|
1
|
Gamble JT, Deisenroth C. Profiling assay performance in the DevTox germ layer reporter platform. Curr Res Toxicol 2025; 8:100223. [PMID: 40017496 PMCID: PMC11867229 DOI: 10.1016/j.crtox.2025.100223] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Revised: 01/22/2025] [Accepted: 01/31/2025] [Indexed: 03/01/2025] Open
Abstract
The U.S. Environmental Protection Agency (U.S. EPA) is mandated to develop new approach methods (NAMs) to detect chemicals risks to susceptible populations, including effects on pregnant women and their offspring. With limited hazard information available for current and new chemicals, NAMs can provide greater relevance to human biology, mechanistic insight, and higher testing capacity than traditional animal models. The DevTox Germ Layer Reporter (GLR) model platform was recently established for high-throughput screening and prioritization of potential developmental hazards. The model platform utilizes the RUES2-GLR pluripotent stem cell reporter line that expresses fluorescent fusion protein biomarkers SOX17 (endoderm), Brachyury (mesoderm), and SOX2 (ectoderm and pluripotency); enabling a multi-lineage readout of gastrulation lineages. The DevTox GLR-Endo assay used the model platform to evaluate chemical effects on differentiating endoderm, yielding a balanced accuracy (BA) of 72% against a training set of 43 developmental toxicants and 23 non-developmental toxicants. To assess the predictivity of additional early embryonic lineages, assays for pluripotency (DevTox GLR-Pluri), ectoderm (DevTox GLR-Ecto), and mesoderm (DevTox GLR-Meso) were developed. Chemical reference set (12 developmental toxicants and 4 non-developmental toxicants) activity for each assay revealed BAs of 92% for DevTox GLR-Endo and DevTox GLR-Pluri, 71% for DevTox GLR-Ecto, and 58% for DevTox GLR-Meso. Expanded testing of the DevTox GLR-Endo and DevTox GLR-Pluri with 63 developmental and non-developmental toxicants yielded BAs of 75% and 68%, respectively. Amongst the four DevTox GLR platform assays, the DevTox GLR-Endo assay maintained the highest degree of efficacy and overall predictive accuracy for the compound set evaluated in this study.
Collapse
Affiliation(s)
- John T. Gamble
- Center for Computational Toxicology and Exposure, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 United States
- Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN 37831, United States
| | - Chad Deisenroth
- Center for Computational Toxicology and Exposure, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 United States
| |
Collapse
|
|
2
|
Smith L, Quelch-Cliffe R, Liu F, Aguilar AH, Przyborski S. Evaluating Strategies to Assess the Differentiation Potential of Human Pluripotent Stem Cells: A Review, Analysis and Call for Innovation. Stem Cell Rev Rep 2025; 21:107-125. [PMID: 39340737 PMCID: PMC11762643 DOI: 10.1007/s12015-024-10793-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/23/2024] [Indexed: 09/30/2024]
Abstract
Pluripotent stem cells have the ability to differentiate into all cells and tissues within the human body, and as a result they are attractive resources for use in basic research, drug discovery and regenerative medicine. In order to successfully achieve this application, starting cell sources ideally require in-depth characterisation to confirm their pluripotent status and their ability to differentiate into tissues representative of the three developmental germ layers. Many different methods to assess potency are employed, each having its own distinct advantages and limitations. Some aspects of this characterisation process are not always well standardised, particularly techniques used to assess pluripotency as a function. In this article, we consider the methods used to establish cellular pluripotency and subsequently analyse characterisation data for over 1590 human pluripotent cell lines from publicly available repositories in the UK and USA. In particular, we focus on the teratoma xenograft assay, its use and protocols, demonstrating the level of variation and the frequency with which it is used. Finally, we reflect on the implications of the findings, and suggest in vitro alternatives using modern innovative technology as a way forward.
Collapse
Affiliation(s)
- Lucy Smith
- Department of Biosciences, Durham University, Durham, England
| | | | - Felicity Liu
- Department of Biosciences, Durham University, Durham, England
| | | | - Stefan Przyborski
- Department of Biosciences, Durham University, Durham, England.
- Reprocell Europe Ltd, NETPark, Sedgefield, England.
| |
Collapse
|
|
3
|
Rodriguez-Lopez A, Huang X, Chen C, Zou J, Zheng W, Chen G. Generation of an induced pluripotent stem cell line (TRNDi042-A) from a Mucopolysaccharidosis type IIIB patient with homozygous p. R626X (c. 1876C > T) mutation in the NAGLU gene. Stem Cell Res 2024; 81:103612. [PMID: 39579553 PMCID: PMC11787772 DOI: 10.1016/j.scr.2024.103612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/09/2024] [Accepted: 11/13/2024] [Indexed: 11/25/2024] Open
Abstract
Mucopolysaccharidosis type IIIB (MPS IIIB), also known as Sanfilippo syndrome, is an autosomal recessive lysosomal storage disorder caused by mutations in the NAGLU gene. It is characterized by progressive neurodegeneration, behavioral problems, and motor function difficulties. A human induced pluripotent stem cell (iPSC) TRNDi042-A line was generated from fibroblasts of a male patient with a homozygous p. R626X (c.1876C > T) in the NAGLU gene producing N-acetyl-glucosaminidase. This iPSC line is a useful resource to study disease pathophysiology and to develop therapeutics treatments. The cell line has a normal karyotype, is free of plasmid integration, and expresses high levels of pluripotency-associated markers.
Collapse
Affiliation(s)
- Alexander Rodriguez-Lopez
- National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Xiuli Huang
- National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Catherine Chen
- National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Jizhong Zou
- iPSC Core, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
| | - Wei Zheng
- National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA
| | - Guibin Chen
- National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
| |
Collapse
|
|
4
|
Tsukamoto M, Kawasaki T, Vemuri MC, Umezawa A, Akutsu H. A passage-free, simplified, and scalable novel method for iPSC generation in three-dimensional culture. Regen Ther 2024; 27:39-47. [PMID: 38496009 PMCID: PMC10940796 DOI: 10.1016/j.reth.2024.02.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 02/17/2024] [Accepted: 02/25/2024] [Indexed: 03/19/2024] Open
Abstract
Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.
Collapse
Affiliation(s)
- Masaya Tsukamoto
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
| | - Tomoyuki Kawasaki
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
| | - Mohan C. Vemuri
- Thermo Fisher Scientific, 7335 Executive Way, Frederick, MD 21702, USA
| | - Akihiro Umezawa
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
| | - Hidenori Akutsu
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
| |
Collapse
|
|
5
|
Boullé M, Leleu A, Schacre S, Banal C, Boucharlat A, Renault S, Hollenstein M, Frosk P, Yates F, Lefort N, Agou F. Generation of IPi002-A/B/C human induced pluripotent stem cell lines from MARCH amniotic fluid cells. Stem Cell Res 2024; 81:103589. [PMID: 39447316 DOI: 10.1016/j.scr.2024.103589] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 10/13/2024] [Accepted: 10/16/2024] [Indexed: 10/26/2024] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) have become a revolutionary tool in biomedical research due to their unique in vitro properties and fate versatility. They offer insights into development or genetic disorders, facilitate drug discovery and hold promise for regenerative medicine. Here we generated three hiPSC cells - IPi002-A/B/C - from primary amniotic fluid cells (AFCs) obtained via amniocentesis for the prenatal diagnosis of MARCH syndrome: Multinucleated neurons, Anhydramnios, Renal dysplasia, Cerebellar hypoplasia, and Hydranencephaly. These AFCs underwent reprogramming through non-integrative viral transduction and the resulting hiPSCs exhibited normal karyotype and expressed typical pluripotency markers.
Collapse
Affiliation(s)
- Mikaël Boullé
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France.
| | - Ambre Leleu
- SupBiotech-CEA/DRF/IBFJ/SEPIA, 92260 Fontenay-aux-Roses, France
| | - Siham Schacre
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France
| | - Céline Banal
- Université Paris-Cité, iPSC Core Facility, Institut Imagine, INSERM UMR U1163, 75015 Paris, France
| | - Alix Boucharlat
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France
| | - Solène Renault
- SupBiotech-CEA/DRF/IBFJ/SEPIA, 92260 Fontenay-aux-Roses, France
| | - Marcel Hollenstein
- Laboratory for Bioorganic Chemistry of Nucleic Acids, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France
| | - Patrick Frosk
- Department of Pediatrics and Child Health, Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Frank Yates
- SupBiotech-CEA/DRF/IBFJ/SEPIA, 92260 Fontenay-aux-Roses, France
| | - Nathalie Lefort
- Université Paris-Cité, iPSC Core Facility, Institut Imagine, INSERM UMR U1163, 75015 Paris, France
| | - Fabrice Agou
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France.
| |
Collapse
|
|
6
|
Mommaerts K, Okawa S, Schmitt M, Kofanova O, Turner TR, Ben RN, Del Sol A, Mathieson W, Schwamborn JC, Acker JP, Betsou F. Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis. Stem Cell Res 2024; 81:103583. [PMID: 39467374 DOI: 10.1016/j.scr.2024.103583] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Revised: 06/28/2024] [Accepted: 10/14/2024] [Indexed: 10/30/2024] Open
Abstract
The successful use of human induced pluripotent stem cells (iPSCs) for research or clinical applications requires the development of robust, efficient, and reproducible cryopreservation protocols. After cryopreservation, the survival rate of iPSCs is suboptimal and cell line-dependent. We assessed the use of ice recrystallization inhibitors (IRIs) for cryopreservation of human iPSCs. A toxicity screening study was performed to assess specific small-molecule carbohydrate-based IRIs and concentrations for further evaluation. Then, a cryopreservation study compared the cryoprotective efficiency of 15 mM IRIs in 5 % or 10 % DMSO-containing solutions and with CryoStor® CS10. Three iPSC lines were cryopreserved as single-cell suspensions in the cryopreservation solutions and post-thaw characteristics, including pluripotency and differential gene expression were assessed. We demonstrate the fitness-for-purpose of 15 mM IRI in 5 % DMSO as an efficient cryoprotective solution for iPSCs in terms of post-thaw recovery, viability, pluripotency, and transcriptomic changes. This mRNA sequencing dataset has the potential to be used for molecular mechanism analysis relating to cryopreservation. Use of IRIs can reduce DMSO concentrations and its associated toxicities, thereby improving the utility, effectiveness, and efficiency of cryopreservation.
