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Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply, and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates thousands-diverse mixtures of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors, or immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in under three months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Background Mesenchymal stem cells have potential applications in inflammatory bowel disease due to their immunomodulatory properties. Our aim was to evaluate the feasibility, safety and efficacy of endoscopic administration of adipose-derived mesenchymal stem cells (ASCs) in a colitis model in rats. Methods Colitis was induced in rats by rectal trinitrobenzenesulfonic acid (TNBS). After 24 h ASCs (107 cells) or saline vehicle were endoscopically injected into the distal colon. Rats were followed for 11 days. Daily weight, endoscopic score at days 1 and 11, macroscopic appearance at necropsy, colon length and mRNA expression of Foxp3 and IL-10 in mesenteric lymph nodes (MLN) were analyzed. Results Endoscopic injection was successful in all the animals. No significant adverse events or mortality due to the procedure occurred. Weight evolution was significantly better in the ASC group, recovering initial weight by day 11 (− 0.8% ± 10.1%, mean ± SD), whereas the vehicle group remained in weight loss (− 6.7% ± 9.2%, p = 0.024). The endoscopic score improved in the ASC group by 47.1% ± 5.3% vs. 21.8% ± 6.6% in the vehicle group (p < 0.01). Stenosis was less frequent in the ASC group (4.8% vs. 41.2%, p < 0.01). Colon length significantly recovered in the ASC group versus the vehicle group (222.6 ± 17.3 mm vs. 193.6 ± 17.9 mm, p < 0.001). The endoscopic score significantly correlated with weight change, macroscopic necropsy score and colon length. Foxp3 and IL-10 mRNA levels in MLN recovered with ASC treatment. Conclusions ASC submucosal endoscopic injection is feasible, safe and ameliorates TNBS-induced colitis in rats, especially stenosis. Electronic supplementary material The online version of this article (10.1186/s13287-018-0837-x) contains supplementary material, which is available to authorized users.

Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the LucieASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58\% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders. European Community's 7th Framework Program for the collaborative project: ``REGENER-AR: Bringing Regenerative Medicine into the market: Allogeneic eASCs Phase IB/IIA clinical trial for treating Rheumatoid Arthritis (contract no. 279174 EC). Sí

Human adipose-derived stem cells (hASCs) are mesenchymal stem cells with reduced immunogenicity and the capability to modulate immune responses. These properties make hASCs of special interest as therapeutic agents in the settings of chronic inflammatory and autoimmune diseases. Exogenous and endogenous toll-like receptor (TLR) ligands have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis because of the permanent exposure of the immune system to TLR-specific stimuli. Therefore, hASCs employed in therapy are potentially exposed to TLR ligands, which may result in the modulation of hASC activity and therapeutic potency. In this study, we demonstrate that hASCs possess active TLR2, TLR3, and TLR4, because activation with specific ligands resulted in induction of nuclear factor kappa B-dependent genes, such as manganese superoxide dismutase and the release of interleukin (IL)-6 and IL-8. TLR3 and TLR4 ligands increased osteogenic differentiation, but no effect on adipogenic differentiation or proliferation was observed. Moreover, we show that TLR activation does not impair the immunogenic and immunosuppressive properties of hASCs. These results may have important implications with respect to the safety and efficacy of hASC-based cell therapies.

Background & Aims: Crohn's disease is a chronic disease characterized by severe T-helper (Th)1 cell-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. Mesenchymal stem cells were recently described to suppress effector T-cell responses and have therapeutic effects in some immune disorders. Here, we investigated the potential therapeutic effects of human adipose-derived mesenchymal stem cells (hASCs) in a model of inflammatory bowel disease. Methods: Mice with trinitrobenzene sulfonic acid-induced colitis were treated with hASCs after onset of disease and clinical scores were evaluated. Inflammatory response was determined by measuring the levels of different inflammatory mediators in colon and serum. Th1-mediated effector responses were evaluated by determining the proliferation and cytokine profile of activated mesenteric lymph node cells. The number of regulatory T cells and the suppressive capacity on Th1 cell responses was determined. Results: Systemic infusion of hASCs or murine ASCs ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation and increasing survival (P < .001). This therapeutic effect was mediated by down-regulating both Th1-driven autoimmune and inflammatory responses. ASCs decreased a wide panel of inflammatory cytokines and chemokines and increased interleukin-10 levels (P < .001), directly acting on activated macrophages. hASCs also impaired Th1 cell expansion and induced/activated CD4+CD25+FoxP3+ regulatory T cells with suppressive capacity on Th1 effector responses in vitro and in vivo (P < .001). Conclusions: hASCs emerge as key regulators of immune tolerance and as attractive candidates for a cell-based therapy for Crohn's disease. © 2009 AGA Institute.

