Lactate and Immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs

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Abstract

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for four weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transients measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.

Authors

Kalina J. Rossler, Willem J. De Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge

Validation of a murine proteome-wide phage display library for identification of autoantibody specificities

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Abstract

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Authors

Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi

A lipid-associated macrophage lineage rewires the spatial landscape of adipose tissue in early obesity

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Abstract

Adipose tissue macrophage (ATM) infiltration is associated with adipose tissue dysfunction and insulin resistance in mice and humans. Recent single cell data highlight increased ATM heterogeneity in obesity, but do not provide a spatial context for ATM phenotype dynamics. We integrated single cell RNA-sequencing, spatial transcriptomics, and imaging of murine adipose tissue in a time course of diet-induced obesity. Overall, proinflammatory immune cells were predominant in early obesity, while non-resident antiinflammatory ATMs predominated in chronic obesity. A subset of these antiinflammatory ATMs were transcriptomically intermediate between monocytes and mature lipid-associated macrophages (LAM) and were consistent with a LAM precursor (pre-LAM). Pre-LAMs were spatially associated with early obesity crown-like structures (CLS), which indicate adipose tissue dysfunction. Spatial data showed colocalization of ligand-receptor transcripts related to lipid signaling among monocytes, pre-LAMs, and LAMs, including Apoe, Lrp1, Lpl and App. Pre-LAM expression of these ligands in early obesity suggested signaling to LAMs in the CLS microenvironment. Our results refine understanding of ATM diversity and provide insight into the dynamics of the LAM lineage during development of metabolic disease.

Authors

Cooper M. Stansbury, Gabrielle A. Dotson, Harrison Pugh, Alnawaz Rehemtulla, Indika Rajapakse, Lindsey A. Muir

Single-cell transcriptome analysis and protein profiling reveal broad immune system activation in IgG4-related disease

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Abstract

IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with unclear pathogenesis. We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naïve IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation. We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with number of involved organs in IgG4-RD. Increased proportions of CD4+ cytotoxic T lymphocytes (CTLs), CD8+ CTLs-GNLY (granulysin) and γδT cells with enhanced chemotaxis and cytotoxicity but with suppressed inhibitory receptors characterize IgG4-RD. Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs) including PRDM1/XBP1 and RUNX3 were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively. Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.

Authors

Chenyang Lu, Shasha Li, Pingying Qing, Qiuping Zhang, Xing Ji, Zhigang Tang, Chunyan Chen, Tong Wu, Yidan Hu, Yi Zhao, Xiaohui Zhang, Qi He, David A. Fox, Chunyu Tan, Yubin Luo, Yi Liu

Transcriptional profiling of rare acantholytic disorders suggests common mechanisms of pathogenesis

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Abstract

Darier, Hailey-Hailey, and Grover’s diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover’s disease were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared upregulation in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover’s disease. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Further, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared to normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover’s disease, and identify decreases in actin organization pathways as a unique signature present in these conditions.

Authors

Quinn R. Roth-Carter, Hope E. Burks, Ziyou Ren, Jennifer L. Koetsier, Lam C. Tsoi, Paul W. Harms, Xianying Xing, Joseph Kirma, Robert M. Harmon, Lisa M. Godsel, Abbey L. Perl, Johann E. Gudjonsson, Kathleen J. Green

Single-cell profiling reveals comparatively inflammatory polarization of human carotid versus femoral plaque leukocytes

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Abstract

Rationale: Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. Objectives: We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 total patients undergoing femoral (N=23) or carotid (N=26) endarterectomy using single-cell ribonucleic acid sequencing (scRNA-seq; N=13), flow cytometry (N=24), and immunohistochemistry (N=12). Findings: Comparative scRNA-seq of CD45 positive-selected leukocytes from femoral (N=9; 35265 cells) and carotid (N=4; 30655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised 2.5- to 4-fold higher proportions of myeloid cells in carotid plaques, whereas non-inflammatory foam cell-like macrophages and LYVE1-overexpressing resident-like macrophages comprised 3.5- to 9-fold higher proportions of myeloid cells in femoral plaque (p<0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus femoral plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively anti-inflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. Conclusion: Human femoral plaques exhibit distinct macrophage profiles and diminished CD8+ T cell populations compared with carotid plaques. Experimental models elucidating determinants of plaque site-specific cell polarization cues are warranted.

