Expression Of Cell-specific Markers Research Articles

The mechanistic role of connexin 43 in maturation of hiPSC cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ECR (European research council) and ReNew (the Novo Nordisk Foundation Center for Stem Cell Medicine supported by Novo Nordisk Foundation grants) Introduction Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are functionally immature and this limits the application of these cells to cardiovascular research. Maturation can be improved by aggregating hiPSC-CMs into three-dimensional (3D) microtissues together with hiPSC-cardiac fibroblasts (hiPSC-CFs) and hiPSC-endothelial cells (hiPSC-ECs). These cardiac microtissues showed improved structural (increase of sarcomere length and organisation) and functional (more mature action potential profile) properties. This effect was only observed when the cardiac microtissues contained hiPSC-CFs. Since connexin 43 (CX43), the most ubiquitous connexin isoform in the heart, was significantly upregulated in hiPSC-CMs from cardiac microtissues, we hypothesise that mechanisms underlying hiPSC-CMs maturation involves the coupling of hiPSC-CMs with hiPSC-CFs through this protein. Purpose The aim of this study is to unravel the role of CX43 in the maturation of hiPSC-CMs in the context of 3D multicellular cardiac tissues. Methods We used the CRISPR/Cas9 technology to generate a CX43 knock-out (KO) hiPSC line. We designed two sgRNAs annealing to exon 2 of GJA1 gene, encoding CX43; indels generated by the non-homologous end joining after the Cas9 double strand break will result in a non-functional protein. Cells were differentiated in vitro into CMs, epicardial cells (EPI) and then CFs and characterized for the expression of cell-specific markers and compared with the wild-type parental control cells. Results and conclusions We screened 31 hiPSC clones and selected those in which both GJA1 alleles contained a deletion. Copy number variation analysis by ddPCR of the flanking regions confirmed that the deletion was specific and confined. The lack of GJA1 expression in the KO clones was confirmed by qPCR, while the lack of the CX43 protein was confirmed by immunofluorescence and Western Blot. This did not affect CX43-KO hiPSCs pluripotency state shown by the presence of pluripotent markers such as NANOG, OCT3/4, SSEA4. Additionally, CX43-KO hiPSCs were able to differentiate into the three germ layers. CX43-KO hiPSCs were successfully differentiated into hiPSC-CMs, -EPI, and -CFs. CX43-KO hiPSC-CMs and controls showed comparable gene expression of cardiac troponin T. Since immunofluorescent staining showed that CX43-KO hiPSC-EPI expressed the epicardial markers WT1 and ZO1, we continued to differentiate them into hiPSC-CFs, which expressed markers such as VIM and COL1A1, similar to the control. Differentiated CX43-KO hiPSC-CMs and CFs will be assembled into cardiac microtissues. Functional and structural assays that include patch-clamp and immunofluorescence will be performed on these microtissue to assess the effect of the CX43 KO on the maturation of hiPSC-CMs. Our results show that the lack of CX43 expression does not impede hiPSCs to differentiate into various cell types originating from the mesoderm germ layer.