Collapse
Affiliation(s)
- Kathleen Mommaerts
- Integrated Biobank of Luxembourg, Luxembourg Institute of Health, 1 rue Louis Rech, L-3555 Dudelange, Luxembourg; Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 2 avenue de Université, L-4365 Esch-sur-Alzette, Luxembourg.
| | - Satoshi Okawa
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 2 avenue de Université, L-4365 Esch-sur-Alzette, Luxembourg
| | - Margaux Schmitt
- Integrated Biobank of Luxembourg, Luxembourg Institute of Health, 1 rue Louis Rech, L-3555 Dudelange, Luxembourg
| | - Olga Kofanova
- Integrated Biobank of Luxembourg, Luxembourg Institute of Health, 1 rue Louis Rech, L-3555 Dudelange, Luxembourg
| | | | - Robert N Ben
- PanTHERA CryoSolutions Inc., Edmonton, Alberta, Canada; Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada
| | - Antonio Del Sol
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 2 avenue de Université, L-4365 Esch-sur-Alzette, Luxembourg; CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain
| | - William Mathieson
- Integrated Biobank of Luxembourg, Luxembourg Institute of Health, 1 rue Louis Rech, L-3555 Dudelange, Luxembourg
| | - Jens C Schwamborn
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 2 avenue de Université, L-4365 Esch-sur-Alzette, Luxembourg
| | - Jason P Acker
- PanTHERA CryoSolutions Inc., Edmonton, Alberta, Canada; Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
| | - Fay Betsou
- Integrated Biobank of Luxembourg, Luxembourg Institute of Health, 1 rue Louis Rech, L-3555 Dudelange, Luxembourg
| |
Collapse
|
|
7
|
De Serres-Bérard T, Pouliot V, Puymirat J, Chahine M. Generation of a control induced pluripotent stem cell line (CBRCULi014-A) derived from the lymphoblastoid cells of a pediatric individual. Stem Cell Res 2024; 81:103587. [PMID: 39471665 DOI: 10.1016/j.scr.2024.103587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Revised: 07/11/2024] [Accepted: 10/15/2024] [Indexed: 11/01/2024] Open
Abstract
Lymphoblastoid cell lines serve as a readily and continuous resource for generating induced pluripotent stem cells (iPSCs), enabling the modeling of various genetic disorders in vitro. When investigating congenital and infantile diseases, age-matched controls derived from pediatric individuals are typically necessary, yet they may be scarce or difficult to obtain. Here, the Sendai virus system was employed to introduce reprogramming factors into lymphoblastoid cells derived from an apparently healthy 4-year-old female. The generated iPSCs strongly expressed pluripotency cell markers and displayed robust trilineage differentiation. CBRCULi014-A is therefore a reliable control iPSC line for pediatric disease investigation.
Collapse
Affiliation(s)
- Thiéry De Serres-Bérard
- CERVO Brain Research Centre, Institut Universitaire en Santé Mentale de Québec, Quebec City, QC G1J 2G3, Canada
| | - Valérie Pouliot
- CERVO Brain Research Centre, Institut Universitaire en Santé Mentale de Québec, Quebec City, QC G1J 2G3, Canada
| | - Jack Puymirat
- LOEX, CHU de Québec-Université Laval Research Center, Quebec City, QC G1J 1Z4, Canada; Department of Medicine, Faculty of Medicine, Université Laval, Quebec City, QC G1V 06A, Canada
| | - Mohamed Chahine
- CERVO Brain Research Centre, Institut Universitaire en Santé Mentale de Québec, Quebec City, QC G1J 2G3, Canada; Department of Medicine, Faculty of Medicine, Université Laval, Quebec City, QC G1V 06A, Canada.
| |
Collapse
|
|
8
|
Dobner J, Diecke S, Krutmann J, Prigione A, Rossi A. Reassessment of marker genes in human induced pluripotent stem cells for enhanced quality control. Nat Commun 2024; 15:8547. [PMID: 39358374 PMCID: PMC11447164 DOI: 10.1038/s41467-024-52922-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Accepted: 09/23/2024] [Indexed: 10/04/2024] Open
Abstract
Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, "hiPSCore", trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications.
Collapse
Affiliation(s)
- Jochen Dobner
- Genome Engineering and Model Development Lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
| | - Sebastian Diecke
- Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany
| | - Jean Krutmann
- Genome Engineering and Model Development Lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
- Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | | | - Andrea Rossi
- Genome Engineering and Model Development Lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany.
| |
Collapse
|
|
9
|
Chu SL, Abe K, Yokota H, Cho D, Hayashi Y, Tsai MD. Deep learning for quantifying spatial patterning and formation process of early differentiated human-induced pluripotent stem cells with micropattern images. J Microsc 2024; 296:79-93. [PMID: 38994744 DOI: 10.1111/jmi.13346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 05/13/2024] [Accepted: 07/01/2024] [Indexed: 07/13/2024]
Abstract
Micropatterning is reliable method for quantifying pluripotency of human-induced pluripotent stem cells (hiPSCs) that differentiate to form a spatial pattern of sorted, ordered and nonoverlapped three germ layers on the micropattern. In this study, we propose a deep learning method to quantify spatial patterning of the germ layers in the early differentiation stage of hiPSCs using micropattern images. We propose decoding and encoding U-net structures learning labelled Hoechst (DNA-stained) hiPSC regions with corresponding Hoechst and bright-field micropattern images to segment hiPSCs on Hoechst or bright-field images. We also propose a U-net structure to extract extraembryonic regions on a micropattern, and an algorithm to compares intensities of the fluorescence images staining respective germ-layer cells and extract their regions. The proposed method thus can quantify the pluripotency of a hiPSC line with spatial patterning including cell numbers, areas and distributions of germ-layer and extraembryonic cells on a micropattern, and reveal the formation process of hiPSCs and germ layers in the early differentiation stage by segmenting live-cell bright-field images. In our assay, the cell-number accuracy achieved 86% and 85%, and the cell region accuracy 89% and 81% for segmenting Hoechst and bright-field micropattern images, respectively. Applications to micropattern images of multiple hiPSC lines, micropattern sizes, groups of markers, living and fixed cells show the proposed method can be expected to be a useful protocol and tool to quantify pluripotency of a new hiPSC line before providing it to the scientific community.
Collapse
Affiliation(s)
- Slo-Li Chu
- Department of Information and Computer Engineering, Chung-Yuan Christian University, Chung-Li, Taoyuan, Taiwan
| | - Kuniya Abe
- BioResource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Hideo Yokota
- Center for Advanced Photonics, RIKEN, Wako, Saitama, Japan
| | - Dooseon Cho
- BioResource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Yohei Hayashi
- BioResource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Ming-Dar Tsai
- Department of Information and Computer Engineering, Chung-Yuan Christian University, Chung-Li, Taoyuan, Taiwan
| |
Collapse
|
|
10
|
Penning A, Snoeck S, Garritsen O, Tosoni G, Hof A, de Boer F, van Hasenbroek J, Zhang L, Thrupp N, Craessaerts K, Fiers M, Salta E. NACC2, a molecular effector of miR-132 regulation at the interface between adult neurogenesis and Alzheimer's disease. Sci Rep 2024; 14:21163. [PMID: 39256511 PMCID: PMC11387632 DOI: 10.1038/s41598-024-72096-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2024] [Accepted: 09/03/2024] [Indexed: 09/12/2024] Open
Abstract
The generation of new neurons at the hippocampal neurogenic niche, known as adult hippocampal neurogenesis (AHN), and its impairment, have been implicated in Alzheimer's disease (AD). MicroRNA-132 (miR-132), the most consistently downregulated microRNA (miRNA) in AD, was recently identified as a potent regulator of AHN, exerting multilayered proneurogenic effects in adult neural stem cells (NSCs) and their progeny. Supplementing miR-132 in AD mouse brain restores AHN and relevant memory deficits, yet the exact mechanisms involved are still unknown. Here, we identify NACC2 as a novel miR-132 target implicated in both AHN and AD. miR-132 deficiency in mouse hippocampus induces Nacc2 expression and inflammatory signaling in adult NSCs. We show that miR-132-dependent regulation of NACC2 is involved in the initial stages of human NSC differentiation towards astrocytes and neurons. Later, NACC2 function in astrocytic maturation becomes uncoupled from miR-132. We demonstrate that NACC2 is present in reactive astrocytes surrounding amyloid plaques in mouse and human AD hippocampus, and that there is an anticorrelation between miR-132 and NACC2 levels in AD and upon induction of inflammation. Unraveling the molecular mechanisms by which miR-132 regulates neurogenesis and cellular reactivity in AD, will provide valuable insights towards its possible application as a therapeutic target.
Collapse
Affiliation(s)
- Amber Penning
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | - Sarah Snoeck
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | - Oxana Garritsen
- UMC Utrecht Brain Center, Utrecht University, Utrecht, The Netherlands
| | - Giorgia Tosoni
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | - Amber Hof
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | - Fleur de Boer
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | | | - Lin Zhang
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
| | - Nicky Thrupp
- VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium
| | | | - Mark Fiers
- VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium
| | - Evgenia Salta
- Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
| |
Collapse
|
|
11
|
Patton MH, Thomas KT, Bayazitov IT, Newman KD, Kurtz NB, Robinson CG, Ramirez CA, Trevisan AJ, Bikoff JB, Peters ST, Pruett-Miller SM, Jiang Y, Schild AB, Nityanandam A, Zakharenko SS. Synaptic plasticity in human thalamocortical assembloids. Cell Rep 2024; 43:114503. [PMID: 39018245 PMCID: PMC11407288 DOI: 10.1016/j.celrep.2024.114503] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2024] [Revised: 04/23/2024] [Accepted: 06/27/2024] [Indexed: 07/19/2024] Open
Abstract
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
Collapse
Affiliation(s)
- Mary H Patton
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Kristen T Thomas
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Ildar T Bayazitov
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Kyle D Newman
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Nathaniel B Kurtz
- Cell and Tissue Imaging Center, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Camenzind G Robinson
- Cell and Tissue Imaging Center, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Cody A Ramirez
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Alexandra J Trevisan
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Jay B Bikoff
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Samuel T Peters
- Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Shondra M Pruett-Miller
- Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Yanbo Jiang
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Andrew B Schild
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Anjana Nityanandam
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Stanislav S Zakharenko
- Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
| |
Collapse
|
|
12
|
Burnight ER, Fenner BJ, Han IC, DeLuca AP, Whitmore SS, Bohrer LR, Andorf JL, Sohn EH, Mullins RF, Tucker BA, Stone EM. Demonstration of the pathogenicity of a common non-exomic mutation in ABCA4 using iPSC-derived retinal organoids and retrospective clinical data. Hum Mol Genet 2024; 33:1379-1390. [PMID: 37930186 PMCID: PMC11305681 DOI: 10.1093/hmg/ddad176] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 09/25/2023] [Accepted: 10/10/2023] [Indexed: 11/07/2023] Open
Abstract
Mutations in ABCA4 are the most common cause of Mendelian retinal disease. Clinical evaluation of this gene is challenging because of its extreme allelic diversity, the large fraction of non-exomic mutations, and the wide range of associated disease. We used patient-derived retinal organoids as well as DNA samples and clinical data from a large cohort of patients with ABCA4-associated retinal disease to investigate the pathogenicity of a variant in ABCA4 (IVS30 + 1321 A>G) that occurs heterozygously in 2% of Europeans. We found that this variant causes mis-splicing of the gene in photoreceptor cells such that the resulting protein contains 36 incorrect amino acids followed by a premature stop. We also investigated the phenotype of 10 patients with compound genotypes that included this mutation. Their median age of first vision loss was 39 years, which is in the mildest quintile of a large cohort of patients with ABCA4 disease. We conclude that the IVS30 + 1321 A>G variant can cause disease when paired with a sufficiently deleterious opposing allele in a sufficiently permissive genetic background.