Objectives:Adult mesenchymal stem cells were recently found to suppress effector T cell and inflammatory responses and have emerged as attractive therapeutic candidates for immune disorders. In rheumatoid arthritis (RA), a loss in the immunological self-tolerance causes the activation of autoreactive T cells against joint components and subsequent chronic inflammation. The aim of this study is to characterise the immunosuppressive activity of human adipose-derived mesenchymal stem cells (hASCs) on collagen-reactive T cells from patients with RA.Methods:The effects of hASCs on collagen-reactive RA human T cell proliferation and cytokine production were investigated, as well as effects on the production of inflammatory mediators by monocytes and fibroblast-like synoviocytes from patients with RA.Results:hASCs suppressed the antigen-specific response of T cells from patients with RA. hASCs inhibited the proliferative response and the production of inflammatory cytokines by collagen-activated CD4 and CD8 T cells. In contrast, the numbers of IL10-producing T cells and monocytes were significantly augmented upon hASC treatment. The suppressive activity of hASCs was cell-to-cell contact dependent and independent. hASCs also stimulated the generation of FoxP3 protein-expressing CD4+CD25+ regulatory T cells, with the capacity to suppress collagen-specific T cell responses. Finally, hASCs downregulated the inflammatory response and the production of matrix-degrading enzymes by synovial cells isolated from patients with RA.Conclusions:The present work identifies hASCs as key regulators of immune tolerance, with the capacity to suppress T cell and inflammatory responses and to induce the generation/activation of antigen-specific regulatory T cells.

A major gap in our knowledge of development is how the growth and identity of tissues and organs are linked during embryogenesis. The vertebrate limb is one of the best models to study these processes. Combining mutant analyses with gain- and loss-of-function approaches in zebrafish and chick embryos, we show that Tbx5 , in addition to its role governing forelimb identity, is both necessary and sufficient for limb outgrowth. We find that Tbx5 functions downstream of WNT signaling to regulate Fgf10 , which, in turn, maintains Tbx5 expression during limb outgrowth. Furthermore, our results indicate that Tbx5 and Wnt2b function together to initiate and specify forelimb outgrowth and identity. The molecular interactions governed by members of the T-box, Wnt and Fgf gene families uncovered in this study provide a framework for understanding not only limb development, but how outgrowth and identity of other tissues and organs of the embryo may be regulated.

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.

IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.

Limb development has become one of the model systems for studying vertebrate development. One crucial aspect in limb development is the origin, differentiation and patterning of muscle. Much progress has been made in recent years towards understanding this process. One of the general observations is that the genes involved in limb muscle development appear to be very similar to those involved in muscle development in other regions of the embryo. In this review, we summarize some of the genes and mechanisms that regulate limb muscle development and discuss various avenues along which a deeper understanding can be gained of how muscle cells originate and differentiate in different tissues during vertebrate development.

We have determined that Strong’s Luxoid (lstJ) mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

Sonic hedgehog (Shh) expression in the developing limb is associated with the zone of polarising activity (ZPA), and both are restricted to the posterior part of the limb bud. We show that the expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor (Fgf), Bone morphogenetic protein (Bmp), and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. This data suggest that Gli3 is a regulator of Shh expression in mouse limb development.

International audience; In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.

