We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct–like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.
Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz
Bone metastases (BoM) are a significant cause of morbidity in patients with estrogen receptor–positive (ER-positive) breast cancer; yet, characterizations of human specimens are limited. In this study, exome-capture RNA sequencing (ecRNA-seq) on aged (8–12 years), formalin-fixed, paraffin-embedded (FFPE), and decalcified cancer specimens was evaluated. Gene expression values and ecRNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared with matched flash-frozen or nondecalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matched synchronous or recurrent BoMs. Overtime, BoMs exhibited gene expression shifts to more Her2 and LumB PAM50 subtype profiles, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets, and longitudinal expression alterations of clinically actionable genes, particularly in the CDK/Rb/E2F and FGFR signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines recurrent longitudinal transcriptional remodeling events in estrogen-deprived breast cancers.
Nolan Priedigkeit, Rebecca J. Watters, Peter C. Lucas, Ahmed Basudan, Rohit Bhargava, William Horne, Jay K. Kolls, Zhou Fang, Margaret Q. Rosenzweig, Adam M. Brufsky, Kurt R. Weiss, Steffi Oesterreich, Adrian V. Lee
The tumor microenvironment imposes physical and functional constraints on the antitumor efficacy of adoptive T cell immunotherapy. Preclinical testing of different T cell preparations can help in the selection of efficient immune therapies, but in vivo models are expensive and cumbersome to develop, while classical in vitro 2D models cannot recapitulate the spatiotemporal dynamics experienced by T cells targeting cancer. Here, we describe an easily customizable 3D model, in which the tumor microenvironment conditions are modulated and the functionality of different T cell preparations is tested. We incorporate human cancer hepatocytes as a single cell or as tumor cell aggregates in a 3D collagen gel region of a microfluidic device. Human T cells engineered to express tumor-specific T cell receptors (TCR–T cells) are then added in adjacent channels. The TCR–T cells’ ability to migrate and kill the tumor target and the profile of soluble factors were investigated under conditions of varying oxygen levels and in the presence of inflammatory cytokines. We show that only the 3D model detects the effect that oxygen levels and the inflammatory environment impose on engineered TCR–T cell function, and we also used the 3D microdevice to analyze the TCR–T cell efficacy in an immunosuppressive scenario. Hence, we show that our microdevice platform enables us to decipher the factors that can alter T cell function in 3D and can serve as a preclinical assay to tailor the most efficient immunotherapy configuration for a specific therapeutic goal.
Andrea Pavesi, Anthony T. Tan, Sarene Koh, Adeline Chia, Marta Colombo, Emanuele Antonecchia, Carlo Miccolis, Erica Ceccarello, Giulia Adriani, Manuela T. Raimondi, Roger D. Kamm, Antonio Bertoletti
Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.
Nataliya P. Buxbaum, Donald E. Farthing, Natella Maglakelidze, Martin Lizak, Hellmut Merkle, Andrea C. Carpenter, Brittany U. Oliver, Veena Kapoor, Ehydel Castro, Gregory A. Swan, Liliane M. dos Santos, Nicolas J. Bouladoux, Catherine V. Bare, Francis A. Flomerfelt, Michael A. Eckhaus, William G. Telford, Yasmine Belkaid, Remy J. Bosselut, Ronald E. Gress
A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver’s natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450–mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.
Soon Seng Ng, Anming Xiong, Khanh Nguyen, Marilyn Masek, Da Yoon No, Menashe Elazar, Eyal Shteyer, Mark A. Winters, Amy Voedisch, Kate Shaw, Sheikh Tamir Rashid, Curtis W. Frank, Nam Joon Cho, Jeffrey S. Glenn
Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical mediator of type 2 immune response to helminth infection. Here, we present advances in segmentation algorithms and analytical tools for multiplex immunofluorescence (MxIF), a platform that enables iterative staining of over 60 antibodies on a single tissue section. These refinements have enabled a comprehensive analysis of tuft cell number, distribution, and protein expression profiles as a function of anatomical location and physiological perturbations. Based solely on DCLK1 immunoreactivity, tuft cell numbers were similar throughout the mouse small intestine and colon. However, multiple subsets of tuft cells were uncovered when protein coexpression signatures were examined, including two new intestinal tuft cell markers, Hopx and EGFR phosphotyrosine 1068. Furthermore, we identified dynamic changes in tuft cell number, composition, and protein expression associated with fasting and refeeding and after introduction of microbiota to germ-free mice. These studies provide a foundational framework for future studies of intestinal tuft cell regulation and demonstrate the utility of our improved MxIF computational methods and workflow for understanding cellular heterogeneity in complex tissues in normal and disease states.
