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The mammalian heart comprises mainly cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells. Cardiomyocyte subtypes are characterized by differential expression of intracellular proteins like MLC-2a, Cx40 or ANF in atrial cardiomyocytes as well as MLC-2v, Hey2 or Irx4 in ventricular cardiomyocytes. Nevertheless, to date the expression of chamber-specific intracellular proteins could not be correlated to corresponding cell surface markers. Therefore, the selective, surface marker-based enrichment of atrial or ventricular cardiomyocytes from whole-heart preparations is currently not possible.In a proof-of-concept study we performed a semi-automated, antibody-based surface marker screen on neonatal mouse heart preparations. By that we were able to identify several candidate markers exclusively expressed by non-cardiomyocytes. Using a magnetic cell sorting approach, we could highly enrich for neonatal cardiomyocytes by efficiently removing non-cardiomyocytes. In a second step, we were able to identify surface markers differentially expressed on atrial and ventricular cardiomyocytes, thereby dividing the pure cardiomyocyte population into two subpopulations. Next, we established cell isolation strategies to specifically isolate atrial and ventricular cardiomyocytes from whole-heart cell preparations with high purities. Expression profiling of the isolated subpopulations confirmed high expression of atrial marker genes such as Nr2f2, Fgf12, Sln, Gja5 and Nppa in the putative atrial fraction as well as high expression of genes associated with a ventricular myocyte identity, e.g. Hey2, Irx4, Lbh, Myh7 in the putative ventricular fraction. The sorted cells were viable, could be cultivated and exhibited spontaneous contractions 24h after plating.For the first time, we found a set of cell surface markers that can be used to specifically isolate atrial and ventricular cardiomyocytes from mouse heart. As the markers were also expressed on mouse embryonic stem cell-derived cardiomyocytes, current work analyzes their relevance for the identification and isolation of pluripotent stem cell-derived cardiomyocyte subpopulations. [ABSTRACT FROM AUTHOR]

Pure cardiovascular cell lineages are a requisite for an unbiased and cell type specific investigation of (patho-) physiological mechanisms involved in heart failure. Moreover pure heart cells are needed as building blocks for engineered heart mimetics. The latter offers the possibility to replace infarcted myocardium and to investigate heart development and cardiomyogenesis in a simplified in vitro model. However, it has to be acknowledged that strategies capable to specifically purify heart cells are not well established. To overcome these experimental limitations we developed a rapid and automated neonatal heart dissociation protocol enabling an immunomagnetic enrichment of the three major heart cell types, namely cardiomyocytes (CMs), cardiac fibroblasts (Fibs) and endothelial cells (Endos) from both, mouse and rat neonatal hearts.First, we established an enzyme mix optimal for the dissociation of neonatal mouse and rat hearts (P0-P3) by screening our enzyme library. Next, we optimized the dissociation by utilizing our gentleMACS technology resulting in a fast (1h), robust, and fully automated heart dissociation protocol. Analysis of the dissociated mouse and rat heart cells showed: (i) high cell vitalities (>90%), (ii) high frequencies of α-Actinin-positive CMs (>60%) and (iii) vimentin-positive non-CMs with a frequency of >40% (P2). In order to selectively enrich various cell-types from these heterogenous cell populations, we performed a cell-surface marker screen. We identified several candidates for the composition of antibody cocktails enabling selective enrichment of mouse and rat CMs, Fibs or Endos with purities of up to 97%. CMs grown in 2D cultures showed spontaneous beating activity and the expression of sarcomeric proteins in a cross-striated pattern (α-Actinin and cTnT). Additionally, heart cells were cultured on organotypic decellularized (1% SDS) mouse heart slices. Analysis of the slice cultures after recellularization showed (i) partial restoration of synchronous contractions and (ii) orientated and elongated heart cells along the microfibrillar heart structure. Purified Fibs were highly proliferative and could be expanded 34-fold over an analyzed culture period of 15 days. Finally, functionality of the purified endothelial cells was proven by Dil-LDL endocytosis.In summary, we established an automated protocol for the dissociation of neonatal mouse and rat hearts, enabling the subsequent immunomagnetic enrichment of CMs, Fibs and Endos which can readily be utilized for 2D cell culture assays or to generate in vitro heart muscle models and surrogate tissue for myocardial repair. [ABSTRACT FROM PUBLISHER]

