therapies. Though the existence of a specific self-renewing stem cell within the pancreas is still far from clear, a surprising variety of cells within the pancreas can differentiate towards a beta-cell phenotype: ductular cells, periductular mesenchymal cells and beta-cells themselves can all give rise to new beta-cells. Extra-pancreatic adult somatic stem cells, in particular, those originating from bone marrow may also be capable of differentiating to beta-cells, though equally well the beneficial effects of bone marrow cells may reside in their contribution to the damaged islet vasculature. Forced expression of the beta-cell-specific transcription factor Pdx1 in hepatocytes also holds promise as a therapeutic strategy to increase insulin levels in diabetic individuals. Embryonic stem (ES) cells are clearly another possible source for generating beta-cells, but ES cells are beyond the scope of this review, which focuses on adult stem and progenitor cells capable of producing beta-cells. Despite considerable endeavour, we still have much to learn in the field of pancreatic regeneration prior to any clinically applicable therapy based upon adult stem cells.

The expectation generated by the pluripotentiality of embryonic stem (ES) cells has initiated a renaissance in stem cell biology. While ES cells can be harvested in abundance and appear to be the most versatile of cells for regenerative medicine, adult stem cells also hold promise, but the identity and subsequent isolation of these comparatively rare cells remains problematic in most tissues, perhaps with the notable exception of the bone marrow. Identifying surface molecules (markers) that would aid in stem cell isolation is thus a major goal for stem cell biologists. Moreover, the characterization of normal stem cells in specific tissues may provide a dividend for the treatment of cancer. There is a growing belief that the successful treatment of neoplastic disease will require specific targeting of the cancer stem cells, cells that may well have many of the characteristics of their normal counterparts.

Background & Aims: Bone marrow (BM) cells form differentiated adult lineages within nonhematopoietic tissues, with a heightened propensity with increasing regenerative pressure dictated by disease. We have previously shown that BM cells engraft into the gut and contribute substantially to the subepithelial intestinal myofibroblast population in the lamina propria. To investigate the reparative capacity of BM in inflammatory bowel disease (IBD), a well-established model of experimental colitis was used. Methods: Lethally irradiated female mice were rescued by a BM transplant from male donors. Colitis was induced 6 weeks posttransplantation by injection of trinitrobenzene sulfonic acid (TNBS), and tissues were analyzed 1–14 days later. Donor-derived cells were detected by in situ hybridization using a Y chromosome-specific probe, and their phenotype was determined by immunohistochemistry. Results: TNBS-induced colitis was manifest as patchy lesions that increased in severity between days 1 and 8, and the mucosa gradually regenerated between days 8 and 14. The contribution of BM to intestinal myofibroblasts was significantly increased in regions of colitis compared with noninflamed regions. Furthermore, BM-derived endothelial cells, pericytes, and vascular smooth muscle cells were frequently interspersed throughout blood vessels, suggesting that these cells facilitate angiogenesis in tissue repair, substantiated by a significant increase in the incidence of BM-derived vascular smooth muscle cells in colitic compared with noninflamed regions. Blood vessels formed entirely from BM-derived cells were also seen, suggesting a role for BM in neovasculogenesis. Conclusions: Our data show that BM contributes to multiple intestinal cell lineages in colitis, with an important function in tissue regeneration and vasculogenesis after injury.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.

The longevity of adult stem cells, and their potential for vast tissue regeneration, makes them a focal point of current research and debate, with future aspirations for the use of stem cells in the treatment of a number of human pathological conditions. Due to the rapid rate of cell turnover in the gastrointestinal tract, the stem cells of this tissue are amongst the most assiduous in the body, although they remain unidentified to this day due to their immature, undifferentiated phenotype. However, our knowledge of the mechanisms regulating gastrointestinal stem cell function is evolving, with the identification of putative cellular markers and the elucidation of signalling pathways which regulate cell behaviour in the normal and neoplastic gastrointestinal tract. This review describes the fundamental properties of the gastrointestinal stem cell including: (i) their number, location and origins, (ii) their primary function of deriving gastrointestinal cell lineages and maintaining tissue homeostasis, (iii) the acquisition of gastrointestinal cell lineages from adult stem cells of extraneous tissues and the consequences of this in a therapeutic context, and (iv) the genetic and morphological phenomena surrounding neoplastic transformation in the gastrointestinal tract.

A large body of evidence supports the idea that certain adult stem cells, particularly those of bone marrow origin, can engraft at alternative locations, particularly when the recipient organ is damaged. Under strong and positive selection pressure these cells will clonally expand/differentiate, making an important contribution to tissue replacement. Similarly, bone marrow derived cells can be amplified in vitro and differentiated into many types of tissue. Despite seemingly irrefutable evidence for stem cell plasticity, a veritable chorus of detractors has emerged, some doubting its very existence, motivated perhaps by more than a little self interest. The issues that have led to this situation include the inability to reproduce certain quite startling observations, and extrapolation from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. Although these issues need resolving and, accepting that cell fusion does appear to allow reprogramming of haemopoietic cells in special circumstances, criticising this whole new field because some areas remain unclear is not good science.

Notwithstanding the fact that adult bone marrow cell engraftment to epithelial organs seems a somewhat uncommon event, there is no doubt it does occur, and under appropriate conditions of a strong and positive selection pressure these cells will expand clonally and make a significant contribution to tissue replacement. Likewise, bone-marrow-derived cells can be amplified in vitro and differentiated into a multitude of tissues. These in essence are the goals of regenerative medicine using any source of stem cells, be it embryonic or adult. Despite such irrefutable evidence of what is possible, a veritable chorus of detractors of adult stem cell plasticity has emerged, some doubting its very existence, motivated perhaps by more than a little self-interest. The issues that have led to this state of affairs have included the inability to reproduce certain widely quoted data, one case where the apparent transdifferentiation was due to contamination of the donor tissue with haematopoietic cells and, most notoriously, extrapolating from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. While these issues need resolving,slamming this whole new field because not everything is crystal clear is not good science. The fact that a phenomenon is quite rare in no way mitigates against its very existence: asteroid collisions with the Earth are rare, but try telling the dinosaurs they do not occur! When such events do occur(transdifferentiation or collision), they certainly can make an impact.