Characterization of Ano1 expressing cells in the mouse corpus cavernosum

Background/objectives: Highly coordinated contraction and relaxation of smooth muscle cells (SMCs) is critical to penile function. The Ca2+-activated Cl− channel, Anoctamin-1 (Ano1) plays a role in this as antagonists of this channel inhibit contractile, electrical and intracellular Ca2+ activity. mRNA encoding Ano1 is highly expressed in whole rabbit corpus cavernosum (CC). Despite its significant contribution to erectile function, the specific cell type that expresses Ano1 has not been determined, with previous studies relying on morphological identification of cells alone. Atypical SMCs (ASMCs) expressing platelet derived growth factor receptor-a (PDGFRa; Pdgfra), myosin heavy chain ( Myh11) and Ano1 have been observed in the renal pelvis where they are thought to drive the activity in adjacent SMCs (Grainger et al. 2020; PMID: 32415739). Therefore, the objective of this study is to identify the cell type that expresses Ano1 in the CC. Methods: Cells were isolated from the CC of mice selectively expressing eGFP in PDGFRa+ cells (PDGFRa+-eGFP mice) and SMCs ( Myh11-eGFP mice). Cell suspensions were sorted using fluorescence activated cell sorting (FACS) and evaluated for expression of cell specific markers ( Pdgfra and Myh11) by qPCR. Enriched PDGFRa+ cells and SMCs were examined with ddPCR to determine the expression of Ano1. Protein expression was evaluated using immunofluorescent labeling of frozen CC sections with an antibody targeting Ano1. Results: FACS sorted Myh11-eGFP+ cells expressed Myh11 and Pdgfra. Sorting of PDGFRa-eGFP+ cells revealed two subpopulations, bright and dim. Examination of these two PDGFRa-eGFP+ subpopulations revealed that while both expressed similar levels of Pdgfra, the dim population also expressed Myh11 suggesting that these cells may be similar to ASMCs of the renal pelvis. ddPCR revealed that the PDGFRa-eGFP+ dim population had higher gene copy numbers of Ano1 compared to Myh11-eGFP+ cells. Immunofluorescent labeling of CC sections revealed that Ano1 protein was present in a subpopulation of PDGFRa+ cells. Conclusion: The data presented in this study demonstrates that Ano1 is expressed in mouse CC PDGFRa-eGFP+ dim populations that also express Pdgfra and Myh11; this is similar to ASMCs in the renal pelvis. Future directions include examining the relationship of PDGFRa-eGFP+ dim cells to SMCs and nerve terminals in the CC and to determine if these cells are also present in human CC. This cell population could represent a novel target for the treatment of erectile dysfunction in patients who do not respond to phosphodiesterase inhibitors. Funding: AUA Research Scholar Award to KIH, NIH DK129528 to CAC, NIH P20GM130459 for imaging and molecular cores. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P037 Modelling gut complexity and disease: development of a next-generation adult stem cell based tubular gut-on-a-chip model

Abstract Background Developing effective therapies for Inflammatory Bowel Disease (IBD) necessitates advanced in vitro models that faithfully replicate the intricacies of the gut. The current challenge is to create models that encompass the 3D morphology, heterogeneity, and boundary characteristics of intestinal tissue while also being amenable to high-throughput screening. Microfluidic techniques are emerging as vital tools to introduce physiologically relevant cues into conventional cell cultures. Methods We present an innovative in vitro model employing human adult stem cell-derived colon organoids cultivated as perfused epithelial tubules within the MIMETAS OrganoPlate®. This microfluidic platform facilitates the simultaneous culture of up to 64 independent gut tubules on an extracellular matrix, offering continuous media perfusion without artificial membrane interference. The barrier function of the epithelial tubules is assessed in real-time through transepithelial electrical resistance (TEER) measurements. Immunostaining and 3D reconstruction of confocal images evaluate the expression of relevant epithelial markers. Results The tubular epithelial model of the intestinal tract demonstrated rapid cell polarization, expression of cell-specific markers, tight junction formation, and the presence of functional transporters. TEER measurements indicated functional and reproducible barrier integrity across tubules generated from different organoid donors for at least 10 days. Incorporation of additional cell types, such as patient-derived immune cells and endothelium, marks a groundbreaking advancement in understanding disease pathogenesis. The plug-and-play modularity of these models allows for flexibility in incorporating specific cell types based on the biological pathway of interest. Conclusion Our next-generation gut-on-a-chip model faithfully mimics the IBD phenotype and facilitates screening for potential drug targets. Combining adult human stem cell-derived intestinal organoids with microfluidic technology provides a robust platform to study physiology and disease mechanisms in patient-specific gut models. The inclusion of IBD patient-derived cells promises further refinement, enabling a more profound exploration of disease phenotypes and treatment responsiveness. Moreover, organoids, as one of the most physiologically relevant cell sources, authentically represent patient biology, enhancing the translational potential of our findings.

Evaluating the Feasibility of Hydrogel-Based Neural Cell Sprays.

