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1
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Westerberg NS, Atneosen-Åsegg M, Melheim M, Chollet ME, Harrison SP, Siller R, Sullivan GJ, Almaas R. Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells. Pediatr Res 2025; 97:195-201. [PMID: 38951656 DOI: 10.1038/s41390-024-03368-0] [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: 02/05/2024] [Revised: 06/03/2024] [Accepted: 06/13/2024] [Indexed: 07/03/2024]
Abstract
OBJECTIVES Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells. METHODS Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation. RESULTS Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls. CONCLUSIONS Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation. IMPACT Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells. Expression of hepatobiliary transporters varies during hypoxia and reoxygenation. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.
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Affiliation(s)
- Niklas Starck Westerberg
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | | | - Maria Melheim
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Maria Eugenia Chollet
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
| | - Sean P Harrison
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Richard Siller
- Department of Molecular Medicine, University of Oslo, Oslo, Norway
| | - Gareth J Sullivan
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
- School of Medicine, University of St Andrews, St Andrews, UK
| | - Runar Almaas
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway.
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
- European Reference Network-Rare Liver, Hamburg, Germany.
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2
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Harrison SP, Baumgarten SF, Chollet ME, Stavik B, Bhattacharya A, Almaas R, Sullivan GJ. Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform. J Pediatr Gastroenterol Nutr 2024; 78:1047-1058. [PMID: 38529852 DOI: 10.1002/jpn3.12195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2022] [Revised: 02/14/2024] [Accepted: 02/28/2024] [Indexed: 03/27/2024]
Abstract
OBJECTIVES Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. METHODS We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. RESULTS Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. CONCLUSIONS Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.
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Affiliation(s)
- Sean P Harrison
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Saphira F Baumgarten
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub-Center of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Research, Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
| | - Maria E Chollet
- Research, Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Benedicte Stavik
- Research, Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Anindita Bhattacharya
- Research, Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Runar Almaas
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | - Gareth J Sullivan
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
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3
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Harrison SP, Siller R, Tanaka Y, Chollet ME, de la Morena-Barrio ME, Xiang Y, Patterson B, Andersen E, Bravo-Pérez C, Kempf H, Åsrud KS, Lunov O, Dejneka A, Mowinckel MC, Stavik B, Sandset PM, Melum E, Baumgarten S, Bonanini F, Kurek D, Mathapati S, Almaas R, Sharma K, Wilson SR, Skottvoll FS, Boger IC, Bogen IL, Nyman TA, Wu JJ, Bezrouk A, Cizkova D, Corral J, Mokry J, Zweigerdt R, Park IH, Sullivan GJ. Scalable production of tissue-like vascularized liver organoids from human PSCs. Exp Mol Med 2023; 55:2005-2024. [PMID: 37653039 PMCID: PMC10545717 DOI: 10.1038/s12276-023-01074-1] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 04/18/2023] [Accepted: 06/02/2023] [Indexed: 09/02/2023] Open
Abstract
The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.
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Affiliation(s)
- Sean P Harrison
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Richard Siller
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Yoshiaki Tanaka
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, USA
- Department of Medicine, Faculty of Medicine, Maisonneuve-Rosemont Hospital Research Center (CRHMR), University of Montreal, Montreal, Canada
| | - Maria Eugenia Chollet
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - María Eugenia de la Morena-Barrio
- Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB, CIBERER, Murcia, Spain
| | - Yangfei Xiang
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, USA
| | - Benjamin Patterson
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, USA
| | - Elisabeth Andersen
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Carlos Bravo-Pérez
- Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB, CIBERER, Murcia, Spain
| | - Henning Kempf
- Department: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany
| | - Kathrine S Åsrud
- Norwegian PSC Research Center, Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Oleg Lunov
- Department of Optical and Biophysical Systems, Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic
| | - Alexandr Dejneka
- Department of Optical and Biophysical Systems, Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic
| | - Marie-Christine Mowinckel
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Benedicte Stavik
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Per Morten Sandset
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Espen Melum
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Department of Haematology, Oslo University Hospital, Oslo, Norway
- Norwegian PSC Research Center, Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
- Section for Gastroenterology, Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway
- European Reference Network RARE-LIVER, Hamburg, Germany
| | - Saphira Baumgarten
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | | | | | - Santosh Mathapati
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Runar Almaas
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
- European Reference Network RARE-LIVER, Hamburg, Germany
| | - Kulbhushan Sharma
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Steven R Wilson
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315, Oslo, Norway
| | - Frøydis S Skottvoll
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
- Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315, Oslo, Norway
| | - Ida C Boger
- Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315, Oslo, Norway
| | - Inger Lise Bogen
- Department of Forensic Sciences, Oslo University Hospital, Oslo, Norway
| | - Tuula A Nyman
- Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway
| | - Jun Jie Wu
- Department of Engineering, Faculty of Science, Durham University, Durham, DH1 3LE, United Kingdom
| | - Ales Bezrouk
- Department of Medical Biophysics, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic
| | - Dana Cizkova
- Department of Histology and Embryology, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic
| | - Javier Corral
- Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB, CIBERER, Murcia, Spain
| | - Jaroslav Mokry
- Department of Histology and Embryology, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic
| | - Robert Zweigerdt
- Department: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany
| | - In-Hyun Park
- Department of Genetics, Yale Stem Cell Center, Child Study Center, Yale School of Medicine, New Haven, USA
| | - Gareth J Sullivan
- Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway.
