Search

Using phage display to generate synthetic therapeutic antibodies targeting JAG1 or JAG2, we identified a selective and potent inhibitor of each ligand that also cross-reacted with the human and mouse [...], Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed (1). Mucociliary clearance (2,3) in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor (4-8). In contrast to such situations in which cell fate decisions are made in rapidly dividing populations (9,10), cells of the homeostatic adult airway epithelium are long-lived (11-13), and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation (14). Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation (15). Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Melengestrol acetate (MGA) has been used in the United States for nearly 40 yr to enhance feedlot heifer performance, yet unequivocal studies have not been conducted to discover the mechanism of action. Our hypothesis was that MGA may induce various populations of muscle-derived cells (MDC) to the adipogenic pathway in both a bovine and murine cell culture model. To determine this, MDC were digested from the semimembranosus muscle tissue of six 14-moold crossbred steers. The addition of insulin, oleic acid, and ciglitizone (IOC) with cultured bovine MDC resulted in morphological differences compared with control cultures. Multilocular lipid droplets stained with Oil Red O were seen not only in single MDC, but also in fused myotubes. An increase (P < 0.05) in relative PPAR[gamma] messenger RNA (mRNA) levels was measured in MDC incubated with IOC. However, rayogenin mRNA levels in MDC incubated with IOC were repressed (P < 0.05) compared with nontreated MDC. Cultures of MDC treated with 10 [micro]M insulin, 10 [micro]M oleic acid, 10 [micro]M ciglitizone, 10 nM estradiol-17[beta] ([E.sub.2]), and 10 nM MGA resulted in cultures with highly dis tributed lipid droplets not only in single cells, but also in the multinucleated myotubes. Relative C/EBP[beta] and PPAR[gamma] mRNA levels in total RNA isolated from MDC treated with MGA increased (P < 0.05) compared with control cultures. Estradiol treatment had no effect (P > 0.05) on these mRNA levels. The addition of both [E.sub.2] and MGA to MDC increased (P < 0.05) C/EBP[beta] mRNA levels and tended (P = 0.06) to increase the PPAR[gamma] mRNA level. There was no difference (P > 0.10) in relative myogenin mRNA among the control, [E.sub.2], and MGA treatments. Relative C/EBP[beta], PPAR[gamma] and myogenin mRNA levels were investigated in murine C2C12, C3H 10T 1/2, and 3T3-L1 cells. Treatment of cultures with 10 nM MGA increased C/EBP[beta] levels (P < 0.05) in C2C12 myoblasts and tended (P = 0.08) to increase C/EBP[beta] levels in 3T3-L1 preadipocytes. These data indicate that populations of cells are present in postnatal skeletal muscle that, under the appropriate stimuli in a culture model, express adipogenic genes and accumulate lipids. In addition, the synthetic progestogen MGA appeared to upregulate the genes necessary for conversion to the adipogenic pathway. Key words: melengestrol acetate, muscle-derived cell, transdifferentiation doi: 10.2527/jas.2008-1645

The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction. myocardial infarction | myocardial regeneration | stem cells I transdifferentiation

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific [beta]-galactosidase ([beta]-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no [beta]-Gal activity. [beta]-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4[alpha], aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, [alpha]-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease. stern cells; cell culture; transgenic mice; transdifferentiation; biliary epithelium

Free fatty acids (FFA) are important extracellular and intracellular signaling molecules and are thought to be involved in [beta]-adrenergic-induced remodeling of adipose tissue, which involves a transient inflammatory response followed by mitochondrial biogenesis and increased oxidative capacity. This work examined the role of hormone-sensitive lipase (HSL), a key enzyme of acylglycerol metabolism, in white adipose tissue (WAT) remodeling using genetic inactivation or pharmacological inhibition. Acute treatment with the [[beta].sub.3]-adrenergic agonist CL-316,243 (CL) induced expression of inflammatory markers and caused extravasation of myeloid cells in WAT of wild-type (WT) mice. HSL-knockout (KO) mice had elevated inflammatory gene expression in the absence of stimulation, and acute injection of CL did not further recruit myeloid cells, nor did it further elevate inflammatory gene expression. Acute pharmacological inhibition of HSL with BAY 59-9435 (BAY) had no effect on inflammatory gene expression in WAT or in cultured 3T3-L1 adipocytes. However, BAY prevented induction of inflammatory cytokines by [beta]-adrenergic stimulation in WAT in vivo and in cultured 3T3-L1 adipocytes. Chronic CL treatment stimulated mitochondrial biogenesis, expanded oxidative capacity, and increased lipid droplet fragmentation in WT mice, and these effects were significantly impaired in HSL-KO mice. In contrast to HSL-KO mice, mice with defective signaling of Toll-like receptor 4, a putative FFA receptor, showed normal [beta]-adrenergic-induced remodeling of adipose tissue. Overall, results reveal the importance of HSL activity in WAT metabolic plasticity and inflammation. transdifferentiation; free fatty acids; Toll-like receptor 4