Neurological injuries have poor prognoses with serious clinical sequelae. Stem cell transplantation enhances neural repair but is hampered by low graft survival (<ca. 5%), necessitating the development of approaches to enhance post-transplant cell viability. Intracerebral injection exerts high mechanical forces on transplant cells with risks of haemorrhage/infection. Transplant cell sprays can offer a non-invasive alternative. This study has assessed if the addition of protective, encapsulating polymer hydrogels to a cell spray format is feasible. Hydrogels (0.1% (1 mg/mL), 0.3% and 0.6% type I rat tail collagen) were trialled for spray deliverability. Cell-enriched hydrogels (containing mouse cortical astrocytes) were sprayed onto culture substrates. Astrocyte viability, cell-specific marker expression, morphology and proliferation were assessed at 24 h and 72 h post spraying. Intra-gel astrocytes and hydrogels could be co-stained using a double immunocytological technique (picrosirius red (PR)/DAB-peroxidase co-labelling). Astrocyte viability remained high post spraying with hydrogel encapsulation (>ca. 80%) and marker expression/proliferative potential of hydrogel-sprayed astrocytes was retained. Combining a cell spray format with polymer encapsulation technologies could form the basis of a non-invasive graft delivery method, offering potential advantages over current cell delivery approaches.

MP27-04 IDENTIFICATION AND CHARACTERIZATION OF NOVEL ATYPICAL SMOOTH MUSCLE CELLS IN THE MOUSE CORPUS CAVERNOSUM

You have accessJournal of UrologyCME1 Apr 2023MP27-04 IDENTIFICATION AND CHARACTERIZATION OF NOVEL ATYPICAL SMOOTH MUSCLE CELLS IN THE MOUSE CORPUS CAVERNOSUM Karen I. Hannigan, Kenton M. Sanders, Sang Don Koh, and Caroline A. Cobine Karen I. HanniganKaren I. Hannigan More articles by this author , Kenton M. SandersKenton M. Sanders More articles by this author , Sang Don KohSang Don Koh More articles by this author , and Caroline A. CobineCaroline A. Cobine More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Highly coordinated relaxation of smooth muscle cells (SMCs) is a requirement for penile erection. Previous studies in the corpus cavernosum (CC) have shown that blocking the Ca2+ activated Cl- channel Ano1 inhibited contractile, electrical and intracellular Ca2+ activity. Penile activity is influenced by neural inputs with SMCs thought to be directly targeted by neurotransmitters due to their expression of soluble guanylate cyclase (sGC), the receptor for nitric oxide (NO). However, as the CC is sparsely innervated, a cellular mediator could be present between nerves and CCSMCs. Despite this, the specific cell type that expresses Ano1 has not been identified, with previous studies relying on morphological identification of SMCs alone. Platelet derived growth factor receptor-α (PDGFRα)+ cells have been identified in other visceral smooth muscles including the gastrointestinal and urinary tracts where they mediate inhibitory neurotransmission. In the renal pelvis, PDGFRα+ cells express Ano1. The aim of this study is to identify and characterize the cell type that expresses Ano1 and to determine whether these cells mediate neurotransmission in the CC. METHODS: Cells were isolated from the CC of mice expressing eGFP in PDGFRα+ cells (PDGFRα+-eGFP mice) and SMCs (SmMHC-eGFP mice). Isolated cells were sorted with FACS. Enriched PDGFRα and SMC populations were examined using qPCR for expression of cell specific markers (Pdfgra and smooth muscle myosin heavy chain (Myh11)) to ensure purity. Purified PDGFRα+ cells and SMCs were evaluated with ddPCR to examine the expression of Ano1 and genes encoding signaling proteins from the NO pathway. RESULTS: PDGFRα+ cells isolated with FACS belonged to bright and dim populations. While qPCR revealed that the bright and dim populations expressed similar levels of Pdgfra, the dim population also expressed Myh11. In contrast, SMCs purified using FACS expressed Myh11 and Pdgfra, similar to the dim population of PDGFRα+ cells. ddPCR revealed that both the dim population of PDGFRα+ cells and SMCs expressed Ano1 and genes encoding signaling proteins from the NO pathway. CONCLUSIONS: These data suggest that Ano1 is expressed by atypical SMCs that also express PDGFRα. Therefore, these cells are a potential novel target for the treatment of erectile dysfunction. Future directions of this project include investigating the interactions between PDGFRa+ cells, SMCs and neurons. Source of Funding: Urology Care Foundation Research Scholar Award, sponsored by the Sexual Medicine Society of North America © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e363 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen I. Hannigan More articles by this author Kenton M. Sanders More articles by this author Sang Don Koh More articles by this author Caroline A. Cobine More articles by this author Expand All Advertisement PDF downloadLoading ...