Collapse
Affiliation(s)
- Erin R Burnight
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Beau J Fenner
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Ian C Han
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Adam P DeLuca
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - S Scott Whitmore
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Laura R Bohrer
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Jeaneen L Andorf
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Elliott H Sohn
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Robert F Mullins
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Budd A Tucker
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| | - Edwin M Stone
- Institute for Vision Research, Carver College of Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52242, United States
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, United States
| |
Collapse
|
|
13
|
Florido MHC, Ziats NP. Endothelial dysfunction and cardiovascular diseases: The role of human induced pluripotent stem cells and tissue engineering. J Biomed Mater Res A 2024; 112:1286-1304. [PMID: 38230548 DOI: 10.1002/jbm.a.37669] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 12/07/2023] [Accepted: 01/02/2024] [Indexed: 01/18/2024]
Abstract
Cardiovascular disease (CVD) remains to be the leading cause of death globally today and therefore the need for the development of novel therapies has become increasingly important in the cardiovascular field. The mechanism(s) behind the pathophysiology of CVD have been laboriously investigated in both stem cell and bioengineering laboratories. Scientific breakthroughs have paved the way to better mimic cell types of interest in recent years, with the ability to generate any cell type from reprogrammed human pluripotent stem cells. Mimicking the native extracellular matrix using both organic and inorganic biomaterials has allowed full organs to be recapitulated in vitro. In this paper, we will review techniques from both stem cell biology and bioengineering which have been fruitfully combined and have fueled advances in the cardiovascular disease field. We will provide a brief introduction to CVD, reviewing some of the recent studies as related to the role of endothelial cells and endothelial cell dysfunction. Recent advances and the techniques widely used in both bioengineering and stem cell biology will be discussed, providing a broad overview of the collaboration between these two fields and their overall impact on tissue engineering in the cardiovascular devices and implications for treatment of cardiovascular disease.
Collapse
Affiliation(s)
- Mary H C Florido
- Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA
- Harvard T.H. Chan School of Public Health, Harvard University, Boston, Massachusetts, USA
- Harvard Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA
| | - Nicholas P Ziats
- Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA
- Departments of Biomedical Engineering and Anatomy, Case Western Reserve University, Cleveland, Ohio, USA
| |
Collapse
|
|
14
|
Kunitomi A, Hirohata R, Osawa M, Washizu K, Arreola V, Saiki N, Kato TM, Nomura M, Kunitomi H, Ohkame T, Ohkame Y, Kawaguchi J, Hara H, Kusano K, Yamamoto T, Takashima Y, Tohyama S, Yuasa S, Fukuda K, Takasu N, Yamanaka S. H1FOO-DD promotes efficiency and uniformity in reprogramming to naive pluripotency. Stem Cell Reports 2024; 19:710-728. [PMID: 38701780 PMCID: PMC11103934 DOI: 10.1016/j.stemcr.2024.04.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 04/06/2024] [Accepted: 04/08/2024] [Indexed: 05/05/2024] Open
Abstract
Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.
Collapse
Affiliation(s)
- Akira Kunitomi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
| | - Ryoko Hirohata
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; CiRA Foundation, Kyoto 606-8397, Japan
| | - Mitsujiro Osawa
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Kaho Washizu
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| | - Vanessa Arreola
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| | - Norikazu Saiki
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Tomoaki M Kato
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; CiRA Foundation, Kyoto 606-8397, Japan
| | - Masaki Nomura
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; CiRA Foundation, Kyoto 606-8397, Japan
| | - Haruko Kunitomi
- Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| | - Tokiko Ohkame
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Yusuke Ohkame
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | | | | | | | - Takuya Yamamoto
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto 606-8501, Japan; Medical-risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto 606-8507, Japan
| | - Yasuhiro Takashima
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Shinsuke Yuasa
- Department of Cardiology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Naoko Takasu
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; CiRA Foundation, Kyoto 606-8397, Japan
| | - Shinya Yamanaka
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; CiRA Foundation, Kyoto 606-8397, Japan; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA
| |
Collapse
|
|
15
|
Pottmeier P, Nikolantonaki D, Lanner F, Peuckert C, Jazin E. Sex-biased gene expression during neural differentiation of human embryonic stem cells. Front Cell Dev Biol 2024; 12:1341373. [PMID: 38764741 PMCID: PMC11101176 DOI: 10.3389/fcell.2024.1341373] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Accepted: 04/16/2024] [Indexed: 05/21/2024] Open
Abstract
Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development.
Collapse
Affiliation(s)
- Philipp Pottmeier
- Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
| | - Danai Nikolantonaki
- Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
| | - Fredrik Lanner
- Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden
| | - Christiane Peuckert
- Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
- The Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
| | - Elena Jazin
- Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
| |
Collapse
|
|
16
|
Boullé M, Boucharlat A, Leleu A, Banal C, Coussement A, Hollenstein M, Yates F, Lefort N, Agou F. Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells. Stem Cell Res 2024; 76:103350. [PMID: 38387169 DOI: 10.1016/j.scr.2024.103350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/04/2023] [Revised: 12/22/2023] [Accepted: 02/14/2024] [Indexed: 02/24/2024] Open
Abstract
Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks.
Collapse
Affiliation(s)
- Mikaël Boullé
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France.
| | - Alix Boucharlat
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France
| | - Ambre Leleu
- Sup'Biotech-CEA/DRF/IBFJ/SEPIA, 92260 Fontenay-aux-Roses, France
| | - Céline Banal
- Université Paris-Cité, iPSC Core Facility, Institut Imagine, INSERM UMR U1163, 75015 Paris, France
| | - Aurélie Coussement
- Service de Cytogénétique, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Université Paris-Cité, 75014 Paris, France
| | - Marcel Hollenstein
- Laboratory for Bioorganic Chemistry of Nucleic Acids, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France
| | - Frank Yates
- Sup'Biotech-CEA/DRF/IBFJ/SEPIA, 92260 Fontenay-aux-Roses, France
| | - Nathalie Lefort
- Université Paris-Cité, iPSC Core Facility, Institut Imagine, INSERM UMR U1163, 75015 Paris, France
| | - Fabrice Agou
- Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris-Cité, CNRS UMR 3523, F-75015 Paris, France.
| |
Collapse
|
|
17
|
Wu Z, Shen S, Mizikovsky D, Cao Y, Naval-Sanchez M, Tan SZ, Alvarez YD, Sun Y, Chen X, Zhao Q, Kim D, Yang P, Hill TA, Jones A, Fairlie DP, Pébay A, Hewitt AW, Tam PPL, White MD, Nefzger CM, Palpant NJ. Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm. Dev Cell 2024; 59:705-722.e8. [PMID: 38354738 DOI: 10.1016/j.devcel.2024.01.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 10/27/2023] [Accepted: 01/23/2024] [Indexed: 02/16/2024]
Abstract
Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.
Collapse
Affiliation(s)
- Zhixuan Wu
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Sophie Shen
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Dalia Mizikovsky
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Yuanzhao Cao
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Marina Naval-Sanchez
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Siew Zhuan Tan
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Yanina D Alvarez
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Yuliangzi Sun
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Xiaoli Chen
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Qiongyi Zhao
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Daniel Kim
- Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia
| | - Pengyi Yang
- Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia
| | - Timothy A Hill
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Alun Jones
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - David P Fairlie
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Alice Pébay
- Department of Anatomy and Physiology, The University of Melbourne, Parkville, VIC 3010, Australia; Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Alex W Hewitt
- Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS 7000, Australia
| | - Patrick P L Tam
- Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia; School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW 2006, Australia
| | - Melanie D White
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Christian M Nefzger
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, QLD 4067, Australia
| | - Nathan J Palpant
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
| |
Collapse
|
|
18
|
Patton MH, Thomas KT, Bayazitov IT, Newman KD, Kurtz NB, Robinson CG, Ramirez CA, Trevisan AJ, Bikoff JB, Peters ST, Pruett-Miller SM, Jiang Y, Schild AB, Nityanandam A, Zakharenko SS. Synaptic plasticity in human thalamocortical assembloids. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.01.578421. [PMID: 38352415 PMCID: PMC10862901 DOI: 10.1101/2024.02.01.578421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/25/2024]
Abstract
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
Collapse
Affiliation(s)
- Mary H. Patton
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Kristen T. Thomas
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Ildar T. Bayazitov
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Kyle D. Newman
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Nathaniel B. Kurtz
- Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Camenzind G. Robinson
- Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Cody A. Ramirez
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Alexandra J. Trevisan
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Jay B. Bikoff
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Samuel T. Peters
- Center for Advanced Genome Engineering, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Shondra M. Pruett-Miller
- Center for Advanced Genome Engineering, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
- Department of Cell & Molecular Biology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Yanbo Jiang
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Andrew B. Schild
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Anjana Nityanandam
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| | - Stanislav S. Zakharenko
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital; Memphis, TN 38105, USA
| |
Collapse
|
|
19
|
Raabe FJ, Hausruckinger A, Gagliardi M, Ahmad R, Almeida V, Galinski S, Hoffmann A, Weigert L, Rummel CK, Murek V, Trastulla L, Jimenez-Barron L, Atella A, Maidl S, Menegaz D, Hauger B, Wagner EM, Gabellini N, Kauschat B, Riccardo S, Cesana M, Papiol S, Sportelli V, Rex-Haffner M, Stolte SJ, Wehr MC, Salcedo TO, Papazova I, Detera-Wadleigh S, McMahon FJ, Schmitt A, Falkai P, Hasan A, Cacchiarelli D, Dannlowski U, Nenadić I, Kircher T, Scheuss V, Eder M, Binder EB, Spengler D, Rossner MJ, Ziller MJ. Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.09.574815. [PMID: 38260577 PMCID: PMC10802452 DOI: 10.1101/2024.01.09.574815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2024]
Abstract
Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.