Human adipose-derived stem cells (hASC) are mesenchymal stem cells with reduced immunogenicity and the ability to modulate immune responses. APRIL and BAFF proteins are overexpressed in inflammatory and autoimmune diseases for which allogeneic hASC therapy is currently under clinical investigation. Modification of hASC properties by the tissue microenvironment could be a critical factor in patient outcome and is still not well understood. Our aim was to characterize the APRIL/BAFF system in hASC by analyzing the ligand and receptor expression patterns, the effects mediated by APRIL and BAFF on hASC, and the underlying signaling. We found that hASC express the tumor necrosis factor proteins APRIL (a proliferation-inducing ligand) and BAFF (B cell-activator factor) as well as their receptors TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), BCMA (B cell maturation antigen) and the BAFF-specific receptor (BAFF-R). APRIL and BAFF secretion was differentially enhanced by CXCL12 and interferon (IFN)-γ, implicated in hASC-mediated migration and immunosuppression, respectively. In addition, APRIL and BAFF induced rapid phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt kinases and promoted an increase in hASC proliferation, without affecting the immunosuppressive capacity of these cells. The use of specific chemical inhibitors indicated that the PI3K transduction pathway is involved in hASC basal growth and that APRIL- and BAFF-mediated effects are ERK-dependent. These results provide new information about the molecular mechanisms that underlie APRIL and BAFF secretion and signaling in hASC, and are of special relevance for the use of allogeneic hASC as therapeutic tools.

et al., Transforming growth factors-beta (TGF-betas) signal through type I and type II serine-threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-beta family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-beta signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI(-/-) mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI(-/-) mice also appeared attenuated through a mechanism involving delta-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI(-/-). Transcript levels of TGF-betas and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-beta family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids., This work was supported by Ministerio de Ciencia e Innovación Grant SAF2007-65451, Instituto de Salud Carlos III Grant RD06/0001/1016, and Fundació La Marató de TV3 Grant 072131 (M.A.H.), by Instituto de Salud Carlos III Grant FIS-PI 060240, by Ministerio de Ciencia e Innovación Grant SAF2005-00811 (R.M.), and by the National Institutes of Health and the G. Harold and Leila Y. Mathers Charitable Foundation (J.C.I.-B.).

Myocardial infarction caused by vascular occlusion results in the formation of nonfunctional fibrous tissue. Cumulative evidence indicates that cell therapy modestly improves cardiac function; thus, novel cell sources with the potential to repair injured tissue are actively sought. Here, we identify and characterize a cell population of cardiac adipose tissue-derived progenitor cells (ATDPCs) from biopsies of human adult cardiac adipose tissue. Cardiac ATDPCs express a mesenchymal stem cell-like marker profile (strongly positive for CD105, CD44, CD166, CD29 and CD90) and have immunosuppressive capacity. Moreover, cardiac ATDPCs have an inherent cardiac-like phenotype and were able to express de nova myocardial and endothelial markers in vitro but not to differentiate into adipocytes. In addition, when cardiac ATDPCs were transplanted into injured myocardium in mouse and rat models of myocardial infarction, the engrafted cells expressed cardiac (troponin I, sarcomeric alpha-actinin) and endothelial (CD31) markers, vascularization increased, and infarct size was reduced in mice and rats. Moreover, significant differences between control and cell-treated groups were found in fractional shortening and ejection fraction, and the anterior wall remained significantly thicker 30 days after cardiac delivery of ATDPCs. Finally, cardiac ATDPCs secreted proangiogenic factors under in vitro hypoxic conditions, suggesting a paracrine effect to promote local vascularization. Our results indicate that the population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) may be valid candidates for future use in cell therapy to regenerate injured myocardium. (C) 2010 Elsevier Ltd. All rights reserved.

Objective Rheumatoid arthritis (RA) is a chronic autoimmune disease caused by loss of immunologic self tolerance and characterized by chronic joint inflammation. Adult mesenchymal stem cells (MSCs) were recently found to suppress effector T cell responses and to have beneficial effects in various immune disorders. The purpose of this study was to examine a new therapeutic strategy for RA based on the administration of human adipose-derived MSCs (AD-MSCs). Methods DBA/1 mice with collagen-induced arthritis were treated with human AD-MSCs after disease onset, and clinical scores were determined. Inflammatory response was determined by measuring the levels of different mediators of inflammation in the joints and serum. The Th1-mediated autoreactive response was evaluated by determining the proliferative response and cytokine profile of draining lymph node cells stimulated with the autoantigen. The number of Treg cells and the suppressive capacity on self-reactive Th1 cells were also determined. Results Systemic infusion of human AD-MSCs significantly reduced the incidence and severity of experimental arthritis. This therapeutic effect was mediated by down-regulating the 2 deleterious disease components: the Th1-driven autoimmune and inflammatory responses. Human AD-MSCs decreased the production of various inflammatory cytokines and chemokines, decreased antigen-specific Th1/Th17 cell expansion, and induced the production of antiinflammatory interleukin-10 in lymph nodes and joints. Human AD-MSCs also induced de novo generation of antigen-specific CD4+CD25+FoxP3+ Treg cells with the capacity to suppress self-reactive T effector responses. Conclusion Human AD-MSCs emerge as key regulators of immune tolerance by inducing the generation/activation of Treg cells and are thus attractive candidates for a cell-based therapy for RA.