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

The Significance of Cancer Stem Cells and Epithelial-Mesenchymal Transition in Metastasis and Anti-Cancer Therapy.

Cancer stem cells (CSCs) have been identified and characterized in both hematopoietic and solid tumors. Their existence was first predicted by Virchow and Cohnheim in the 1870s. Later, many studies showed that CSCs can be identified and isolated by their expression of specific cell markers. The significance of CSCs with respect to tumor biology and anti-cancer treatment lies in their ability to maintain quiescence with very slow proliferation, indefinite self-renewal, differentiation, and trans-differentiation such as epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET). The ability for detachment, migration, extra- and intravasation, invasion and thereby of completing all necessary steps of the metastatic cascade highlights their significance for metastasis. CSCs comprise the cancer cell populations responsible for tumor growth, resistance to therapies and cancer metastasis. In this review, the history of the CSC theory, their identification and characterization and their biology are described. The contribution of the CSC ability to undergo EMT for cancer metastasis is discussed. Recently, novel strategies for drug development have focused on the elimination of the CSCs specifically. The unique functional and molecular properties of CSCs are discussed as possible therapeutic vulnerabilities for the development of novel anti-metastasis treatments. Prospectively, this may provide precise personalized anti-cancer treatments with improved therapeutic efficiency with fewer side effects and leading to better prognosis.

A Single Cell Atlas and Interactive Web-Resource of Pediatric Cancers and Healthy Bone Marrow

Single-cell profiling allows for in-depth characterization of the tumor microenvironment and revealing the heterogeneity of pediatric cancers. Ease of access and analysis of the primary tumor single-cell data from studies on solid and blood pediatric tumors is essential for improving diagnostics and therapies. We have developed the Pediatric Single Cell Cancer Atlas (PedSCAtlas), with the goal of highlighting the heterogeneity of malignant and microenvironment cells across pediatric cancers. The atlas allows for quick exploration, comparative analysis, visualization, and prognostic assessment of genes/pathways across pediatric cancers without requiring bioinformatics analysis or computational support. The PedSCAtlas currently contains expression data of >1.2 million single cell (sc) and single nuclei (sn) from 134 pediatric hematological and solid-organ cancer samples and healthy bone marrow (BM). The hematological cancer samples are dominated by acute leukemias containing scRNA-seq data of acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL), and mixed phenotype acute leukemia (MPAL). The scRNA-seq data are generated from the BM samples collected during the clinical diagnosis of the disease. The data for the atlas was either generated at the Bhasin lab or collected from public resources including the GEO database, ScPCA (https://scpca.alexslemonade.org/), and HCA (https://data.humancellatlas.org/). Additionally, the atlas also contains bulk RNA-sequencing data and clinical information retrieved from the TARGET Initiative (https://ocg.cancer.gov/programs/target). The single cell/nuclei datasets are processed using a uniform workflow that includes quality control and filtering, normalization, integration with batch correction (if needed), dimensionality reduction, clustering, and cellular annotations. The data normalization and integration are performed using sctransform (Hao and Hao et al, Cell 2021) and harmony (Korsunsky et al, Nature Methods 2019) algorithms respectively. The visualization of integrated pediatric cancers single cell was achieved using Uniform Manifold Approximations and Projection (UMAP) method (Becht et al, Nature Biotech 2018). The clusters were annotated based on the expression of cell-specific markers. Differential expression analysis was used to identify potential biomarker sets for malignant cells of disease subtypes. To further ascertain that these biomarkers were malignant cell (blast) specific in the acute leukemia subtypes, the genes were filtered to exclude those with consistent expression in healthy BM cell types. To compare the expression profile of genes between single cells and bulk data, the raw counts of bulk RNA-seq datasets were filtered and normalized using the Voom algorithm (Ritchie et al, Nucleic Acids Research 2015). The resource has modules for biomarker analysis, bulk RNA deconvolution, and survival analysis to facilitate easy analysis of single-cell data. Survival analysis is available for the bulkRNA-seq dataset; the survival is calculated using the overall survival values and gene expression/enrichment input by the user. The SingleCell module allows users to visualize the pediatric cancer dataset on a UMAP by selecting the aspect (i.e., cell type) to group the dataset by; it also allows users to enter a gene name or select a pathway of their interest and view its expression or enrichment in a feature or violin plot. The ImmuneCell module allows users to interact with the adult healthy BM dataset by inputting a gene and viewing its expression on a feature plot. The Biomarkers module contains pre-defined biomarker sets for each disease subtype in the selected cancer. The final module, BulkExpression, contains the bulk RNA-seq data for the selected cancer type and allows users to view gene expression or analyze the survival associations of an input gene or gene set. The web resource source code is written in R programming language and the interactive web server has been implemented using the R Shiny and ShinyDashboard packages. The PedScAtlas resource provides a unique and straightforward tool for biomarker identification, analysis of pediatric cancer subtype heterogeneity, and transcriptome profile of the tumor microenvironment. The resource is available online at https://bhasinlab.bmi.emory.edu/PediatricSC/. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NIMG-106. WHAT’S BEHIND TSPO PET LABELLING: DIGGING FOR THE HISTOLOGICAL, CELLULAR AND MOLECULAR CORRELATES

