Clonal populations of lineage-uncommitted pluripotent mesenchymal stem cells have been identified in prenatal avians and rodents. These cells reside in the connective tissue matrices of many organs and tissues. They demonstrate extended capabilities for selfrenewal and the ability to differentiate into multiple separate tissues within the mesodermal germ line. This study was designed to determine whether such cells are present in the connective tissues of postnatal mammals. This report describes a cell clone derived by isolation from postnatal rat connective tissues, cryopreservation, extended propagation, and serial dilution clonogenic analysis. In the undifferentiated state, this clone demonstrates a high nuclear-to-cytoplasmic ratio and extended capacity for self-renewal. Subsequent morphological, histochemical, and immunochemical analysis after the induction of differentiation revealed phenotypic markers characteristic of multiple cell types of mesodermal origin, such as skeletal muscle, smooth muscle, fat cells, cartilage, and bone. These results indicate that this clone consists of pluripotent mesenchymal stem cells. This report demonstrates that clonal populations of reserve stem cells are present in mammals after birth. Potential roles for such cells in the maintenance, repair, and regeneration of mesodermal tissues are discussed.

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Intestinal epithelial cells (IEC) comprise diverse lineages that serve distinct roles necessary for regulation of nutrient absorption, regeneration, immunity, and homeostasis 1,2 . Goblet cells secrete Trefoil factor 3 (TFF3) to maintain mucus viscosity and drive mucosal healing by inhibiting cell death and influencing tight junction protein expression 3 . However, whether TFF3 signaling relies upon conventional ligand-receptor interactions has been unclear for decades. This study demonstrates that the orphan transmembrane protein leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) immunoprecipitates with TFF3, that LINGO2 and TFF3 co-localize at the IEC cell surface, and that TFF3/LINGO2 interactions block IEC apoptosis. Loss of function studies show that TFF3-driven STAT3 and EGFR activation are both LINGO2 dependent. Importantly, we demonstrate that TFF3 disrupts LINGO2/EGFR interactions that normally restrict EGFR activity, resulting in enhanced EGFR signaling. Excessive EGFR activation in Lingo2 gene deficient mice exacerbates colitic disease and accelerates host resistance to parasitic nematodes, whereas TFF3 deficiency results in host susceptibility. Thus, our data demonstrating that TFF3 functions through a previously unrecognized ligandreceptor interaction with LINGO2 to de-repress LINGO2-dependent inhibition of EGFR activation provides a novel conceptual framework explaining how TFF3-mediates mucosal wound healing through enhanced activation of the EGFR pathway.3 Although Trefoil factor 3 (TFF3) is well known to drive reparative pathways at respiratory, ocular, genitourinary and gastrointestinal mucosa, the identity of a potential TFF3 receptor has remained elusive for several decades 4-8 . In addition to enhancing mucous viscosity 9 , TFF3 induces epithelial cell survival and proliferation, activates EGFR, β-catenin, and STAT3 signaling pathways, and controls tight junction protein expression through ill-defined mechanisms to protect against gastrointestinal (GI) tissue injury and inflammation 3,10 . Since the anti-inflammatory activity of TFF3 suggested that leukocytes may be directly responsive to TFF3, we utilized a human macrophage/monocyte cell line (U937) to first ask whether cytokine release could be regulated in response to rTFF3 exposure in order to isolate the TFF3 receptor. We found that rhTFF3 caused a dose-dependent reduction in endotoxin-mediated TNF release over a range of 1-100 ng/ml and that TFF3 suppression was lost at a higher range of rhTFF3 (100-1000 ng/ml) ( Figure S1A). TFF3-induced interleukin 10 (IL-10) production was found over several orders of magnitude, but was lost at higher doses of rhTFF3 ( Figure 1A). As the dose response is similar to the established range of activity for most cytokine and growth factor receptors 11 , these observations suggested that TFF3 was interacting with a receptor in a conventional manner.TFF3 was likely to interact with its receptor through low-affinity interactions because glycosylation has been shown critical for biological...