CAR T cell therapy has been an effective treatment option for hematological malignancies. However, the therapeutic potential of CAR T cells can be reduced by several constraints, partly due to immunogenicity and toxicities. The lack of established workflows enabling thorough evaluation of new candidates, limits comprehensive CAR assessment. To improve the selection of lead CAR candidates, we established a stringent, multistep workflow based on specificity assessments, employing multiple assays and technologies. Moreover, we characterized a human FOLR1-directed CAR binding domain. Selection of binding domains was based on extensive specificity assessment by flow cytometry and imaging, to determine on-/off-target and off-tumor reactivity. CAR T cell functionality and specificity were assessed by high-throughput screening and advanced in vitro assays. Our validation strategy highlights that assays comprehensively characterizing CAR functionality and binding specificity complement each other. Thereby, critical specificity considerations can be addressed early in the development process to overcome current limitations for future CAR T cell therapies.

Summary: The ever-growing amount of genome-wide omics data paved the way for solving life science problems in a data-driven manner. Among others, enrichment analysis is part of the standard analysis arsenal to determine systemic signals in any given transcriptomic or proteomic data. Only a part of the members of the fungal kingdom, however, can be analyzed via public web applications, despite the global rise of fungal pathogens and their increasing resistance to antimycotics. We present FungiFun3, a major update of our user-friendly gene set enrichment web application dedicated to fungi. FungiFun3 was rebuilt from scratch to support a modern and easy-to-use web interface and supports >4-fold more fungal strains (n = 1287 in total) than its predecessor. In addition, it also allows ranked gene set enrichment analysis at the genomic scale. FungiFun3 thus serves as a starting hub for identifying molecular signals in omics datasets related to a vast amount of available fungal strains including human fungal pathogens of the WHO's priority list and far beyond., Availability and Implementation: FungiFun3, including sample data and FAQ, is freely available at https://fungifun3.hki-jena.de/., (© The Author(s) 2024. Published by Oxford University Press.)

Treatment options for ovarian cancer patients are limited, and a high unmet clinical need remains for targeted and long-lasting, efficient drugs. Genetically modified T cells expressing chimeric antigen receptors (CAR), are promising new drugs that can be directed towards a defined target and have shown efficient, as well as persisting, anti-tumor responses in many patients. We sought to develop novel CAR T cells targeting ovarian cancer and to assess these candidates preclinically. First, we identified potential CAR targets on ovarian cancer samples. We confirmed high and consistent expressions of the tumor-associated antigen FOLR1 on primary ovarian cancer samples. Subsequently, we designed a series of CAR T cell candidates against the identified target and demonstrated their functionality against ovarian cancer cell lines in vitro and in an in vivo xenograft model. Finally, we performed additional in vitro assays recapitulating immune suppressive mechanisms present in solid tumors and developed a process for the automated manufacturing of our CAR T cell candidate. These findings demonstrate the feasibility of anti-FOLR1 CAR T cells for ovarian cancer and potentially other FOLR1-expressing tumors.

Introduction: Racism, a known social determinant of health, affects the mental health and well-being of pregnant and postpartum women and their children. Convincing evidence highlights the urgent need to better identify the mechanisms and the ways in which young children's development and mental health are adversely impacted by their mothers' experiences of racism. With the additional stressor of the COVID-19 pandemic, the criticality of improving knowledge of these domains has never been starker. The proposed project will address these questions and move the field forward to create targeted, culturally informed preventative interventions, thus achieving mental health equity for all children and families., Methods: This prospective research is a cohort study that will longitudinally observe the outcomes of a cohort of mothers and their children recruited during the initial phase of the COVID-19 pandemic. Data will be parent/caregiver questionnaires assessing mental health, racism, support, and resilience at multiple time points with the first beginning at 24 months, clinical interviews with mothers, electronic medical records of mothers, and videotaped dyadic interactions at child age 24 and 48 months. A subset of Black participants will be asked to participate in qualitative interviews at child age 36 months., Results: Analyze will be performed within and across Black and Non-Latino/a/e/x white (NLW) groups, and comparing mothers and fathers/secondary caregivers. Descriptive and multivariate analyzes will be run to better characterize how young children's development and mental health may be adversely impacted by their caregiver's experiences of racism., Discussion: This prospective longitudinal mixed-methods study evaluates the simultaneous effects of the COVID-19 pandemic and racism on mothers and their developing children to characterize cross-racial differences, providing insight into risk and resilience factors in early development and the peripartum period., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Njoroge, Tieu, Eckardt, Himes, Alexandre, Hall, Wisniewski, Popoola, Holloway, Rodriguez, Kornfield, Momplaisir, Wang, Gur and Waller.)