Collapse
Affiliation(s)
- Florian J. Raabe
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), 80804 Munich, Germany
| | - Anna Hausruckinger
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
- Department of Psychiatry, University of Münster, 48149 Münster, Germany
| | - Miriam Gagliardi
- Department of Psychiatry, University of Münster, 48149 Münster, Germany
- Center for Soft Nanoscience, University of Münster, 48149 Münster, Germany
| | - Ruhel Ahmad
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Valeria Almeida
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Institute of Biology, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
| | - Sabrina Galinski
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Systasy Bioscience GmbH, 81669 Munich, Germany
| | - Anke Hoffmann
- Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Liesa Weigert
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Christine K. Rummel
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), 80804 Munich, Germany
| | - Vanessa Murek
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Lucia Trastulla
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Laura Jimenez-Barron
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Alessia Atella
- Department of Psychiatry, University of Münster, 48149 Münster, Germany
- Center for Soft Nanoscience, University of Münster, 48149 Münster, Germany
| | - Susanne Maidl
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Danusa Menegaz
- Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Barbara Hauger
- Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | | | - Nadia Gabellini
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
| | - Beate Kauschat
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
| | - Sara Riccardo
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- NEGEDIA (Next Generation Diagnostic), Pozzuoli, Italy
| | - Marcella Cesana
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- Department of Advanced Biomedical Sciences, University of Naples “Federico II”, Naples, Italy
| | - Sergi Papiol
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Max Planck Institute of Psychiatry, 80804 Munich, Germany
- Institute of Psychiatric Phenomics and Genomics (IPPG), University Hospital, LMU Munich, 80336 Munich, Germany
| | - Vincenza Sportelli
- Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Monika Rex-Haffner
- Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Sebastian J. Stolte
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
| | - Michael C. Wehr
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Systasy Bioscience GmbH, 81669 Munich, Germany
| | - Tatiana Oviedo Salcedo
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
| | - Irina Papazova
- Department of Psychiatry, Psychotherapy, and Psychosomatics, Medical Faculty, University of Augsburg, 86156 Augsburg, Germany
| | - Sevilla Detera-Wadleigh
- Human Genetics Branch, National Institute of Mental Health Intramural Research Program (NIMH-IRP), Bethesda, MD, 20892, USA
| | - Francis J McMahon
- Human Genetics Branch, National Institute of Mental Health Intramural Research Program (NIMH-IRP), Bethesda, MD, 20892, USA
| | - Andrea Schmitt
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Laboratory of Neuroscience (LIM27), Institute of Psychiatry, University of São Paulo, São Paulo-SP 05403-903, Brazil
| | - Peter Falkai
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Alkomiet Hasan
- Department of Psychiatry, Psychotherapy, and Psychosomatics, Medical Faculty, University of Augsburg, 86156 Augsburg, Germany
| | - Davide Cacchiarelli
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- School for Advanced Studies, Genomics and Experimental Medicine Program, University of Naples “Federico II”, Naples, Italy
- Department of Translational Medicine, University of Naples “Federico II”, Naples, Italy
| | - Udo Dannlowski
- Institute for Translational Psychiatry, University of Münster, 48149 Münster, Germany
| | - Igor Nenadić
- Department of Psychiatry and Psychotherapy, Philipps-University and University Hospital Marburg, UKGM, 35039 Marburg, Germany
| | - Tilo Kircher
- Department of Psychiatry and Psychotherapy, Philipps-University and University Hospital Marburg, UKGM, 35039 Marburg, Germany
| | - Volker Scheuss
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
- MSH Medical School Hamburg, Hamburg, Germany
| | - Matthias Eder
- Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Elisabeth B. Binder
- Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Dietmar Spengler
- Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, 80804 Munich, Germany
| | - Moritz J. Rossner
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU Munich, 80336 Munich, Germany
| | - Michael J. Ziller
- Lab for Genomics of Complex Diseases, Max Planck Institute of Psychiatry, 80804 Munich, Germany
- Department of Psychiatry, University of Münster, 48149 Münster, Germany
- Center for Soft Nanoscience, University of Münster, 48149 Münster, Germany
| |
Collapse
|
|
20
|
Furukawa Y, Ishii M, Ando J, Ikeda K, Igarashi KJ, Kinoshita S, Azusawa Y, Toyota T, Honda T, Nakanishi M, Ohshima K, Masuda A, Yoshida E, Kitade M, Porteus M, Terao Y, Nakauchi H, Ando M. iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer. Cell Rep Med 2023; 4:101327. [PMID: 38091985 PMCID: PMC10772465 DOI: 10.1016/j.xcrm.2023.101327] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 07/20/2023] [Accepted: 11/17/2023] [Indexed: 12/22/2023]
Abstract
Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory T cells, which enhance cytotoxicity against cervical cancer through TGFβR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of T cell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" T cell therapy with robust anti-cervical cancer effects.
Collapse
Affiliation(s)
- Yoshiki Furukawa
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Midori Ishii
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Jun Ando
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; Division of Cell Therapy & Blood Transfusion Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Kazuya Ikeda
- Department of Pediatrics, School of Medicine, Stanford University, 291 Campus Drive, Stanford, CA 94305, USA
| | - Kyomi J Igarashi
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA
| | - Shintaro Kinoshita
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Yoko Azusawa
- Division of Cell Therapy & Blood Transfusion Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Tokuko Toyota
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Tadahiro Honda
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Mahito Nakanishi
- TOKIWA-Bio, Inc., Tsukuba Center Inc. (TCI), Building G, 2-1-6 Sengen, Tsukuba, Ibaraki 305-0047, Japan
| | - Koichi Ohshima
- Department of Pathology, School of Medicine, Kurume University, Fukuoka 830-0011, Japan
| | - Ayako Masuda
- Department of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Emiko Yoshida
- Department of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Mari Kitade
- Department of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Matthew Porteus
- Department of Pediatrics, School of Medicine, Stanford University, 291 Campus Drive, Stanford, CA 94305, USA
| | - Yasuhisa Terao
- Department of Obstetrics and Gynecology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
| | - Hiromitsu Nakauchi
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA.
| | - Miki Ando
- Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
| |
Collapse
|
|
21
|
Ryu S, Weber C, Chu PH, Ernest B, Jovanovic VM, Deng T, Slamecka J, Hong H, Jethmalani Y, Baskir HM, Inman J, Braisted J, Hirst MB, Simeonov A, Voss TC, Tristan CA, Singeç I. Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness. Biofabrication 2023; 16:015016. [PMID: 37972398 DOI: 10.1088/1758-5090/ad0d13] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Accepted: 11/16/2023] [Indexed: 11/19/2023]
Abstract
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
Collapse
Affiliation(s)
- Seungmi Ryu
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Claire Weber
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Pei-Hsuan Chu
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Ben Ernest
- Rancho Biosciences, 16955 Via Del Campo, #200, San Diego, CA 92127, United States of America
| | - Vukasin M Jovanovic
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Tao Deng
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Jaroslav Slamecka
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Hyenjong Hong
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Yogita Jethmalani
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Hannah M Baskir
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Jason Inman
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - John Braisted
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Marissa B Hirst
- Rancho Biosciences, 16955 Via Del Campo, #200, San Diego, CA 92127, United States of America
| | - Anton Simeonov
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Ty C Voss
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Carlos A Tristan
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| | - Ilyas Singeç
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America
| |
Collapse
|
|
22
|
Li B, Jin X, Chan HM. Effects of low doses of methylmercury (MeHg) exposure on definitive endoderm cell differentiation in human embryonic stem cells. Arch Toxicol 2023; 97:2625-2641. [PMID: 37612375 PMCID: PMC10475006 DOI: 10.1007/s00204-023-03580-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Accepted: 08/03/2023] [Indexed: 08/25/2023]
Abstract
Fetal development is one of the most sensitive windows to methylmercury (MeHg) toxicity. Laboratory and epidemiological studies have shown a dose-response relationship between fetal MeHg exposure and neuro performance in different life stages from infants to adults. In addition, MeHg exposure has been reported to be associated with disorders in endoderm-derived organs, such as morphological changes in liver cells and pancreatic cell dysfunctions. However, the mechanisms of the effects of MeHg on non-neuronal organs or systems, especially during the early development of endoderm-derived organs, remain unclear. Here we determined the effects of low concentrations of MeHg exposure during the differentiation of definitive endoderm (DE) cells from human embryonic stem cells (hESCs). hESCs were exposed to MeHg (0, 10, 100, and 200 nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction. Transcriptomic analysis showed that sub-lethal doses of MeHg exposure could alter global gene expression patterns during hESC to DE cell differentiation, leading to increased expression of endodermal genes/proteins and the over-promotion of endodermal fate, mainly through disrupting calcium homeostasis and generating ROS. Bioinformatic analysis results suggested that MeHg exerts its developmental toxicity mainly by disrupting ribosome biogenesis during early cell lineage differentiation. This disruption could lead to aberrant growth or dysfunctions of the developing endoderm-derived organs, and it may be the underlying mechanism for the observed congenital diseases later in life. Based on the results, we proposed an adverse outcome pathway for the effects of MeHg exposure during human embryonic stem cells to definitive endoderm differentiation.
Collapse
Affiliation(s)
- Bai Li
- Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, K1N 6N5, Canada
| | - Xiaolei Jin
- Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON, K1A 0K9, Canada.
| | - Hing Man Chan
- Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
| |
Collapse
|
|
23
|
Battaglia RA, Faridounnia M, Beltran A, Robinson J, Kinghorn K, Ezzell JA, Bharucha-Goebel D, Bönnemann CG, Hooper JE, Opal P, Bouldin TW, Armao D, Snider NT. Intermediate filament dysregulation in astrocytes in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN). Mol Biol Cell 2023; 34:mbcE23030094. [PMID: 37672338 PMCID: PMC10846626 DOI: 10.1091/mbc.e23-03-0094] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2023] [Revised: 08/29/2023] [Accepted: 08/30/2023] [Indexed: 09/08/2023] Open
Abstract
Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament (IF) turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically-relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient-specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared to isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinuclear vimentin and GFAP accumulations and abnormal nuclear morphology. In over-expression systems, GFAP oligomerization and perinuclear aggregation were augmented in the presence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared to cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.
Collapse
Affiliation(s)
- Rachel A. Battaglia
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Maryam Faridounnia
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Adriana Beltran
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Jasmine Robinson
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Karina Kinghorn
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - J. Ashley Ezzell
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | | | | | - Jody E. Hooper
- Department of Pathology, Stanford University, Palo Alto, CA 94305
| | - Puneet Opal
- Departments of Neurology and Cell and Developmental Biology, Northwestern University, Chicago, IL 60611
| | - Thomas W. Bouldin
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Diane Armao
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
- Department of Radiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Natasha T. Snider
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| |
Collapse
|
|
24
|
Lo EKW, Velazquez JJ, Peng D, Kwon C, Ebrahimkhani MR, Cahan P. Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols. Stem Cell Reports 2023; 18:1721-1742. [PMID: 37478860 PMCID: PMC10444577 DOI: 10.1016/j.stemcr.2023.06.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Revised: 06/16/2023] [Accepted: 06/17/2023] [Indexed: 07/23/2023] Open
Abstract
Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.