Abstract TSPO is frequently upregulated in glioblastoma. Despite its use as an imaging biomarker for PET in glioma, the interpretation is challenging due to the heterogeneity of cell populations that could contribute to the TSPO-PET signal. We therefore dissected TSPO labelling in connection with the underlying histopathological and molecular features in biopsy samples from glioma patients. Comparing regions of high and low TSPO-PET signal in 58 patients, we assessed TSPO protein expression and expression of cell differentiation markers by use of immunohistochemistry on consecutive sections and by multiplex stains. Our results suggest that apart from microglia and macrophages the glial tumor cells relevantly contribute to the overall TSPO signal in glioma patients. To identify hallmarks and GO terms associated with TSPO labelling/expression we next performed RNAseq in 33 patients. Using DESeq2 followed by FUMA and Reactome as well as GSEA with normalized counts, we identified three TSPO-dependent functional clusters, i.e. apoptosis/DNA repair, extracellular matrix organization and immune system. On the DNA level, we analyzed the TSPO promoter by direct bisulfite sequencing suggesting that a loss of TSPO methylation may mechanistically contribute to the TSPO overexpression observed in high-grade gliomas. Exceeding our investigations on glioma biopsies we stained tissue microarrays (130 individuals) for TSPO that cover a broader spectrum of both human brain pathologies (13 different entities) and non-neoplastic tissues (14 different brain regions). We here found a high degree of heterogeneity of TSPO expression between entities and regions. Merging this information with TSPO-PETs from respective patients/brain regions allows to generate a map of TSPO expression in healthy and diseased brain for clinical use. Altogether, our approach of integrating histological, molecular and imaging data will provide unique insights into TSPO-PET enrichment patterns which may help to better understand and to comprehensively describe the clinical relevance of this novel imaging biomarker.

Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina-cervix-decidua-organ-on-a-chip and feto-maternal interface-organ-on-a-chip.