Human adipose-derived mesenchymal stem cells (hASCs) are mesenchymal stem cells (MSCs) with reduced immunogenicity and capability to modulate immune responses. Whereas the immunosuppressive activity of bone marrow-MSCs has received considerable attention during the last few years, the specific mechanisms underlying hASC-mediated immunosuppression have been poorly studied. Recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that hASCs and bone marrow-MSCs, while having similarities, are quite different. This suggests that different mechanisms of immunosuppression may apply. Here, we report that hASCs inhibit peripheral blood mononuclear cells (PBMCs), and CD4(+) and CD8(+) T cell proliferation in both cell-cell contact and transwell conditions, which is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASCs stimulated by IFN-gamma, PBMCs, or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. Many factors have been involved in MSC-mediated immunosuppression, including IFN-gamma, IL-10, hepatocyte growth factor, prostaglandin E2, transforming growth factor-beta1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, and IL-10. Using pharmacological inhibitors, neutralizing antibodies, and genetically modified hASCs that constitutively express or silence IDO enzyme, we demonstrate that, in the case of hASCs, the IFN-gamma/IDO axis is essential. Taken together, our data support the key role of IDO in the therapeutic use of hASC on immunomediated diseases.

Background and aims: Inflammatory bowel diseases (IBDs) are associated with uncontrolled innate and adaptive immunity against normal constituents, including commensal bacteria and microbial products. Mesenchymal stem cells (MSCs) suppress effector T cell responses and have beneficial effects in various immune disorders. This work investigates the therapeutic effects of human adipose-derived MSCs (hASCs) in various models of IBD and sepsis. Methods: Acute and chronic colitis was induced in mice with dextran sulfate sodium. Sepsis was induced by caecal ligation and puncture or by endotoxin injection. Colitic and septic mice were treated intraperitoneally with hASCs or murine ASCs, and diverse disease clinical signs and mortality were determined. The levels of various inflammatory cytokines and chemokines, T helper 1(Th1)-type response and generation of regulatory T cells (Treg) were determined in affected organs. Results: Systemic infusion of ASCs significantly ameliorated the clinical and histopathological severity of colitis, abrogating weight loss, diarrhoea and inflammation, and increasing survival. The therapeutic effect was associated with downregulation of the Th1-driven inflammatory responses. ASCs decreased a wide panel of inflammatory cytokines and chemokines and increased interleuklin 10 (IL10), acting on macrophages. hASCs also impaired Th1 cell activation in both colonic mucosa and draining lymph nodes. The induction of IL10-secreting Treg was partially involved in the therapeutic effect of hASCs. Moreover, ASCs protected from severe sepsis by reducing the infiltration of inflammatory cells in various target organs and by downregulating the production of various inflammatory mediators. Conclusions: hASCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for IBD and sepsis.

Although the T-box family of transcription factors function in many different tissues, their role in liver development is unknown. Here we show that Tbx3, the T-box gene that is mutated in human ulnar-mammary syndrome, is specifically expressed in multipotent hepatic progenitor cells,`hepatoblasts', isolated from the developing mouse liver. Tbx3-deficient hepatoblasts presented severe defects in proliferation as well as uncontrollable hepatobiliary lineage segregation, including the promotion of cholangiocyte (biliary epithelial cell) differentiation, which thereby caused abnormal liver development. Deletion of Tbx3 resulted in the increased expression of the tumor suppressor p19ARF(Cdkn2a), which in turn induced a growth arrest in hepatoblasts and activated a program of cholangiocyte differentiation. Thus, Tbx3 plays a crucial role in controlling hepatoblast proliferation and cell-fate determination by suppressing p19ARF expression and thereby promoting liver organogenesis.