Collapse
Affiliation(s)
- Emily K W Lo
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Jeremy J Velazquez
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Da Peng
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Chulan Kwon
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Mo R Ebrahimkhani
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
| | - Patrick Cahan
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
| |
Collapse
|
|
25
|
Chu SL, Abe K, Lin KT, Yokota H, Cho D, Tsai MD. Quantitative Analyses for Early Tempo-spatial Patterning of Differentiated Human Induced Pluripotent Stem Cells on Micropatterns using Time-lapse Bright-field Microscopy Images. ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. ANNUAL INTERNATIONAL CONFERENCE 2023; 2023:1-4. [PMID: 38082741 DOI: 10.1109/embc40787.2023.10340990] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2023]
Abstract
Three germ layer formation on micropatterns are extremely useful for quantitative analysis of hiPSC (human induced pluripotent stem cells) pluripotency. Spatial patterns of stem cells differentiated on the micropatterns will be formed from about 24 hours after differentiation induction and usually quantitated near 48 hours. To delineate the germ layer formation process, temporal changes in spatial patterning of germ layers should be analyzed by noninvasive microscopy. This study proposed a series of image processing methods combined with a U-net automatic segmentation to segment differentiated hiPSCs captured by bright-field microscopy. High segmentation accuracy (83.3%) for the test bright-field images compared with their concurrent Hoechst images (85%) was achieved. Tempo-spatial patterning and formation process of germ layers on the micropatterns can be visualized and quantified by segmenting time-lapse bright-field microscopy images using our method.
Collapse
|
|
26
|
Cooke JA, Voigt AP, Collingwood MA, Stone NE, Whitmore SS, DeLuca AP, Burnight ER, Anfinson KR, Vakulskas CA, Reutzel AJ, Daggett HT, Andorf JL, Stone EM, Mullins RF, Tucker BA. Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement. Stem Cells Transl Med 2023; 12:365-378. [PMID: 37221451 PMCID: PMC10267581 DOI: 10.1093/stcltm/szad028] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Accepted: 01/26/2023] [Indexed: 05/25/2023] Open
Abstract
Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.
Collapse
Affiliation(s)
- Jessica A Cooke
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Andrew P Voigt
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | | | - Nicholas E Stone
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - S Scott Whitmore
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Adam P DeLuca
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Erin R Burnight
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Kristin R Anfinson
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | | | - Austin J Reutzel
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Heather T Daggett
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Jeaneen L Andorf
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Edwin M Stone
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Robert F Mullins
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Budd A Tucker
- Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| |
Collapse
|
|
27
|
Cohen PJR, Luquet E, Pletenka J, Leonard A, Warter E, Gurchenkov B, Carrere J, Rieu C, Hardouin J, Moncaubeig F, Lanero M, Quelennec E, Wurtz H, Jamet E, Demarco M, Banal C, Van Liedekerke P, Nassoy P, Feyeux M, Lefort N, Alessandri K. Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors. Biomaterials 2023; 295:122033. [PMID: 36764194 DOI: 10.1016/j.biomaterials.2023.122033] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2022] [Revised: 12/12/2022] [Accepted: 01/31/2023] [Indexed: 02/05/2023]
Abstract
Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome the scale-up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need for large-scale culture in regenerative medicine. Despite constant improvements, current protocols that use microcarriers or generate cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies significantly improve viability and expansion rates while maintaining pluripotency compared to standard hPSC culture platforms such as 2D cultures, microcarriers, and aggregates. By further tuning capsule size and culture conditions, we scale up this method to industrial-scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 277-fold in 6.5 days. In brief, our findings indicate that our 3D culture system offers a suitable strategy both for basic stem cell biology experiments and for clinical applications.
Collapse
Affiliation(s)
- Philippe J R Cohen
- Université Paris Cité, Imagine Institute, IPSC Core Facility, INSERM UMR U1163, F-75015, Paris, France; Treefrog Therapeutics, F-33600, Pessac, France.
| | | | | | | | | | | | | | | | | | | | | | - Eddy Quelennec
- Université Paris Cité, Imagine Institute, IPSC Core Facility, INSERM UMR U1163, F-75015, Paris, France; Treefrog Therapeutics, F-33600, Pessac, France
| | | | | | | | - Celine Banal
- Université Paris Cité, Imagine Institute, IPSC Core Facility, INSERM UMR U1163, F-75015, Paris, France
| | - Paul Van Liedekerke
- Inria Paris & Sorbonne Université LJLL, 2 Rue Simone IFF, F-75012, Paris, France
| | - Pierre Nassoy
- LP2N, Laboratoire Photonique Numérique et Nanosciences, Univ. Bordeaux, F-33400, Talence, France; Institut D'Optique Graduate School & CNRS UMR 5298, F-33400, Talence, France
| | | | - Nathalie Lefort
- Université Paris Cité, Imagine Institute, IPSC Core Facility, INSERM UMR U1163, F-75015, Paris, France
| | | |
Collapse
|
|
28
|
Weber C, Hirst MB, Ernest B, Schaub NJ, Wilson KM, Wang K, Baskir HM, Chu PH, Tristan CA, Singeç I. SEQUIN is an R/Shiny framework for rapid and reproducible analysis of RNA-seq data. CELL REPORTS METHODS 2023; 3:100420. [PMID: 37056373 PMCID: PMC10088091 DOI: 10.1016/j.crmeth.2023.100420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 12/23/2022] [Accepted: 02/10/2023] [Indexed: 03/08/2023]
Abstract
SEQUIN is a web-based application (app) that allows fast and intuitive analysis of RNA sequencing data derived for model organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also developed the iPSC Profiler, a practical gene module scoring tool that helps measure and compare pluripotent and differentiated cell types. Benchmarking to other commercial and non-commercial products underscored several advantages of SEQUIN. Freely available to the public, SEQUIN empowers scientists using interdisciplinary methods to investigate and present transcriptome data firsthand with state-of-the-art statistical methods. Hence, SEQUIN helps democratize and increase the throughput of interrogating biological questions using next-generation sequencing data with single-cell resolution.
Collapse
Affiliation(s)
- Claire Weber
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation, Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Marissa B. Hirst
- Rancho Biosciences, 16955 Via Del Campo, #200, San Diego, CA 92127, USA
| | - Ben Ernest
- Rancho Biosciences, 16955 Via Del Campo, #200, San Diego, CA 92127, USA
| | - Nicholas J. Schaub
- National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Kelli M. Wilson
- National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Ke Wang
- National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Hannah M. Baskir
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation, Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Pei-Hsuan Chu
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation, Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Carlos A. Tristan
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation, Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Ilyas Singeç
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation, Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| |
Collapse
|
|
29
|
Teratoma Assay for Testing Pluripotency and Malignancy of Stem Cells: Insufficient Reporting and Uptake of Animal-Free Methods-A Systematic Review. Int J Mol Sci 2023; 24:ijms24043879. [PMID: 36835305 PMCID: PMC9967860 DOI: 10.3390/ijms24043879] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2022] [Revised: 01/28/2023] [Accepted: 02/06/2023] [Indexed: 02/17/2023] Open
Abstract
Pluripotency describes the ability of stem cells to differentiate into derivatives of the three germ layers. In reporting new human pluripotent stem cell lines, their clonal derivatives or the safety of differentiated derivatives for transplantation, assessment of pluripotency is essential. Historically, the ability to form teratomas in vivo containing different somatic cell types following injection into immunodeficient mice has been regarded as functional evidence of pluripotency. In addition, the teratomas formed can be analyzed for the presence of malignant cells. However, use of this assay has been subject to scrutiny for ethical reasons on animal use and due to the lack of standardization in how it is used, therefore questioning its accuracy. In vitro alternatives for assessing pluripotency have been developed such as ScoreCard and PluriTest. However, it is unknown whether this has resulted in reduced use of the teratoma assay. Here, we systematically reviewed how the teratoma assay was reported in publications between 1998 (when the first human embryonic stem cell line was described) and 2021. Our analysis of >400 publications showed that in contrast to expectations, reporting of the teratoma assay has not improved: methods are not yet standardized, and malignancy was examined in only a relatively small percentage of assays. In addition, its use has not decreased since the implementation of the ARRIVE guidelines on reduction of animal use (2010) or the introduction of ScoreCard (2015) and PluriTest (2011). The teratoma assay is still the preferred method to assess the presence of undifferentiated cells in a differentiated cell product for transplantation since the in vitro assays alone are not generally accepted by the regulatory authorities for safety assessment. This highlights the remaining need for an in vitro assay to test malignancy of stem cells.
Collapse
|
|
30
|
Suresh Babu S, Duvvuru H, Baker J, Switalski S, Shafa M, Panchalingam KM, Dadgar S, Beller J, Ahmadian Baghbaderani B. Characterization of human induced pluripotent stems cells: Current approaches, challenges, and future solutions. BIOTECHNOLOGY REPORTS (AMSTERDAM, NETHERLANDS) 2023; 37:e00784. [PMID: 36818379 PMCID: PMC9929203 DOI: 10.1016/j.btre.2023.e00784] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 01/09/2023] [Accepted: 01/19/2023] [Indexed: 01/25/2023]
Abstract
Human induced pluripotent stem cells (iPSC) have demonstrated massive potentials for use in regenerative and personalized medicine due to their ability to expand in culture and differentiate into specialized cells with therapeutic benefits. However, in order to industrialize iPSC-derived therapies, it is necessary to address the existing challenges surrounding the analytics implemented in the manufacturing process to evaluate and monitor cell expansion, differentiation, and quality of the final products. Here, we review some of the key analytical methods used as part of identity, potency, or safety for in-process or final product release testing and highlighted the challenges and potential solutions for consideration in the Chemistry, Manufacturing and Controls (CMC) strategy for iPSC-based therapies. Some of the challenges associated with characterization and testing of iPSC-based products are related to the choice of analytical technology (to ensure fit-for-purpose), assay reliability and robustness. Automation of analytical methods may be required to reduce hands on time, and improve reliability of the methods through reducing assay variability. Indeed, we have shown that automation of analytical methods is feasible (evaluated using an ELISA based assay) and would result in more precise measurements (demonstrated by lower co-efficient of Variation and standard deviation), less hands-on time, and swift compared to a manually run assay. Therefore, in order to support commercialization of iPSC-based therapies we suggest a well-designed testing strategy to be established in the development phase while incorporating robust, reproducible, reliable, and potentially automated analytics in the manufacturing process.
Collapse
|
|
31
|
Schmidt M, Zeevaert K, Elsafi Mabrouk MH, Goetzke R, Wagner W. Epigenetic biomarkers to track differentiation of pluripotent stem cells. Stem Cell Reports 2023; 18:145-158. [PMID: 36460001 PMCID: PMC9860076 DOI: 10.1016/j.stemcr.2022.11.001] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 10/31/2022] [Accepted: 11/01/2022] [Indexed: 12/03/2022] Open
Abstract
Quality control of induced pluripotent stem cells remains a challenge. For validation of the pluripotent state, it is crucial to determine trilineage differentiation potential toward endoderm, mesoderm, and ectoderm. Here, we report GermLayerTracker, a combination of site-specific DNA methylation (DNAm) assays that serve as biomarker for early germ layer specification. CG dinucleotides (CpGs) were identified with characteristic DNAm at pluripotent state and after differentiation into endoderm, mesoderm, and ectoderm. Based on this, a pluripotency score was derived that tracks reprogramming and may indicate differentiation capacity, as well as lineage-specific scores to monitor either directed differentiation or self-organized multilineage differentiation in embryoid bodies. Furthermore, we established pyrosequencing assays for fast and cost-effective analysis. In the future, the GermLayerTracker could be used for quality control of pluripotent cells and to estimate lineage-specific commitment during initial differentiation events.