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

Models of heart disease and drug responses are increasingly based on human pluripotent stem cells (hPSCs) since their ability to capture human heart (dys-)function is often better than animal models. Simple monolayer cultures of hPSC-derived cardiomyocytes, however, have shortcomings. Some of these can be overcome using more complex, multi cell-type models in 3D. Here we review modalities that address this, describe efforts to tailor readouts and sensors for monitoring tissue- and cell physiology (exogenously and in situ) and discuss perspectives for implementation in industry and academia., Competing Interests: J.N and D.E. are employees of Miltenyi Biotec B.V. & Co. KG., (© 2022 The Authors.)

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine., (© 2022. The Author(s).)

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1 , which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1 , overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies. IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans ., (Copyright © 2020 Ruben et al.)

Intestinal epithelial cells (IEC) form a tight barrier to the gut lumen. Paracellular permeability of the intestinal barrier is regulated by tight junction proteins and can be modulated by microorganisms and other stimuli. The polymorphic fungus Candida albicans, a frequent commensal of the human mucosa, has the capacity of traversing this barrier and establishing systemic disease within the host. Infection of polarized C2BBe1 IEC with wild-type C. albicans led to a transient increase of transepithelial electric resistance (TEER) before subsequent barrier disruption, accompanied by a strong decline of junctional protein levels and substantial, but considerably delayed cytotoxicity. Time-resolved microarray-based transcriptome analysis of C. albicans challenged IEC revealed a prominent role of NF-κB and MAPK signalling pathways in the response to infection. Hence, we inferred a gene regulatory network based on differentially expressed NF-κB and MAPK pathway components and their predicted transcriptional targets. The network model predicted activation of GDF15 by NF-κB was experimentally validated. Furthermore, inhibition of NF-κB activation in C. albicans infected C2BBe1 cells led to enhanced cytotoxicity in the epithelial cells. Taken together our study identifies NF-κB activation as an important protective signalling pathway in the response of epithelial cells to C. albicans., (© 2016 John Wiley & Sons Ltd.)

As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin.

Rationale: Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers., Methods and Results: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis., Conclusion: Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

Unlabelled: Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance., Importance: Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor., (Copyright © 2015 Leonhardt et al.)

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues.

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Although morphological plasticity is a central virulence trait of Candida albicans, the number of filament-associated genes and the interplay of mechanisms regulating their expression remain unknown. By correlation-based network modeling of the transcriptional response to different defined external stimuli for morphogenesis we identified a set of eight genes with highly correlated expression patterns, forming a core filamentation response. This group of genes included ALS3, ECE1, HGT2, HWP1, IHD1 and RBT1 which are known or supposed to encode for cell- wall associated proteins as well as the Rac1 guanine nucleotide exchange factor encoding gene DCK1 and the unknown function open reading frame orf19.2457. The validity of network modeling was confirmed using a dataset of advanced complexity that describes the transcriptional response of C. albicans during epithelial invasion as well as comparing our results with other previously published transcriptome studies. Although the set of core filamentation response genes was quite small, several transcriptional regulators are involved in the control of their expression, depending on the environmental condition.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of uterine contractions during labour. Expression of connexin43 (Cx43), the major myometrial gap junction protein, is increased during delivery. We have generated a mouse mutant (Cx43fl/fl:SM-CreERT2), in which the coding region of Cx43 can be specifically deleted in smooth muscle cells at any given time point by application of tamoxifen. By this approach, we were able to study long-term effects on myometrial functions that are necessary for parturition as well as gap junction intercellular communication in primary myometrial cell cultures. We found a prolongation of the pregnancy in 82% of tamoxifen-treated Cx43fl/fl:SM-CreERT2 mice as well as decreased dye coupling in cultured primary myocytes of these animals. Other parturition-specific parameters such as the regulation of oxytocin receptor, prostaglandin F receptor or progesterone remained unchanged. Our results indicate the important function of Cx43 during parturition in the living animal and suggest further strategies to investigate the role of connexins in uterine contractility in transgenic mice.

