Ivan Bertoncello

Tissue Hyperplasia and Enhanced T-Cell Signalling via ZAP-70 in c-Cbl-Deficient Mice

ABSTRACT The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, theCaenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3ε cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.

Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial–mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colony-forming potential in vitro. We describe a population of EpCAMhi CD49fpos CD104pos CD24low epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAMneg Sca-1pos lung mesenchymal cells. We show that soluble fibroblast growth factor-10 and hepatocyte growth factor partially replace the requirement for mesenchymal support of epithelial colony formation, allowing clonal passaging and demonstration of their capacity for self-renewal. These data support a model in which the adult mouse lung contains a minor population of multipotent epithelial stem/progenitor cells with the capacity for self-renewal and whose descendants give rise to airway and alveolar epithelial cell lineages in vitro.

Endogenous Fibroblastic Progenitor Cells in the Adult Mouse Lung Are Highly Enriched in the Sca-1 Positive Cell Fraction†‡§

Originally identified as a marker specifying murine hematopoietic stem cells, the Sca‐1 antigen has since been shown to be differentially expressed by candidate stem cells in tissues including vascular endothelium, skeletal muscle, mammary gland, and prostate of adult mice. In the adult murine lung, Sca‐1 has previously been identified as a selectable marker for the isolation of candidate nonhematopoietic (CD45−), nonendothelial (CD31−) bronchioalveolar stem cells (BASC) located at the bronchioalveolar duct junction that coexpress surfactant protein C and the Clara cell specific protein. Our systematic analysis of CD45−CD31−Sca‐1+ cells in fetal, neonatal, and adult lung shows that very few of these cells are detectable prior to birth but expand exponentially postnatally coinciding with the transition from the saccular to the alveolar stage of lung development. Unlike candidate BASCs, the CD45−CD31−Sca‐1+CD34+ cell fraction we describe coexpresses immunophenotypic markers (Thy‐1 and platelet‐derived growth factor receptor α) that define lung fibroblastic rather than epithelial cells. The mesenchymal “signature” of the CD45−CD31−Sca‐1+CD34+ cell fraction is further confirmed by transcriptional profiling, by cell culture studies demonstrating enrichment for clonogenic lipofibroblastic and nonlipofibroblastic progenitors, and by immunohistochemical localization of Sca‐1 in perivascular cells of the lung parenchyma. Although the CD45−CD31−Sca‐1+CD34+ cell phenotype does define endogenous clonogenic progenitor cells in the adult murine lung, our data indicate that these progenitors are predominantly representative of mesenchymal cell lineages, and highlights the pressing need for the identification of alternative markers and robust functional assays for the identification and characterization of epithelial and fibroblastic stem and progenitor cell populations in the adult lung. STEM CELLS 2009;27:623–633

The relationship between bone, hemopoietic stem cells, and vasculature

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.

Hemopoietic Stem Cells with Higher Hemopoietic Potential Reside at the Bone Marrow Endosteum

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage−Sca+Kit+ [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8‐fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long‐term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays.

Functional Analysis of Two Distinct Bronchiolar Progenitors during Lung Injury and Repair

Air spaces of the mammalian lung are lined by a specialized epithelium that is maintained by endogenous progenitor cells. Within bronchioles, the abundance and distribution of progenitor cells that contribute to epithelial homeostasis change as a function of maintenance versus repair. It is unclear whether functionally distinct progenitor pools or a single progenitor cell type maintain the epithelium and how the behavior is regulated in normal or disease states. To address these questions, we applied fractionation methods for the enrichment of distal airway progenitors. We show that bronchiolar progenitor cells can be subdivided into two functionally distinct populations that differ in their susceptibility to injury and contribution to repair. The proliferative capacity of these progenitors is confirmed in a novel in vitro assay. We show that both populations give rise to colonies with a similar dependence on stromal cell interactions and regulation by TGF-β. These findings provide additional insights into mechanisms of epithelial remodeling in the setting of chronic lung disease and offer hope that pharmacologic interventions may be developed to mitigate tissue remodeling.

Identification of Human Embryonic Stem Cell Surface Markers by Combined Membrane-Polysome Translation State Array Analysis and Immunotranscriptional Profiling†‡§

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently, there are few well‐defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane‐polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2, H9, and MEL1) grown under feeder and feeder‐free conditions were sorted into subpopulations by fluorescence‐activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM‐2 and CD9. Colony‐forming assays confirmed that cells displaying high coimmunoreactivity to GCTM‐2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling, 820 genes were identified that were common to the GCTM‐2high/CD9high stem cell‐like subpopulation. Membrane‐polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets, 88 genes encode proteins that mark the pluripotent subpopulation, of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P‐Cadherin, with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation‐associated transcripts and survey of translated membrane‐bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. STEM CELLS 2009;27:2446–2456

c-Myb is critical for murine colon development.

The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.

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 CD166(neg) and CD166(pos) lung stromal cells have different proliferative and differentiative potential. CD166(neg) lung stromal cells exhibit high proliferative potential with the capacity to differentiate along the lipofibroblastic and myofibroblastic lineages, whereas CD166(pos) lung stromal cells have limited proliferative potential and are committed to the myofibroblastic lineage. Moreover, we show that CD166(pos) lung stromal cells do not share the same epithelial-supportive capacity as their CD166(neg) 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.

Colony Stimulating Factor-1 Dependence of Paneth Cell Development in the Mouse Small Intestine

BACKGROUND & AIMS Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development. METHODS CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1. RESULTS Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages. CONCLUSIONS These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohns disease and ulcerative colitis.