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
- Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.
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4
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Pardo-Palacios FJ, Arzalluz-Luque A, Kondratova L, Salguero P, Mestre-Tomás J, Amorín R, Estevan-Morió E, Liu T, Nanni A, McIntyre L, Tseng E, Conesa A. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.17.541248. [PMID: 37398077 PMCID: PMC10312485 DOI: 10.1101/2023.05.17.541248] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/04/2023]
Abstract
The emergence of long-read RNA sequencing (lrRNA-seq) has provided an unprecedented opportunity to analyze transcriptomes at isoform resolution. However, the technology is not free from biases, and transcript models inferred from these data require quality control and curation. In this study, we introduce SQANTI3, a tool specifically designed to perform quality analysis on transcriptomes constructed using lrRNA-seq data. SQANTI3 provides an extensive naming framework to describe transcript model diversity in comparison to the reference transcriptome. Additionally, the tool incorporates a wide range of metrics to characterize various structural properties of transcript models, such as transcription start and end sites, splice junctions, and other structural features. These metrics can be utilized to filter out potential artifacts. Moreover, SQANTI3 includes a Rescue module that prevents the loss of known genes and transcripts exhibiting evidence of expression but displaying low-quality features. Lastly, SQANTI3 incorporates IsoAnnotLite, which enables functional annotation at the isoform level and facilitates functional iso-transcriptomics analyses. We demonstrate the versatility of SQANTI3 in analyzing different data types, isoform reconstruction pipelines, and sequencing platforms, and how it provides novel biological insights into isoform biology. The SQANTI3 software is available at https://github.com/ConesaLab/SQANTI3 .
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5
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The Exciting Realities and Possibilities of iPS-Derived Cardiomyocytes. Bioengineering (Basel) 2023; 10:bioengineering10020237. [PMID: 36829731 PMCID: PMC9952364 DOI: 10.3390/bioengineering10020237] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Revised: 02/03/2023] [Accepted: 02/09/2023] [Indexed: 02/12/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) have become a prevalent topic after their discovery, advertised as an ethical alternative to embryonic stem cells (ESCs). Due to their ability to differentiate into several kinds of cells, including cardiomyocytes, researchers quickly realized the potential for differentiated cardiomyocytes to be used in the treatment of heart failure, a research area with few alternatives. This paper discusses the differentiation process for human iPSC-derived cardiomyocytes and the possible applications of said cells while answering some questions regarding ethical issues.
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6
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Amel A, Rossouw S, Goolam M. Gastruloids: A Novel System for Disease Modelling and Drug Testing. Stem Cell Rev Rep 2023; 19:104-113. [PMID: 36308705 DOI: 10.1007/s12015-022-10462-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/03/2022] [Indexed: 01/29/2023]
Abstract
By virtue of its inaccessible nature, mammalian implantation stage development has remained one of the most enigmatic and hard to investigate periods of embryogenesis. Derived from pluripotent stem cells, gastruloids recapitulate key aspects of gastrula-stage embryos and have emerged as a powerful in vitro tool to study the architectural features of early post-implantation embryos. While the majority of the work in this emerging field has focused on the use of gastruloids to model embryogenesis, their tractable nature and suitability for high-throughput scaling, has presented an unprecedented opportunity to investigate both developmental and environmental aberrations to the embryo as they occur in vitro. This review summarises the recent developments in the use of gastruloids to model congenital anomalies, their usage in teratogenicity testing, and the current limitations of this emerging field.
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Affiliation(s)
- Atoosa Amel
- Department of Human Biology, University of Cape Town, 7925, Cape Town, South Africa
| | - Simoné Rossouw
- Department of Human Biology, University of Cape Town, 7925, Cape Town, South Africa
| | - Mubeen Goolam
- Department of Human Biology, University of Cape Town, 7925, Cape Town, South Africa. .,UCT Neuroscience Institute, Cape Town, South Africa.