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.

The molecular mechanisms regulating skeletal muscle regeneration and differentiation are not well understood. We analyzed the expression of connexins (Cxs) 40, 43 and 45 in normal and regenerating tibialis anterior muscle and in primary cultures of differentiating myoblasts in adult and newborn mice, respectively. Cxs 45 and 43, but not 40, were strongly expressed in normal muscle and their expression was upregulated during regeneration. Furthermore, the functional role of Cx43 during differentiation and regeneration was examined after induced deletion of Cx43 in transgenic mice. In vivo, the inducible deletion of Cx43 delayed the formation of myofibers and prolonged the expression of myogenin during regeneration. In primary cultures of satellite cell-derived myoblasts, induced deletion of Cx43 led to decreased expression of myogenin and MyoD, dye coupling, creatine kinase activity and myoblast fusion. Thus, the expression of Cx45 and Cx43 is upregulated during skeletal muscle regeneration and Cx43 is required for normal myogenesis in vitro and adult muscle regeneration in vivo.

Background: Connexin 43 (Cx43) is a major determinant of conduction in the ventricular working myocardium of mammals. We investigated the effect of decreased Cx43 expression on conduction velocity and arrhythmogenesis using adult mice with inducible deletion of Cx43., Methods and Results: Cx43Cre-ER(T)/+ mice, in which 1 coding region of the Cx43 gene was replaced by Cre-ER(T), were mated to Cx43fl/fl mice, generating Cx43Cre-ER(T)/fl mice. Application of 4-hydroxytamoxifen (4-OHT) induced Cre-ER(T)-mediated deletion of the floxed Cx43 allele. Epicardial ventricular mapping using a 13x19 multiterminal electrode grid (300-microm spacing) was performed on Langendorff-perfused hearts from Cx43fl/fl plus carrier (n=10), Cx43fl/fl plus 4-OHT (n=10), Cx43 Cre-ER(T)/fl plus carrier (n=9), and Cx43Cre-ER(T)/fl plus 4-OHT (n=10). Cx43 protein amount in group 3 hearts was decreased by 50% compared with group 1. 4-OHT did not affect cardiac protein amounts in group 2 but decreased Cx43 expression up to 95% in group 4 compared with group 3. Epicardial activation of both left ventricle (LV) and right ventricle (RV) during sinus rhythm was similar in all groups. Conduction velocity (CV) changed only in group 4 animals. For RV (LV), longitudinal CV decreased from 38 (35) to 31.6 (33.6) and transverse CV from 24.4 (16.8) to 10.1 (11.3) cm/s. Dispersion of conduction in RV (LV) was increased by 91% (38%). Programmed stimulation resulted in ventricular arrhythmias in group 4 (7 of 10 mice) but never in groups 1 through 3., Conclusions: Heterozygous expression of Cx43 did not affect ventricular conduction velocity. Up to 95% decrease of Cx43 protein in 4-OHT-treated Cx43(Cre-ER(T)/fl) mice reduced conduction velocity and increased dispersion of conduction and propensity for ventricular arrhythmias.

To analyze the effect of connexin loss on the repair of wounded tail skin, we have studied the following transgenic mouse mutants: connexin30-/-, connexin31-/- and connexin43Cre-ER(T)/fl (for inducible deletion of the connexin43 coding region). Connexin43 and connexin31 are expressed in the basal and spinous layers of wild-type epidermis, whereas connexin31 and small amounts of connexin30, as well as connexin26 proteins, were found in the granulous layer. Connexin43 was downregulated in connexin31-deficient mice, whereas mice with reduced connexin43 exhibited an upregulation of connexin30. During wound healing, connexin30 and connexin26 proteins were upregulated in all epidermal layers, whereas connexin43 and connexin31 protein expression were downregulated. In connexin31-/- mice, reduced levels of connexin30 protein were observed on days 1 and 2 after wounding. The closure of epidermal wounds in mice with decreased amounts of connexin43 protein occurred one day earlier. Under these conditions the expression profiles of connexin30 and connexin31 were also temporarily shifted by one day. Furthermore, dye transfer between keratinocytes in skin sections from connexin43-deficient mice was decreased by 40%. These results suggest that downregulation of connexin43 appears to be a prerequisite for the coordinated proliferation and mobilization of keratinocytes during wound healing.

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.