Authors

Joshua Slysz, Arjun Sinha, Matthew DeBerge, Shalini Singh, Harris Avgousti, Inhyeok Lee, Kristofor Glinton, Reina Nagasaka, Prarthana J. Dalal, Shaina J. Alexandria, Ching Man Wai, Ricardo Tellez, Mariavittoria Vescovo, Ashwin Sunderraj, Xinkun Wang, Matthew J. Schipma, Ryan K Sisk, Rishab Gulati, Jenifer Vallejo, Ryosuke Saigusa, Donald M. Lloyd-Jones, Jon Lomasney, Samuel E. Weinberg, Karen J. Ho, Klaus Ley, Chiara Giannarelli, Edward B. Thorp, Matthew J. Feinstein

Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells

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Abstract

Specific and efficient smooth muscle cell (SMC)-targeted gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing Bacterial Artificial Chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the 1) CreNLSP2A or 2) CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre knock-in mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our new models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Authors

Yang Zhao, Guizhen Zhao, Ziyi Chang, Tianqing Zhu, Ying Zhao, Haocheng Lu, Chao Xue, Thomas L. Saunders, Yanhong Guo, Lin Chang, Y. Eugene Chen, Jifeng Zhang

Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics

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Abstract

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long-lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here we investigated qualitatively the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a unique droplet microfluidic and imaging approach, we analyzed >4,000 single IgG-secreting cells revealing high inter-individual variability in affinity for RBD with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented >65% of the plasmablast response at all timepoints. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

Authors

Matteo Broketa, Aurélien Sokal, Michael Mor, Pablo Canales-Herrerias, Angga Perima, Annalisa Meola, Ignacio Fernández, Bruno Iannascoli, Guilhem Chenon, Alexis Vandenberghe, Laetitia Languille, Marc Michel, Bertrand Godeau, Sebastien Gallien, Giovanna Melica, Marija Backovic, Felix A. Rey, Jean Baudry, Natalia T. Freund, Matthieu Mahevas, Pierre Bruhns

A modeling-based approach to optimize COVID-19 vaccine dosing schedules for improved protection

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Abstract

While the development of different vaccines slowed the dissemination of SARS-CoV-2, the occurrence of breakthrough infections has continued to fuel the COVID-19 pandemic. To at least secure partial protection in majority of the population through one dose of a COVID-19 vaccine, delayed administration of boosters has been implemented in many countries. However, waning immunity and emergence of new variants of SARS-CoV-2 suggest that such measures may induce breakthrough infections due to intermittent lapses in protection. Optimizing vaccine dosing schedules to ensure prolonged continuity in protection could thus help control the pandemic. We developed a mechanistic model of immune response to vaccines as an in-silico tool for dosing schedule optimization. The model was calibrated with clinical datasets of acquired immunity to COVID-19 mRNA vaccines in healthy and immunocompromised subjects and showed robust validation by accurately predicting neutralizing antibody kinetics in response to multiple doses of COVID-19 mRNA vaccines. Importantly, by estimating population vulnerability to breakthrough infections, we predicted tailored vaccination dosing schedules to minimize breakthrough infections, especially for immunocompromised subjects. We identified that the optimal vaccination schedules vary from CDC-recommended dosing, suggesting that the model is a valuable tool to optimize vaccine efficacy outcomes during future outbreaks.

Authors

Prashant Dogra, Carmine Schiavone, Zhihui Wang, Javier Ruiz-Ramírez, Sergio Caserta, Daniela I. Staquicini, Christopher Markosian, Jin Wang, H. Dirk Sostman, Renata Pasqualini, Wadih Arap, Vittorio Cristini

Foramen lacerum impingement of trigeminal nerve root (FLIT) as a rodent model for trigeminal neuralgia

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Abstract

Trigeminal neuralgia (TN) is a classical neuralgic pain condition with distinct clinical characteristics. Modeling TN in rodents proves challenging. Recently, we found that a foramen in rodent skull base, the foramen lacerum, provides direct access to the trigeminal nerve root. Using this access, we developed FLIT (Foramen Lacerum Impingement of Trigeminal nerve root) model and observed distinct pain-like behaviors in rodents, including paroxysmal asymmetric facial grimaces, head tilt when eating, avoidance of solid chew, lack of wood chewing, etc. The FLIT model recapitulated key clinical features of TN, including lancinating pain-like behavior, and dental pain-like behavior. Importantly, when compared with a trigeminal neuropathic pain model (infraorbital nerve chronic constriction injury, IoN-CCI), the FLIT model was associated with significantly higher numbers of c-Fos positive cells in the primary somatosensory cortex (S1), unraveling robust cortical activation in the FLIT model. Using intravital two-photon calcium imaging, synchronized S1 neural dynamics were only present in the FLIT but not the IoN-CCI model, revealing differential implication of cortical activation in different pain models. Taken together, FLIT is a clinically relevant rodent model of TN which could facilitate pain research and therapeutics development.

Authors

Weihua Ding, Liuyue Yang, Qian Chen, Kun Hu, Yan Liu, Eric Bao, Changning Wang, Jianren Mao, Shiqian Shen