Genital mycoplasmas can break the cervical barrier and cause intraamniotic infection and preterm birth. This study developed a six-chamber vagina-cervix-decidua-organ-on-a-chip (VCD-OOC) that recapitulates the female reproductive tract during pregnancy with culture chambers populated by vaginal epithelial cells, cervical epithelial and stromal cells, and decidual cells. Cells cultured in VCD-OOC were characterized by morphology and immunostaining for cell-specific markers. We transferred the media from the decidual cell chamber of the VCD-OOC to decidual cell chamber in feto-maternal interface organ-on-a-chip (FMi-OOC), which contains the fetal membrane layers. An ascending Ureaplasma parvum infection was created in VCD-OOC. U. parvum was monitored for 48 h post-infection with their cytotoxicity (LDH assay) and inflammatory effects (multiplex cytokine assay) in the cells tested. An ascending U. parvum infection model of PTB was developed using CD-1 mice. The cell morphology and expression of cell-specific markers in the VCD-OOC mimicked those seen in lower genital tract tissues. U. parvum reached the cervical epithelial cells and decidua within 48 h and did not cause cell death in VCD-OOC or FMi-OOC cells. U. parvum infection promoted minimal inflammation, while the combination of U. parvum and LPS promoted massive inflammation in the VCD-OOC and FMi-OOC cells. In the animal model, U. parvum vaginal inoculation of low-dose U. parvum did not result in PTB, and even a high dose had only some effects on PTB (20%). However, intra-amniotic injection of U. parvum resulted in 67% PTB. We report the colonization of U. parvum in various cell types; however, inconsistent, and low-grade inflammation across multiple cell types suggests poor immunogenicity induced by U. parvum.

DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.

Neuroblastoma is the most common cancer in infants and the most common extracranial solid tumor in childhood. DRR1 was identified to be downregulated in poorly differentiated ganglion cells from neuroblastoma model mice. However, the roles of DRR1 in neuroblastoma remain largely unclear. The neuroblastoma cells were induced to differentiate, and the expression of DRR1 was detected. The expression of the neuroblastoma cell differentiation markers was analyzed in DRR1 shRNA- or DRR1-expressing vector-treated neuroblastoma cells. The downstream genes of DRR1 were screened with ChIP-seq assay. Finally, TNB1 cells were infected with DRR1 shRNA and CREB expressing vector containing lentivirus, and the expression of the cell differentiation markers, cell cycle distribution and tumor growth were analyzed. The expression of DRR1 was increased in differentiated neuroblastoma cells, and downregulation of DRR1 expression inhibited the differentiation of neuroblastoma cells. Further experiments indicated that CREB is a candidate downstream gene of DRR1, and it mediates neuroblastoma cell differentiation. Moreover, overexpression of CREB rescued the effect of DRR1 shRNA on cell differentiation, cell cycle distribution and tumor growth in neuroblastoma. DRR1-CREB axis modulates the differentiation of neuroblastoma cells and is associated with the outcome of neuroblastoma patients. DRR1 is involved in regulation of the differentiation of neuroblastoma. Binding with actin is essential for DRR1 to regulate neuroblastoma cell differentiation. CREB is a candidate downstream gene of DRR1 in regulating of the differentiation of neuroblastoma.

Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants

Gene therapy is a rapidly developing field, and adeno-associated viruses (AAVs) are a leading viral-vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wild-type and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA sequencing (RNA-seq) AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers except for a photoreceptor population with altered expression of photoreceptor-specific genes. The efficiency and tropism of AAVs in human explants were quantified with single-cell resolution. The top-performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

Cervix-on-a-chip for investigating ascending Ureaplasma parvum infection in pregnancy