Collapse
Affiliation(s)
- Marco Schmidt
- Institute for Stem Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany; Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Kira Zeevaert
- Institute for Stem Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany; Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Mohamed H Elsafi Mabrouk
- Institute for Stem Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany; Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Roman Goetzke
- Institute for Stem Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany; Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Wolfgang Wagner
- Institute for Stem Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany; Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany.
| |
Collapse
|
|
32
|
The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test. TOXICS 2022; 10:toxics10070392. [PMID: 35878297 PMCID: PMC9321663 DOI: 10.3390/toxics10070392] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Revised: 06/14/2022] [Accepted: 06/30/2022] [Indexed: 11/16/2022]
Abstract
Environmental chemical exposures are a contributing factor to birth defects affecting infant morbidity and mortality. The USA EPA is committed to developing new approach methods (NAMs) to detect chemical risks to susceptible populations, including pregnant women. NAM-based coverage for cellular mechanisms associated with early human development could enhance identification of potential developmental toxicants (DevTox) for new and existing data-poor chemicals. The human pluripotent stem cell test (hPST) is an in vitro test method for rapidly identifying potential human developmental toxicants that employs directed differentiation of embryonic stem cells to measure reductions in SOX17 biomarker expression and nuclear localization. The objective of this study was to expand on the hPST principles to develop a model platform (DevTox GLR) that utilizes the transgenic RUES2-GLR cell line expressing fluorescent reporter fusion protein biomarkers for SOX17 (endoderm marker), BRA (mesoderm marker), and SOX2 (ectoderm and pluripotency marker). Initial assay adaption to definitive endoderm (DevTox GLR-Endo) was performed to emulate the hPST SOX17 endpoint and enable comparative evaluation of concordant chemical effects. Assay duration was reduced to two days and screening throughput scaled to 384-well format for enhanced speed and efficiency. Assay performance for 66 chemicals derived from reference and training set data resulted in a balanced accuracy of 72% (79% sensitivity and 65% specificity). The DevTox GLR-Endo assay demonstrates successful adaptation of the hPST concept with increased throughput, shorter assay duration, and minimal endpoint processing. The DevTox GLR model platform expands the in vitro NAM toolbox to rapidly identify potential developmental hazards and mechanistically characterize toxicant effects on pathways and processes associated with early human development.
Collapse
|
|
33
|
Piersma AH, Baker NC, Daston GP, Flick B, Fujiwara M, Knudsen TB, Spielmann H, Suzuki N, Tsaioun K, Kojima H. Pluripotent stem cell assays: Modalities and applications for predictive developmental toxicity. Curr Res Toxicol 2022; 3:100074. [PMID: 35633891 PMCID: PMC9130094 DOI: 10.1016/j.crtox.2022.100074] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Revised: 04/21/2022] [Accepted: 05/09/2022] [Indexed: 12/02/2022] Open
Abstract
This manuscript provides a review focused on embryonic stem cell-based models and their place within the landscape of alternative developmental toxicity assays. Against the background of the principles of developmental toxicology, the wide diversity of alternative methods using pluripotent stem cells developed in this area over the past half century is reviewed. In order to provide an overview of available models, a systematic scoping review was conducted following a published protocol with inclusion criteria, which were applied to select the assays. Critical aspects including biological domain, readout endpoint, availability of standardized protocols, chemical domain, reproducibility and predictive power of each assay are described in detail, in order to review the applicability and limitations of the platform in general and progress moving forward to implementation. The horizon of innovative routes of promoting regulatory implementation of alternative methods is scanned, and recommendations for further work are given.
Collapse
Affiliation(s)
- Aldert H. Piersma
- Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
| | | | - George P. Daston
- Global Product Stewardship, The Procter & Gamble Company, Cincinnati, OH, USA
| | - Burkhard Flick
- Experimental Toxicology and Ecology, BASF SE, Ludwigshafen am Rhein, Germany
| | - Michio Fujiwara
- Drug Safety Research Labs, Astellas Pharma Inc., Tsukuba-shi, Japan
| | - Thomas B. Knudsen
- Center for Computational Toxicology and Exposure, U.S. Environmental Protection Agency, Research Triangle Park, USA
| | - Horst Spielmann
- Institute for Pharmacy, Faculty of Biology, Chemistry, and Pharmacy, Freie Universität, Berlin, Germany
| | - Noriyuki Suzuki
- Cell Science Group Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan
| | - Katya Tsaioun
- Evidence-Based Toxicology Collaboration at Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Hajime Kojima
- National Institute of Health Sciences, Kawasaki, Japan
| |
Collapse
|
|
34
|
Manuela J, David ZJ, Nicole S, Nicole C, Paul B, Erich K, Lisa SP, Claudia M, Marcel L, Stefan K. Optimization of the TeraTox assay for preclinical teratogenicity assessment. Toxicol Sci 2022; 188:17-33. [PMID: 35485993 PMCID: PMC9237991 DOI: 10.1093/toxsci/kfac046] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives. To improve the sensitivity of human teratogenicity prediction, we characterized the TeraTox test, a newly developed multilineage differentiation assay using 3D human-induced pluripotent stem cells. TeraTox produces primary output concentration-dependent cytotoxicity and altered gene expression induced by each test compound. These data are fed into an interpretable machine-learning model to perform prediction, which relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Comparing TeraTox predictions with known human or animal toxicity, we report an accuracy of 69% (specificity: 53%, sensitivity: 79%). TeraTox performed better than 2 quantitative structure-activity relationship models and had a higher sensitivity than the murine embryonic stem cell test (accuracy: 58%, specificity: 76%, and sensitivity: 46%) run in the same laboratory. The overall prediction accuracy could be further improved by combining TeraTox and mouse embryonic stem cell test results. Furthermore, patterns of altered gene expression revealed by TeraTox may help grouping toxicologically similar compounds and possibly deducing common modes of action. The TeraTox assay and the dataset described here therefore represent a new tool and a valuable resource for drug teratogenicity assessment.
Collapse
Affiliation(s)
- Jaklin Manuela
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland.,Department for In Vitro Toxicology and Biomedicine Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, Germany
| | - Zhang Jitao David
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | - Schäfer Nicole
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | - Clemann Nicole
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | - Barrow Paul
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | - Küng Erich
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | - Sach-Peltason Lisa
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| | | | - Leist Marcel
- Department for In Vitro Toxicology and Biomedicine Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, Germany
| | - Kustermann Stefan
- Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Switzerland
| |
Collapse
|
|
35
|
Bialecka M, Montilla-Rojo J, Roelen BAJ, Gillis AJ, Looijenga LHJ, Salvatori DCF. Humanised Mice and Immunodeficient Mice (NSG) Are Equally Sensitive for Prediction of Stem Cell Malignancy in the Teratoma Assay. Int J Mol Sci 2022; 23:ijms23094680. [PMID: 35563071 PMCID: PMC9105268 DOI: 10.3390/ijms23094680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 04/18/2022] [Accepted: 04/21/2022] [Indexed: 11/26/2022] Open
Abstract
The use of human pluripotent stem cells (hPSCs) in regenerative medicine has great potential. However, it is important to exclude that these cells can undergo malignant transformation, which could lead to the development of malignant tumours. This property of hPSCs is currently being tested using the teratoma assay, through which cells are injected into immunodeficient mice. Transplantation of stem cells in immunocompromised recipient animals certainly has a much higher incidence of tumour formation. On the other hand, the results obtained in immunodeficient mice could indicate a risk of tumour formation that is practically not present in the human immunocompetent recipient. The presence of a humanised immune system might be more representative of the human situation; therefore, we investigated if the demonstrated malignant features of chosen and well-characterised stem cell lines could be retrieved and if new features could arise in a humanised mouse model. Hu-CD34NSGTM (HIS) mice were compared side by side with immunocompromised mice (NSG) after injection of a set of benign (LU07) and malignant (LU07+dox and 2102Ep) cell lines. Analysis of the tumour development, histological composition, pathology evaluation, and malignancy-associated miRNA expression levels, both in tumour and plasma samples, revealed no differences among mouse groups. This indicates that the HIS mouse model is comparable to, but not more sensitive than, the NSG immunodeficient model for studying the malignancy of stem cells. Since in vivo teratoma assay is cumbersome, in vitro methods for the detection of malignancy are urgently needed.
Collapse
Affiliation(s)
- Monika Bialecka
- Anatomy and Physiology, Department Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands; (M.B.); (J.M.-R.); (B.A.J.R.)
| | - Joaquin Montilla-Rojo
- Anatomy and Physiology, Department Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands; (M.B.); (J.M.-R.); (B.A.J.R.)
| | - Bernard A. J. Roelen
- Anatomy and Physiology, Department Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands; (M.B.); (J.M.-R.); (B.A.J.R.)
| | - Ad J. Gillis
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, The Netherlands; (A.J.G.); (L.H.J.L.)
| | - Leendert H. J. Looijenga
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, The Netherlands; (A.J.G.); (L.H.J.L.)
| | - Daniela. C. F. Salvatori
- Anatomy and Physiology, Department Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands; (M.B.); (J.M.-R.); (B.A.J.R.)
- Correspondence:
| |
Collapse
|
|
36
|
Banal C, Lefort N. Generation and characterization of IMAGINi013-A, an induced pluripotent stem cell line generated from a healthy donor. Stem Cell Res 2022; 61:102755. [PMID: 35334405 DOI: 10.1016/j.scr.2022.102755] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Revised: 03/18/2022] [Accepted: 03/19/2022] [Indexed: 12/01/2022] Open
Abstract
Human pluripotent stem cells are a powerful tool to study development, to model diseases or as cellular substrates for drug screening. We generated a human induced pluripotent stem cell (hiPSC) line from a healthy control donor. Peripheral blood mononuclear cells (PBMCs) from this donor were reprogrammed using integration-free Sendai virus. This cell line had normal karyotype, expressed pluripotency hallmarks and differentiated into the three primary germ layers.