Eliot T. McKinley, Yunxia Sui, Yousef Al-Kofahi, Bryan A. Millis, Matthew J. Tyska, Joseph T. Roland, Alberto Santamaria-Pang, Christina L. Ohland, Christian Jobin, Jeffrey L. Franklin, Ken S. Lau, Michael J. Gerdes, Robert J. Coffey
Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (>60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research. We designed LipidFinder, an open-source Python workflow. An algorithm is included that optimizes analysis based on users’ own data, and outputs are screened against online databases and categorized into LIPID MAPS classes. LipidFinder outperformed three widely used metabolomics packages using data from human platelets. We show a family of three 12-hydroxyeicosatetraenoic acid phosphoinositides (16:0/, 18:1/, 18:0/12-HETE-PI) generated by thrombin-activated platelets, indicating crosstalk between eicosanoid and phosphoinositide pathways in human cells. The software is available on GitHub (https://github.com/cjbrasher/LipidFinder), with full userguides.
Anne O’Connor, Christopher J. Brasher, David A. Slatter, Sven W. Meckelmann, Jade I. Hawksworth, Stuart M. Allen, Valerie B. O’Donnell
Quantification of stable isotope tracers has revealed the dynamic state of living tissues. A new form of imaging mass spectrometry quantifies isotope ratios in domains much smaller than a cubic micron, enabling measurement of cell turnover and metabolism with stable isotope tracers at the single-cell level with a methodology we refer to as multi-isotope imaging mass spectrometry. In a first-in-human study, we utilize stable isotope tracers of DNA synthesis and de novo lipogenesis to prospectively measure cell birth and adipocyte lipid turnover. In a study of healthy adults, we elucidate an age-dependent decline in new adipocyte generation and adipocyte lipid turnover. A linear regression model suggests that the aging effect could be mediated by a decline in insulin-like growth factor-1 (IGF-1). This study therefore establishes a method for measurement of cell turnover and metabolism in humans with subcellular resolution while implicating the growth hormone/IGF-1 axis in adipose tissue aging.
Christelle Guillermier, Pouneh K. Fazeli, Soomin Kim, Mingyue Lun, Jonah P. Zuflacht, Jessica Milian, Hang Lee, Hugues Francois-Saint-Cyr, Francois Horreard, David Larson, Evan D. Rosen, Richard T. Lee, Claude P. Lechene, Matthew L. Steinhauser
Idiopathic pulmonary fibrosis (IPF) is a fatal progressive fibrotic lung disease characterized by the presence of invasive myofibroblasts in the lung. Currently, there are only two FDA-approved drugs (pirfenidone and nintedanib) for the treatment of IPF. There are no defined criteria to guide specific drug therapy. New methodologies are needed not only to predict personalized drug therapy, but also to screen novel molecules that are on the horizon for treatment of IPF. We have developed a model system that exploits the invasive phenotype of IPF lung tissue. This ex vivo 3D model uses lung tissue from patients to develop pulmospheres. Pulmospheres are 3D spheroids composed of cells derived exclusively from primary lung biopsies and inclusive of lung cell types reflective of those in situ, in the patient. We tested the pulmospheres of 20 subjects with IPF and 9 control subjects to evaluate the responsiveness of individual patients to antifibrotic drugs. Clinical parameters and outcomes were also followed in the same patients. Our results suggest that pulmospheres simulate the microenvironment in the lung and serve as a personalized and predictive model for assessing responsiveness to antifibrotic drugs in patients with IPF.
Ranu Surolia, Fu Jun Li, Zheng Wang, Huashi Li, Gang Liu, Yong Zhou, Tracy Luckhardt, Sejong Bae, Rui-ming Liu, Sunad Rangarajan, Joao de Andrade, Victor J. Thannickal, Veena B. Antony
Functional intestines are composed of cell types from all 3 primary germ layers and are generated through a highly orchestrated and serial developmental process. Directed differentiation of human pluripotent stem cells (hPSCs) has been shown to yield gut-specific cell types; however, these structures do not reproduce critical functional interactions between cell types of different germ layers. Here, we developed a simple protocol for the generation of mature functional intestinal organoids from hPSCs under xenogeneic-free conditions. The stem cell–derived gut organoids produced here were found to contain distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, that were derived from all 3 germ layers; moreover, they demonstrated intestinal functions, including peptide absorption, and showed innervated bowel movements in response to stimulation with histamine and anticholinergic drugs. Importantly, the gut organoids obtained using this xenogeneic-free system could be stably maintained in culture for prolonged periods and were successfully engrafted in vivo. Our xenogeneic-free approach for generating gut organoids from hPSCs provides a platform for studying human intestinal diseases and for pharmacological testing.
Hajime Uchida, Masakazu Machida, Takumi Miura, Tomoyuki Kawasaki, Takuya Okazaki, Kengo Sasaki, Seisuke Sakamoto, Noriaki Ohuchi, Mureo Kasahara, Akihiro Umezawa, Hidenori Akutsu
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