To develop a microfluidic cervix-on-a-chip (COC) to study ascending bacterial infection in pregnancy. COC with 6 interconnected culture chambers connected by microchannels containing cells from vagina (VEC), cervical epithelium and stroma, transition zone, and decidua was developed. Cells in COC were characterized by morphology and expression of cell-specific markers. An ascending Ureaplasma parvum (UP) infection was tested by inoculating UP (107 CCU) in VEC. Propagation of infection in COC (fluorescent microscopy), cytotoxicity (LDH assay) and inflammation (cytokine assays) by UP (107 or 1010 CCU) were determined. For physiological validation of in vitro data, we inoculated UP vaginally (107 or 1010 CCU) in pregnant mice and monitored for preterm birth (PTB). Cellular characteristics (morphology, collagen production, and expression of markers) in the COC were as seen in vivo, validated devices’ physiological relevance (Fig. 1). In vitro, ascending U. parvum colonized the cervical epithelial and decidual cells within 48 h. Both low and high dose UP colonization did not promote cell death in any of the COC cells. Low dose UP increased IL-8 (stroma) while high dose UP increased GM-CSF (ectocervix) and IL-8 (stroma), and IL-6 and IL-8 (decidua) (Fig. 1). Vaginal inoculation of low and high doses of UP infection had no or 20% PTB rate, respectively compared to 90% PTB rate in E. coli models (positive control). Intraamniotic injection of low dose UP increased PTB rate to 67% (Fig. 2). In vitro cervix-on-a-chip model of ascending infection showed UP colonization with poor immunogenicity. This finding was recapitulated in vivo. Validated COC is useful for in vitro preclinical studies in pregnancy and parturition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Pediatric Single Cell Cancer Atlas: An Integrative Web-Based Resource for Single Cell Transcriptome Data from Pediatric Leukemias

Introduction: The emergence and optimization of single cell profiling as a powerful tool to characterize the tumor microenvironment has revealed the heterogeneity of pediatric cancers, particularly different leukemia types/subtypes. Ease of access and analysis of the data from studies on different leukemia types is critical for improving diagnosis as well as therapy.Currently, there are no single cell data based resources available for pediatric leukemias. We have developed a comprehensive resource, Pediatric Single Cell Cancer Atlas (PedScAtlas), with the goal of developing a pan-leukemia genomics signature as well as highlighting the heterogeneity of different types of leukemia. This resource facilitates exploration and visualization of expression signatures in different leukemias without requiring extensive analysis and bioinformatics support.Methods: The PedScAtlas was built based on single cell data from various leukemias and normal bone marrow (BM) cells that have been pre-processed and analyzed using a uniform approach to generate normalized expression data (M. Bhasin et al. Blood 2020 (ASH), S. S. Bhasin et al. Blood 2020 (ASH), Panigraphy et al. JCI 2019, Stroopinsky et al. Haematologica 2021, Thomas et al. Blood 2020 (ASH)). The current version of PedScAtlas contains data from 30 local leukemia samples (Bhasin, et al. Blood 2020 (ASH), Thomas et al. Blood 2020 (ASH)) and those available on public resources such as GEO (Bailur et al. JCI Insight 2020). The PedScAtlas dataset and the Immune cell dataset each underwent quality control, integration, normalization, and dimensionality reduction using the Uniform Manifold Approximation and Projection (UMAP) method. Unsupervised UMAP analysis identified cellular clusters with similar transcriptome profiles that were annotated based on expression of cell-specific markers. Differential expression comparing different types of leukemia including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and mixed phenotype acute leukemia (MPAL) samples was performed. To further ascertain genes specifically expressed in malignant blasts, the atlas also contains data from normal BM samples (Bailur et al. JCI Insight 2020) and healthy immune cells from the census of Immune Cells by the Human Cell Atlas Project (https://data.humancellatlas.org/). The web resource source code is written in R programming language and the interactive webserver has been implemented using the R Shiny package (Fig 1). The tool has been extensively tested on multiple operating systems (Linux, Mac, Windows) and web-browsers (Chrome, FireFox, and Safari). The tool is currently hosted on a 64bit CentOS 6 backend server running the Shiny Server program designed to host R Shiny applications.Results: The PedScAtlas contains data that facilitate exploration of gene expression profiles across leukemia types/subtypes and tumor microenvironment (TME) cell types. The atlas includes data from 33,930 AML, 25,744 T-ALL, 13,404 MPAL, and 6,252 B-ALL blast cells. It also contains single cell profiles of healthy BM samples from publicly available studies. The user can select data sets from the 5 major types of leukemia and normal BM in any combination of their choice to explore the expression profile of a gene of interest. The data can be visualized as UMAP (Fig. 1), or violin plots with annotations based on cluster ID, cell type, disease type, sample ID, and future continuous remission or relapse outcome. The UMAP with gene expression analyses allows the user to visualize the distribution of cell expressing a given gene on the UMAP plot. The Biomarker tool shows expression of different leukemia biomarker gene sets in the entire leukemia dataset. The Immune Cell section contains BM data from the Human Cell Atlas Project; the purpose of this section is to validate leukemia biomarkers by checking that the gene does not have significant expression in the healthy immune microenvironment.Conclusions: The PedScAtlas resource provides a unique and straightforward tool for biomarker identification, analysis of leukemia subtype heterogeneity, and transcriptome profile of the immune cell microenvironment. The resource is available online at https://bhasinlab.bmi.emory.edu/PediatricSC/. [Display omitted] DisclosuresDeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors.

Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1β and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses.

Gleevec and Rapamycin Synergistically Reduce Cell Viability and Inhibit Proliferation and Angiogenic Function of Mouse Bone Marrow-Derived Endothelial Progenitor Cells

Objective: This study investigates the synergistic effects of Gleevec (imatinib) and rapamycin on the proliferative and angiogenic properties of mouse bone marrow-derived endothelial progenitor cells (EPCs). Materials and Methods: EPCs were isolated from mouse bone marrow and treated with different concentrations of Gleevec or rapamycin individually or in combination. The cell viability and proliferation were examined using the MTT assay. An analysis of cell cycle and apoptosis was performed using flow cytometry. Formation of capillary-like tubes was examined in vitro, and the protein expression of cell differentiation markers was determined using Western blot analysis. Results: Gleevec significantly reduced cell viability, cell proliferation, and induced cell apoptosis in EPCs. Rapamycin had similar effects on EPCs, but it did not induce cell apoptosis. The combination of Gleevec and rapamycin reduced the cell proliferation but increased cell apoptosis. Although rapamycin had no demonstratable effect on tube formation, the combined therapy of Gleevec and rapamycin significantly reduced tube formation when compared with Gleevec alone. Mechanistically, Gleevec, but not rapamycin, induced a significant elevation in caspase-3 activity in EPCs, and it attenuated the expression of the endothelial protein marker platelet-derived growth factor receptor α. Functionally, rapamycin, but not Gleevec, significantly enhanced the expression of endothelial differentiation marker proteins, while attenuating the expression of mammalian target of rapamycin signaling-related proteins. Conclusions: Gleevec and rapamycin synergistically suppress cell proliferation and tube formation of EPCs by inducing cell apoptosis and endothelial differentiation. Mechanistically, it is likely that rapamycin enhances the proapoptotic and antiangiogenic effects of Gleevec by promoting the endothelial differentiation of EPCs. Given that EPCs are involved in the pathogenesis of some cardiovascular diseases and critical to angiogenesis, pharmacological inhibition of EPC proliferation by combined Gleevec and rapamycin therapy may be a promising approach for suppressing cardiovascular disease pathologies associated with angiogenesis.

Measurement of the Adipose Stem Cells Cell Sheets Transmittance.

In the field of cell therapy, the interest in cell sheet technology is increasing. To determine the cell sheet harvesting time requires experience and practice, and different factors could change the harvesting time (variability among donors and culture media, between cell culture dishes, initial cell seeding density). We have developed a device that can measure the transmittance of the multilayer cell sheets, using a light emitting diode and a light detector, to estimate the harvesting time. The transmittance of the adipose stromal cells cell sheets (ASCCS) was measured every other day as soon as the cells were confluent, up to 12 days. The ASCCS, from three different initial seeding densities, were harvested at 8, 10, and 12 days after seeding. Real-time PCR and immunostaining confirmed the expression of specific cell markers (CD29, CD73, CD90, CD105, HLA-A, HLA-DR), but less than the isolated adipose stromal cells. The number of cells per cell sheets, the average thickness per cell sheet, and the corresponding transmittance showed no correlation. Decrease of the transmittance seems to be correlated with the cell sheet maturation. For the first time, we are reporting the success development of a device to estimate ASCCS harvesting time based on their transmittance.

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron

BackgroundTherapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). Methods and materialshADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. ResultsMatrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. ConclusionsOverall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine.

Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.

Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient’s healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient’s iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.