Inflammatory injury to the pancreas results in regeneration of normal tissue and formation of metaplastic lesions of a ductal phenotype. These metaplastic ductal lesions (MDL) are called tubular complexes (TC), mucinous metaplasia, or pancreatic intraepithelial neoplasia. Because they are regularly found in chronic pancreatitis and pancreatic cancer, their formation is thought to represent a step in inflammation-mediated carcinogenesis. Despite these lesions' ductal character, their origin is controversial. All known pancreatic cell lineages have been suggested as the origin. In vitro studies suggest that differentiated cells in the pancreas remain highly plastic and can transdifferentiate as a mechanism of regeneration and metaplasia. In vivo studies suggest that islets, specifically [beta] cells, may be the cell of origin. However, in vitro studies are subject to ductal cell contamination, and previous in vivo studies interpret static data rather than direct evidence. Using genetic lineage tracing in vivo, we investigate whether transdifferentiation of [beta] cells contributes to regeneration or metaplasia in pancreatitis. RIP-CreER;Z/AP mice were used to heritably tag [beta] cells in the adult pancreas. Injury by cerulein pancreatitis resulted in regeneration of normal tissue and metaplasia with formation of two distinct types of TC and mucinous lesions. Lineage tracing revealed that none of these MDL are of [beta] cell origin; nor do [beta] cells contribute to regeneration of normal acinar and ductal tissue, which indicates that the plasticity of differentiated pancreatic islet cells, suggested by earlier static and in vitro studies, plays no role in regeneration, metaplasia, and carcinogenesis in vivo. metaplasia | pancreatitis | pancreatic intraepithelial neoplasia

Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-[[beta].sup.1] promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-[[beta].sup.1] stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-[[beta].sup.1] with or without preincubation with ZVAD-FMK (pancaspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-[[beta].sup.1] caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-[[beta].sup.1] treatment. EGF inhibited apoptosis and facilitated TGF-[[beta].sup.1] induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-[[beta].sup.1]-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-[[beta].sup.1] induces EMT independently of its proapoptotic effects. TGF-[[beta].sup.1] and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type. proliferation: fibrosis; transdifferentiation; tubular epithelium

Differentiated cells of the corneal epithelium are converted to hair, along with their associated stem cells, then interfollicular epidermis, by means of a multistep process triggered by dermal developmental signals. The committed basal cells of the adult corneal epithelium dedifferentiate under the control of signals from an associated embryonic hair-forming dermis, likely Wnts, and revert to a limbal basal cell phenotype. This initial process involves the down-regulation of Pax6 and the loss of expression of corneal-specific keratins and the induction of basal keratinocyte markers. These dedifferentiated cells are able to reinduce dermal condensations, which in turn induce the formation of hair follicles from cells that have lost Pax6 expression, by means of a Noggin-dependent mechanism, An epidermis is subsequently formed by cells derived from the newly segregated hair stem cells. cornea | dermal-epidermal interactions | Wnt | [beta]-catenin | Noggin

The molecular mechanism of retinal ganglion cell (RGC) genesis and development is not well understood. Published data suggest that the process may involve two bHLH genes, ath5 and NSCL1. Gain-of-function studies show that ath5 increases RGC production in the developing retina. We examined whether two chick genes, cath5 and cNSCL1, can guide retinal pigment epithelial (RPE) cells to transdifferentiate toward RGCs. Ectopic expression of cath5 and cNSCL1 in cultured chick RPE cells was achieved through retroviral transduction, cath5 alone was unable to induce de novo expression of early RGC markers, such as RA4 antigen, neurofilament (160 kDa), and a neurofilament-associated antigen. However, cath5 induced the expression of these proteins when the RPE cells were cultured with medium supplemented with bFGF. Since bFGF alone can induce only RA4 antigen, the expression of the additional RGC markers reflects a synergism between cath5 and bFGF in promoting RPE transdifferentiation toward RGCs. Morphologically, the RA4+ cells in bFGF + cath5 cultures appeared more neuron-like than those generated by bFGF alone. cNSCL1 also promoted bFGF-stimulated RPE cells to transdifferentiate toward RGCs that expressed RA4 antigen, N-CAM, Islet-1, neurofilament, and neurofilament-associated antigen. We found that cath5 induced cNSCL1 expression, but not vice versa. Our data suggest that cath5 or cNSCL1 alone was insufficient to induce RPE transdifferentiation into RGCs, but could further neural differentiation initiated by bFGF. We propose that intrinsic factors act synergistically with extrinsic factors during RGC genesis and development. Keywords: Cell fate; Development; Neuron differentiation; RPE transdifferentiation; bHLH; cath5: cNSCL1; Retinal ganglion cells

As many recent reports have suggested that BM HSCs may harbor unexpected developmental plasticity (1-14), we set out to test rigorously the cell fate potential of prospectively isolated, long-term reconstituting [...], To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral brood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GF[P.sup.+]:GF[P.sup.-] parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that 'transdifferentiation' of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.