Background. The heart forms from a linear tube that is subject to complex remodeling during embryonic development. Hallmarks of this remodeling are the looping of the heart tube and the regionalization into chamber and non-chamber myocardium. Cardiomyocytes in the future chamber myocardium acquire different cellular and physiological characteristics through activation of a chamber-specific genetic program, which is in part mediated by T-box genes. Methodology/Principal Finding. We characterize two new zebrafish T-box transcription factors, tbx3b and tbx2a, and analyze their role during the development of the atrioventricular canal. Loss- and gain-of-function analyses demonstrate that tbx3b and tbx2a are necessary to repress the chamber-genetic program in the non-chamber myocardium. We also show that tbx3b and tbx2a are required to control cell proliferation in the atrioventricular canal and that misregulation of cell proliferation in the heart tube influences looping. Furthermore, we characterize the heart phenotype of a novel Tbx3 mutation in mice and show that both the control of cell proliferation and the repression of chamber-specific genetic program in the non-chamber myocardium are conserved roles of Tbx3 in this species. Conclusions/Significance. Taken together, our results uncover an evolutionarily conserved role of Tbx2/3 transcription factors during remodeling of the heart myocardium and highlight the importance of controlling cell proliferation as a driving force of morphogenesis.

Adult stem cells are considered the present and the future in gene therapy. One of the sources for adult stem cells is adipose tissue where CMDL (lipoaspiration derived stem cells) are obtained, CMDL are well known for their autorenovation capacity, long term viability and potential to differentiate to diverse cellular types in vitro, like adipogenic, condrogenic, miogenic, osteogenic eventhough neurons.

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs/fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC , which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine.

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left-right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.

Left-sided expression ofNodalin the lateral plate mesoderm is a conserved feature necessary for the establishment of normal left–right asymmetry during vertebrate embryogenesis. By using gain- and loss-of-function experiments in zebrafish and mouse, we show that the activity of the Notch pathway is necessary and sufficient forNodalexpression around the node, and for proper left–right determination. We identify Notch-responsive elements in theNodalpromoter, and unveil a direct relationship between Notch activity andNodalexpression around the node. Our findings provide evidence for a mechanism involving Notch activity that translates an initial symmetry-breaking event into asymmetric gene expression.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.

The body plan is generated by cells and tissues that become arranged precisely in the embryo. This process, termed pattern formation, involves cell interactions in which a particular group of cells produce signals that specify new cell types or patterns of differentiation in responding cells. These patterning signals emanate from very discrete centers called "organizer centers," such as the Hensen's node or Spemann organizer, the midbrain-hindbrain junction, the notochord, or in the case of the limb, the zone of polarizing activity (ZPA) or the apical ectodermal ridge (AER). The developing vertebrate limb is an ideal model system for the study of pattern formation because, in addition to surgical manipulations, molecular manipulations are now feasible. In this review we summarize early experiments that established, by means of surgical manipulations, the different organizer centers of the vertebrate limb: the ectoderm covering the limb bud, the apical ectodermal ridge, the zone of polarizing activity, and the distal mesoderm (progress zone) underlying the AER. We then describe the domains of expression of various genes present during the development of the limb and discuss some of the functional approaches (overexpression and lack of function studies) undertaken to ascertain their role in limb outgrowth. The knowledge acquired in the last few years has had an enormous impact not only on our view of how limbs develop (perhaps now one of the most approachable vertebrate model systems) but also in a more general sense of how the embryo is organized in space and time.

The zinc finger gene Gli 3 plays a role in limb and brain development. To facilitate the molecular analysis of different mouse mutations of this gene, the murine cDNA was isolated and sequenced. This 5113 by cDNA encodes a putative protein of 1596 amino acids. Comparison of the murine and human GL13 cDNA revealed an overall homology of 85% between the deduced amino acid sequences. More importantly, several regions of the protein, including the zinc fingers, are more highly conserved ( > 95%), suggesting that these represent functional domains in the Gli3 protein.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.