Turnover of the epithelial cell lineages within the gastrointestinal tract is a constant process, occurring every 2-7 days under normal homeostasis and increasing after damage. This process is regulated by multipotent stem cells, which give rise to all gastrointestinal epithelial cell lineages and can regenerate whole intestinal crypts and gastric glands. The stem cells of the gastrointestinal tract are as yet undefined, although it is generally agreed that they are located within a 'niche' in the intestinal crypts and gastric glands. Studies of allophenic tetraparental chimeric mice and targeted stem cell mutations suggest that a single stem cell undergoes asymmetrical division to produce an identical daughter cell, and thus replicate itself, and a committed progenitor cell which further differentiates into an adult epithelial cell type. The discovery of stem cell plasticity in many tissues, including the ability of transplanted bone marrow to transdifferentiate into intestinal subepithelial myofibroblasts, provides a potential use of bone marrow cells to deliver therapeutic genes to damaged tissues, for example, in treatment of mesenchymal diseases in the gastrointestinal tract, such as fibrosis and Crohn's disease. Studies are beginning to identify the molecular pathways that regulate stem cell proliferation and differentiation into adult gastrointestinal cell lineages, such as the Wnt and Notch/Delta signalling pathways, and the importance of mesenchymal-epithelial interactions in normal gastrointestinal epithelium and in development and disease.

Background and aims: In order to establish whether extraintestinal cells contribute to the turnover and repair of gastrointestinal tissues, we studied the colons and small intestines of female mice that had received a male bone marrow transplant, together with gastrointestinal biopsies from female patients that had developed graft versus host disease after receiving a bone marrow transplant from male donors. Methods: Using in situ hybridisation to detect Y chromosomes and immunohistochemistry, we demonstrated that cells derived from injected bone marrow frequently engrafted into the intestine and differentiated into pericryptal myofibroblasts. Results: In the human intestine, we confirmed by combining in situ hybridisation with immunostaining for smooth muscle actin that the bone marrow derived cells within the intestine exhibited a myofibroblast phenotype. In female mouse recipients of male bone marrow grafts, we observed colocalisation of Y chromosomes and clusters of newly formed marrow derived myofibroblasts. While few of these were present at seven days after bone marrow transplantation, they were numerous at 14 days, and by six weeks entire columns of pericryptal myofibroblasts could be seen running up the sides of crypts in both the small intestine and colon. These columns appeared to extend into the villi in the small intestine. Within the intestinal lamina propria, these Y chromosome positive cells were negative for the mouse macrophage marker F4/80 antigen and CD34. Conclusions: Bone marrow derived pericryptal myofibroblasts were present in the mouse intestine following irradiation and bone marrow transplant, and in the intestines of human patients suffering graft versus host disease following a bone marrow transplant. Our data indicate that bone marrow cells contribute to the regeneration of intestinal myofibroblasts and epithelium after damage, and we suggest that this could be exploited therapeutically.

Haemophilia B arises from mutations in the factor IX gene. Its treatment in humans, by recombinant protein substitution, is expensive thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of this disease without development of immune reactions by introduction of HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent haemophiliac and normal outbred mice. Plasma factor IX persists at therapeutic concentrations in all treated mice to date (ten months). Functional normalisation of the blood clotting has been achieved in two haemophiliac mice and significant correction in a third. In comparison, administration of an adenoviral vector encoding human factor IX resulted in transient and diminishing factor IX expression which was nearly undetectable by 8 months. Following lentiviral administration, no anti-factor IX antibodies were detected by ELISA and no cellular immunity was detected by immunohistochemical staining of macrophages, neutrophils, CD4-positive or CD8-positive cells. There was no elevation of serum liver enzymes or evidence of vector spread to the maternal circulation. No vector spread to sperm was detected by TaqMan analysis. This is the first demonstration of complete phenotypic correction of a severe genetic disease by in utero gene therapy with no evidence of toxicity.

The intestinal tract has a rapid epithelial cell turnover, which continues throughout life. The process is regulated and maintained by a population of stem cells, which give rise to all the intestinal epithelial cell lineages. Studies in both the mouse and the human show that these cells are capable of forming clonal crypt populations. Stem cells remain hard to identify, however it is thought that they reside in a 'niche' towards the base of the crypt and their activity is regulated by the paracrine secretion of growth factors and cytokines from surrounding mesenchymal cells. Stem cell division is usually asymmetric with the formation of an identical daughter stem cell and committed progenitor cells. Progenitor cells retain the ability to divide until they terminally differentiate. Occasional symmetric division produces either 2 daughter cells with stem cell loss, or 2 stem cells and eventual clone dominance. This stochastic extinction of stem cell lines with eventual dominance of one cell line is called 'niche succession'. The discovery of plasticity, the ability of stem cells to engraft into, and in some cases replace the function of damaged host tissues has generated a large amount of scientific and clinical interest: however the concept remains controversial and is still a subject of hot debate. Studies are beginning to identify the complex molecular, genetic and cellular pathways underlying stem cell function such as Wnt signalling, bone morphogenetic protein (BMP) and Notch/Delta pathways. The derangement of these pathways within stem cells plays an integral part in the development of malignancy within the intestinal tract.