Collapse
Affiliation(s)
- Céline Banal
- Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France
| | - Nathalie Lefort
- Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.
| |
Collapse
|
|
37
|
Urrutia-Cabrera D, Hsiang-Chi Liou R, Lin J, Shi Y, Liu K, Hung SSC, Hewitt AW, Wang PY, Ching-Bong Wong R. Combinatorial Approach of Binary Colloidal Crystals and CRISPR Activation to Improve Induced Pluripotent Stem Cell Differentiation into Neurons. ACS APPLIED MATERIALS & INTERFACES 2022; 14:8669-8679. [PMID: 35166105 DOI: 10.1021/acsami.1c17975] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Conventional methods of neuronal differentiation in human induced pluripotent stem cells (iPSCs) are tedious and complicated, involving multistage protocols with complex cocktails of growth factors and small molecules. Artificial extracellular matrices with a defined surface topography and chemistry represent a promising venue to improve neuronal differentiation in vitro. In the present study, we test the impact of a type of colloidal self-assembled patterns (cSAPs) called binary colloidal crystals (BCCs) on neuronal differentiation. We developed a CRISPR activation (CRISPRa) iPSC platform that constitutively expresses the dCas9-VPR system, which allows robust activation of the proneural transcription factor NEUROD1 to rapidly induce neuronal differentiation within 7 days. We show that the combinatorial use of BCCs can further improve this neuronal differentiation system. In particular, our results indicate that fine tuning of silica (Si) and polystyrene (PS) particle size is critical to generate specific topographies to improve neuronal differentiation and branching. BCCs with 5 μm silica and 100 nm carboxylated PS (PSC) have the most prominent effect on increasing neurite outgrowth and more complex ramification, while BCCs with 2 μm Si and 65 nm PSC particles are better at promoting neuronal enrichment. These results indicate that biophysical cues can support rapid differentiation and improve neuronal maturation. In summary, our combinatorial approach of CRISPRa and BCCs provides a robust and rapid pipeline for the in vitro production of human neurons. Specific BCCs can be adapted to the late stages of neuronal differentiation protocols to improve neuronal maturation, which has important implications in tissue engineering, in vitro biological studies, and disease modeling.
Collapse
Affiliation(s)
- Daniel Urrutia-Cabrera
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia
| | - Roxanne Hsiang-Chi Liou
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia
| | - Jiao Lin
- Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 510810, China
| | - Yue Shi
- Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 510810, China
| | - Kun Liu
- Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 510810, China
| | - Sandy S C Hung
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia
| | - Alex W Hewitt
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia
| | - Peng-Yuan Wang
- Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 510810, China
- Oujiang Laboratory, Wenzhou, Zhejiang 325016, China
| | - Raymond Ching-Bong Wong
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia
- Shenzhen Eye Hospital, Shenzhen University School of Medicine, Shenzhen 510810, China
| |
Collapse
|
|
38
|
Novak G, Kyriakis D, Grzyb K, Bernini M, Rodius S, Dittmar G, Finkbeiner S, Skupin A. Single-cell transcriptomics of human iPSC differentiation dynamics reveal a core molecular network of Parkinson's disease. Commun Biol 2022; 5:49. [PMID: 35027645 PMCID: PMC8758783 DOI: 10.1038/s42003-021-02973-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Accepted: 12/14/2021] [Indexed: 01/02/2023] Open
Abstract
Parkinson's disease (PD) is the second-most prevalent neurodegenerative disorder, characterized by the loss of dopaminergic neurons (mDA) in the midbrain. The underlying mechanisms are only partly understood and there is no treatment to reverse PD progression. Here, we investigated the disease mechanism using mDA neurons differentiated from human induced pluripotent stem cells (hiPSCs) carrying the ILE368ASN mutation within the PINK1 gene, which is strongly associated with PD. Single-cell RNA sequencing (RNAseq) and gene expression analysis of a PINK1-ILE368ASN and a control cell line identified genes differentially expressed during mDA neuron differentiation. Network analysis revealed that these genes form a core network, members of which interact with all known 19 protein-coding Parkinson's disease-associated genes. This core network encompasses key PD-associated pathways, including ubiquitination, mitochondrial function, protein processing, RNA metabolism, and vesicular transport. Proteomics analysis showed a consistent alteration in proteins of dopamine metabolism, indicating a defect of dopaminergic metabolism in PINK1-ILE368ASN neurons. Our findings suggest the existence of a network onto which pathways associated with PD pathology converge, and offers an inclusive interpretation of the phenotypic heterogeneity of PD.
Collapse
Affiliation(s)
- Gabriela Novak
- The Integrative Cell Signalling Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.
- Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg.
- Center for Systems and Therapeutics, the Gladstone Institutes and Departments of Neurology and Physiology, University of California, San Francisco, San Francisco, CA, 94158, USA.
| | - Dimitrios Kyriakis
- The Integrative Cell Signalling Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Kamil Grzyb
- The Integrative Cell Signalling Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Michela Bernini
- The Integrative Cell Signalling Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Sophie Rodius
- Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg
| | - Gunnar Dittmar
- Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg
- Department of Life Sciences and Medicine, University of Luxembourg, Belvaux, Luxembourg
| | - Steven Finkbeiner
- Center for Systems and Therapeutics, the Gladstone Institutes and Departments of Neurology and Physiology, University of California, San Francisco, San Francisco, CA, 94158, USA
| | - Alexander Skupin
- The Integrative Cell Signalling Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.
- University of California San Diego, La Jolla, CA, 92093, USA.
| |
Collapse
|
|
39
|
Rodriguez-Polo I, Behr R. Non-human primate pluripotent stem cells for the preclinical testing of regenerative therapies. Neural Regen Res 2022; 17:1867-1874. [PMID: 35142660 PMCID: PMC8848615 DOI: 10.4103/1673-5374.335689] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies. Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases. Therefore, the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years. For the preclinical validation of cell replacement therapies in non-human primates, it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts. However, pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems. In recent years, however, relevant progress has also been made with non-human primate pluripotent stem cells. This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies. We focus on the critical domains of (1) reprogramming and embryonic stem cell line derivation, (2) cell line maintenance and characterization and, (3) application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.
Collapse
|
|
40
|
Keller A, Krivec N, Markouli C, Spits C. Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells. Methods Mol Biol 2022; 2429:57-72. [PMID: 35507155 DOI: 10.1007/978-1-0716-1979-7_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Human pluripotent stem cells have a wide variety of potential applications, ranging from clinical translation to in vitro disease modeling. However, there is significant variation in the potential of individual cell lines to differentiate towards each of the three germ layers as a result of (epi)genetic background, culture conditions, and other factors. We describe here in detail a methodology to evaluate this bias using short directed differentiation towards neuroectoderm, mesendoderm, and definitive endoderm in combination with quantification by RT-qPCR and immunofluorescent stains.
Collapse
Affiliation(s)
- Alexander Keller
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Nuša Krivec
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Christina Markouli
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium
| | - Claudia Spits
- Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium.
| |
Collapse
|
|
41
|
Andrews PW. Human pluripotent stem cells: tools for regenerative medicine. BIOMATERIALS TRANSLATIONAL 2021; 2:294-300. [PMID: 35837419 PMCID: PMC9255800 DOI: 10.12336/biomatertransl.2021.04.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Subscribe] [Scholar Register] [Received: 11/13/2021] [Revised: 12/12/2021] [Accepted: 12/20/2021] [Indexed: 01/17/2023]
Abstract
Human embryonic stem cells and induced pluripotent stem cells, together denoted as pluripotent stem cells have opened up unprecedented opportunities for developments in human healthcare over the past 20 years. Although much about the properties and behaviour of these cells required to underpin their applications has been discovered over this time, a number of issues remain. This brief review considers the history of these developments and some of the underlying biology, pointing out some of the problems still to be resolved, particularly in relation to their genetic stability and possible malignancy.
Collapse
Affiliation(s)
- Peter W. Andrews
- The Centre for Stem Cell Biology, The School of Bioscience, The University of Sheffield, Sheffield, UK
| |
Collapse
|
|
42
|
Tristan CA, Ormanoglu P, Slamecka J, Malley C, Chu PH, Jovanovic VM, Gedik Y, Jethmalani Y, Bonney C, Barnaeva E, Braisted J, Mallanna SK, Dorjsuren D, Iannotti MJ, Voss TC, Michael S, Simeonov A, Singeç I. Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells. Stem Cell Reports 2021; 16:3076-3092. [PMID: 34861164 PMCID: PMC8693769 DOI: 10.1016/j.stemcr.2021.11.004] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Revised: 11/02/2021] [Accepted: 11/04/2021] [Indexed: 12/21/2022] Open
Abstract
Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.
Collapse
Affiliation(s)
- Carlos A Tristan
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Pinar Ormanoglu
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Jaroslav Slamecka
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Claire Malley
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Pei-Hsuan Chu
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Vukasin M Jovanovic
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Yeliz Gedik
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Yogita Jethmalani
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Charles Bonney
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Elena Barnaeva
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - John Braisted
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Sunil K Mallanna
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Dorjbal Dorjsuren
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Michael J Iannotti
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Ty C Voss
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Sam Michael
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Anton Simeonov
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA
| | - Ilyas Singeç
- National Center for Advancing Translational Sciences (NCATS), Division of Preclinical Innovation (DPI), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA.
| |
Collapse
|
|
43
|
Vickers A, Tewary M, Laddach A, Poletti M, Salameti V, Fraternali F, Danovi D, Watt FM. Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation. Stem Cell Reports 2021; 16:2628-2641. [PMID: 34678211 PMCID: PMC8581167 DOI: 10.1016/j.stemcr.2021.09.017] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Revised: 09/22/2021] [Accepted: 09/23/2021] [Indexed: 12/03/2022] Open
Abstract
Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation.
Collapse
Affiliation(s)
- Alice Vickers
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Mukul Tewary
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Anna Laddach
- Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, Great Maze Pond, London SE1 9RT, UK; Development and Homeostasis of the Nervous System Laboratory, The Francis Crick Institute, London NW1 1AT, UK
| | - Martina Poletti
- Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK; Quadram Institute, Norwich Research Park, Norwich NR4 7UZ, UK
| | - Vasiliki Salameti
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK
| | - Franca Fraternali
- Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, Great Maze Pond, London SE1 9RT, UK
| | - Davide Danovi
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK; bit.bio, Babraham Research Campus, The Dorothy Hodgkin Building, Cambridge CB22 3FH, UK
| | - Fiona M Watt
- Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK.
| |
Collapse
|
|
44
|
Chu SL, Abe K, Yokota H, Cho D, Chen YH, Tsai MD. High Resolution U-Net for Quantitatively Analyzing Early Spatial Patterning of Human Induced Pluripotent Stem Cells on Micropatterns. ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. ANNUAL INTERNATIONAL CONFERENCE 2021; 2021:3713-3716. [PMID: 34892043 DOI: 10.1109/embc46164.2021.9630956] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Human induced pluripotent stem cells (hiPSCs) can differentiate into three germ layer cells, i.e. ectoderm, mesoderm and endoderm, on micropatterned chips in highly synchronous and reproducible manners. The cells are confined within the chip, expanding two-dimensionally as almost in the form of monolayer, thus to be ideal for serving quantitative analysis of their pluripotency. We present a new U-Net (MP-UNet) structure for cell segmentation of early spatial patterning of hiPSCs on micropattern chips using Hoechst fluorescence images. In this structure, the encoding/decoding layers can be dynamically adjusted to extract sufficient image features and be flexible to image sizes. Dice and weight loss functions are designed to identify slight difference in low signal-to-noise ratio, high boundary-to-area ratio and compacted cell images. Several sizes of Hoechst images were tested to show MP-UNet can achieve high accuracy in cell regions and number counting for various sizes of micropattern chips, thus to be excellent quantitative tool for early spatial patterning of hiPSCs.
Collapse
|
|
45
|
Siehler J, Blöchinger AK, Meier M, Lickert H. Engineering islets from stem cells for advanced therapies of diabetes. Nat Rev Drug Discov 2021; 20:920-940. [PMID: 34376833 DOI: 10.1038/s41573-021-00262-w] [Citation(s) in RCA: 61] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/22/2021] [Indexed: 12/20/2022]
Abstract
Diabetes mellitus is a metabolic disorder that affects more than 460 million people worldwide. Type 1 diabetes (T1D) is caused by autoimmune destruction of β-cells, whereas type 2 diabetes (T2D) is caused by a hostile metabolic environment that leads to β-cell exhaustion and dysfunction. Currently, first-line medications treat the symptomatic insulin resistance and hyperglycaemia, but do not prevent the progressive decline of β-cell mass and function. Thus, advanced therapies need to be developed that either protect or regenerate endogenous β-cell mass early in disease progression or replace lost β-cells with stem cell-derived β-like cells or engineered islet-like clusters. In this Review, we discuss the state of the art of stem cell differentiation and islet engineering, reflect on current and future challenges in the area and highlight the potential for cell replacement therapies, disease modelling and drug development using these cells. These efforts in stem cell and regenerative medicine will lay the foundations for future biomedical breakthroughs and potentially curative treatments for diabetes.
Collapse
Affiliation(s)
- Johanna Siehler
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany.,Technical University of Munich, Medical Faculty, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Anna Karolina Blöchinger
- Technical University of Munich, Medical Faculty, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany.,Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
| | - Matthias Meier
- Technical University of Munich, Medical Faculty, Munich, Germany.,Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany
| | - Heiko Lickert
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany. .,Technical University of Munich, Medical Faculty, Munich, Germany. .,German Center for Diabetes Research (DZD), Neuherberg, Germany. .,Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
| |
Collapse
|
|
46
|
Zeng Y, Tao F, Cui Z, Wu L, Xu J, Dong W, Liu C, Yang Z, Qin S. Dynamic integration and segregation of amygdala subregional functional circuits linking to physiological arousal. Neuroimage 2021; 238:118224. [PMID: 34087364 DOI: 10.1016/j.neuroimage.2021.118224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 04/23/2021] [Accepted: 05/29/2021] [Indexed: 11/18/2022] Open
Abstract
The dynamical organization of brain networks is essential to support human cognition and emotion for rapid adaption to ever-changing environment. As the core nodes of emotion-related brain circuitry, the basolateral amygdala (BLA) and centromedial amygdala (CMA) as two major amygdalar nuclei, are recognized to play distinct roles in affective functions and internal states, via their unique connections with cortical and subcortical structures in rodents. However, little is known how the dynamical organization of emotion-related brain circuitry reflects internal autonomic responses in humans. Using resting-state functional magnetic resonance imaging (fMRI) with K-means clustering approach in a total of 79 young healthy individuals (cohort 1: 42; cohort 2: 37), we identified two distinct states of BLA- and CMA-based intrinsic connectivity patterns, with one state (integration) showing generally stronger BLA- and CMA-based intrinsic connectivity with multiple brain networks, while the other (segregation) exhibiting weaker yet dissociable connectivity patterns. In an independent cohort 2 of fMRI data with concurrent recording of skin conductance, we replicated two similar dynamic states and further found higher skin conductance level in the integration than segregation state. Moreover, machine learning-based Elastic-net regression analyses revealed that time-varying BLA and CMA intrinsic connectivity with distinct network configurations yield higher predictive values for spontaneous fluctuations of skin conductance level in the integration than segregation state. Our findings highlight dynamic functional organization of emotion-related amygdala nuclei circuits and networks and its links to spontaneous autonomic arousal in humans.
Collapse
Affiliation(s)
- Yimeng Zeng
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China; Beijing Key Laboratory of Brain Imaging and Connectomics, Beijing Normal University, Beijing, China
| | - Fuxiang Tao
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China; Beijing Key Laboratory of Brain Imaging and Connectomics, Beijing Normal University, Beijing, China
| | - Zaixu Cui
- Chinese Institute for Brain Research, Beijing, China
| | - Liyun Wu
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China
| | - Jiahua Xu
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China; Beijing Key Laboratory of Brain Imaging and Connectomics, Beijing Normal University, Beijing, China
| | - Wenshan Dong
- School of Psychology, South China Normal University, Guangzhou, China
| | - Chao Liu
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China
| | - Zhi Yang
- Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Shaozheng Qin
- State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China; IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China; Beijing Key Laboratory of Brain Imaging and Connectomics, Beijing Normal University, Beijing, China.
| |
Collapse
|
|
47
|
Chen Y, Tristan CA, Chen L, Jovanovic VM, Malley C, Chu PH, Ryu S, Deng T, Ormanoglu P, Tao D, Fang Y, Slamecka J, Hong H, LeClair CA, Michael S, Austin CP, Simeonov A, Singeç I. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat Methods 2021; 18:528-541. [PMID: 33941937 PMCID: PMC8314867 DOI: 10.1038/s41592-021-01126-2] [Citation(s) in RCA: 77] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2020] [Accepted: 03/22/2021] [Indexed: 12/27/2022]
Abstract
Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. Pluripotent cells are capable of extensive self-renewal, yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. Here, we deployed innovative high-throughput screening strategies to identify a small molecule cocktail that dramatically improves viability of hPSCs and their differentiated progeny. The combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.
Collapse
Affiliation(s)
- Yu Chen
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Carlos A Tristan
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Lu Chen
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Vukasin M Jovanovic
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Claire Malley
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Pei-Hsuan Chu
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Seungmi Ryu
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Tao Deng
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Pinar Ormanoglu
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Dingyin Tao
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Yuhong Fang
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Jaroslav Slamecka
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Hyenjong Hong
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Christopher A LeClair
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Sam Michael
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Christopher P Austin
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Anton Simeonov
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA
| | - Ilyas Singeç
- National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD, USA.
| |
Collapse
|
|
48
|
Sasamata M, Shimojo D, Fuse H, Nishi Y, Sakurai H, Nakahata T, Yamagishi Y, Sasaki-Iwaoka H. Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo LabDroid. SLAS Technol 2021; 26:441-453. [PMID: 33775154 DOI: 10.1177/24726303211000690] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Induced pluripotent stem cells (iPSCs) are attractive for use in early drug discovery because they can differentiate into any cell type. Maintenance cultures and differentiation processes for iPSCs, however, require a high level of technical expertise. To overcome this problem, technological developments such as enhanced automation are necessary to replace manual operation. In addition, a robot system with the flexibility and expandability to carry out maintenance culture and each of the required differentiation processes would also be important. In this study, we established a platform to enable the multiple processes required for iPSC experiments using the Maholo LabDroid, which is a humanoid robotic system with excellent reproducibility and flexibility. The accuracy and robustness of Maholo LabDroid enabled us to cultivate undifferentiated iPSCs for 63 days while maintaining their ability to differentiate into the three embryonic germ layers. Maholo LabDroid maintained and harvested iPSCs in six-well plates, then seeded them into 96-well plates, induced differentiation, and implemented immunocytochemistry. As a result, Maholo LabDroid was confirmed to be able to perform the processes required for myogenic differentiation of iPSCs isolated from a patient with muscular disease and achieved a high differentiation rate with a coefficient of variation (CV) <10% in the first trial. Furthermore, the expandability and flexibility of Maholo LabDroid allowed us to experiment with multiple cell lines simultaneously.
Collapse
Affiliation(s)
- Miho Sasamata
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan
| | - Daisuke Shimojo
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan
| | - Hiromitsu Fuse
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Yohei Nishi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Tatsutoshi Nakahata
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Yukiko Yamagishi
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | | |
Collapse
|
|
49
|
Dziedzicka D, Tewary M, Keller A, Tilleman L, Prochazka L, Östblom J, Couvreu De Deckersberg E, Markouli C, Franck S, Van Nieuwerburgh F, Spits C, Zandstra PW, Sermon K, Geens M. Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm. Sci Rep 2021; 11:6137. [PMID: 33731744 PMCID: PMC7969605 DOI: 10.1038/s41598-021-85447-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 02/23/2021] [Indexed: 02/06/2023] Open
Abstract
Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.
Collapse
Affiliation(s)
- Dominika Dziedzicka
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mukul Tewary
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada ,grid.13097.3c0000 0001 2322 6764Centre for Stem Cells and Regenerative Medicine, King’s College London, Guy’s Hospital, London, SE1 9RT UK
| | - Alexander Keller
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Laurentijn Tilleman
- grid.5342.00000 0001 2069 7798Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Ghent, Belgium
| | - Laura Prochazka
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada
| | - Joel Östblom
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada
| | - Edouard Couvreu De Deckersberg
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Christina Markouli
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Silvie Franck
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Filip Van Nieuwerburgh
- grid.5342.00000 0001 2069 7798Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Ghent, Belgium
| | - Claudia Spits
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Peter W. Zandstra
- grid.17063.330000 0001 2157 2938Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1 Canada ,grid.17091.3e0000 0001 2288 9830Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4 Canada ,grid.17091.3e0000 0001 2288 9830School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3 Canada
| | - Karen Sermon
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mieke Geens
- grid.8767.e0000 0001 2290 8069Research Group Reproduction and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| |
Collapse
|
|
50
|
Jerber J, Seaton DD, Cuomo ASE, Kumasaka N, Haldane J, Steer J, Patel M, Pearce D, Andersson M, Bonder MJ, Mountjoy E, Ghoussaini M, Lancaster MA, Marioni JC, Merkle FT, Gaffney DJ, Stegle O. Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation. Nat Genet 2021; 53:304-312. [PMID: 33664506 PMCID: PMC7610897 DOI: 10.1038/s41588-021-00801-6] [Citation(s) in RCA: 156] [Impact Index Per Article: 39.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 01/25/2021] [Indexed: 02/06/2023]
Abstract
Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.
Collapse
Affiliation(s)
- Julie Jerber
- Open Targets, Wellcome Genome Campus, Hinxton, Cambridge, UK
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Daniel D Seaton
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Anna S E Cuomo
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Natsuhiko Kumasaka
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - James Haldane
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Juliette Steer
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Minal Patel
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Daniel Pearce
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Malin Andersson
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Marc Jan Bonder
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Ed Mountjoy
- Open Targets, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Maya Ghoussaini
- Open Targets, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | | | - John C Marioni
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
- Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
| | - Florian T Merkle
- Metabolic Research Laboratories and Medical Research Council Metabolic Diseases Unit, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK.
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
| | - Daniel J Gaffney
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
| | - Oliver Stegle
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
- Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
- Division of Computational Genomics and Systems Genetics, German Cancer Research Center, Heidelberg, Germany.
| |
Collapse
|