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7
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Kristiansen CK, Chen A, Høyland LE, Ziegler M, Sullivan GJ, Bindoff LA, Liang KX. Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations. Cell Cycle 2022; 21:2206-2221. [PMID: 35815665 DOI: 10.1080/15384101.2022.2092185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages - pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.Abbreviation: BSA: Bovine serum albumin; DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3β; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20.
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Affiliation(s)
- Cecilie Katrin Kristiansen
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway.,b Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
| | - Anbin Chen
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway.,Department of Neurosurgery, Qilu Hospital and Institute of Brain and Brain-Inspired Science, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Key Laboratory of Brain Function Remodeling, Jinan, China
| | | | - Mathias Ziegler
- Department of Biomedicine, University of Bergen, Bergen, Norway
| | - Gareth John Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Institute of Immunology, Oslo University Hospital, Oslo, Norway.,Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Laurence A Bindoff
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
| | - Kristina Xiao Liang
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
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8
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Sharma K, Asp NT, Harrison SP, Siller R, Baumgarten SF, Gupta S, Chollet ME, Andersen E, Sullivan GJ, Simonsen A. Autophagy modulates cell fate decisions during lineage commitment. Autophagy 2021; 18:1915-1931. [PMID: 34923909 PMCID: PMC9450964 DOI: 10.1080/15548627.2021.2008691] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Early events during development leading to exit from a pluripotent state and commitment toward a specific germ layer still need in depth understanding. Autophagy has been shown to play a crucial role in both development and differentiation. This study employs human embryonic and induced pluripotent stem cells to understand the early events of lineage commitment with respect to the role of autophagy in this process. Our data indicate that a dip in autophagy facilitates exit from pluripotency. Upon exit, we demonstrate that the modulation of autophagy affects SOX2 levels and lineage commitment, with induction of autophagy promoting SOX2 degradation and mesendoderm formation, whereas inhibition of autophagy causes SOX2 accumulation and neuroectoderm formation. Thus, our results indicate that autophagy-mediated SOX2 turnover is a determining factor for lineage commitment. These findings will deepen our understanding of development and lead to improved methods to derive different lineages and cell types.Abbreviations: ACTB: Actin, beta; ATG: Autophagy-related; BafA1: Bafilomycin A1; CAS9: CRISPR associated protein 9; CQ: Chloroquine; DE: Definitive endoderm; hESCs: Human Embryonic Stem Cells; hiPSCs: Human Induced Pluripotent Stem Cells; LAMP1: Lysosomal Associated Membrane Protein 1; MAP1LC3: Microtubule-Associated Protein 1 Light Chain 3; MTOR: Mechanistic Target Of Rapamycin Kinase; NANOG: Nanog Homeobox; PAX6: Paired Box 6; PE: Phosphatidylethanolamine; POU5F1: POU class 5 Homeobox 1; PRKAA2: Protein Kinase AMP-Activated Catalytic Subunit Alpha 2; SOX2: SRY-box Transcription Factor 2; SQSTM1: Sequestosome 1; ULK1: unc-51 like Autophagy Activating Kinase 1; WDFY3: WD Repeat and FYVE Domain Containing 3.
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Affiliation(s)
- Kulbhushan Sharma
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.,Division of Stem Cell and Gene Therapy Research, Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi, India.,Department of Neurology, Akershus University Hospital, Lørenskog, Norway
| | - Nagham T Asp
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | - Sean P Harrison
- Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Richard Siller
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Saphira F Baumgarten
- Hybrid Technology Hub, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Swapnil Gupta
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Neurology, Akershus University Hospital, Lørenskog, Norway
| | - Maria E Chollet
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway.,Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Elisabeth Andersen
- Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway.,Department of Haematology, Oslo University Hospital, Oslo, Norway
| | - Gareth J Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Pediatric Research, Oslo University Hospital, Oslo, Norway.,Hybrid Technology Hub, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Norwegian Center for Stem Cell Research, Oslo University Hospital and University of Oslo, Oslo, Norway.,Institute of Immunology, Oslo University Hospital, Oslo, Norway
| | - Anne Simonsen
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.,Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital Montebello, Oslo, Norway
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9
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Mennen RH, Oldenburger MM, Piersma AH. Endoderm and mesoderm derivatives in embryonic stem cell differentiation and their use in developmental toxicity testing. Reprod Toxicol 2021; 107:44-59. [PMID: 34861400 DOI: 10.1016/j.reprotox.2021.11.009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 11/25/2021] [Accepted: 11/29/2021] [Indexed: 02/06/2023]
Abstract
Embryonic stem cell differentiation models have increasingly been applied in non-animal test systems for developmental toxicity. After the initial focus on cardiac differentiation, attention has also included an array of neuro-ectodermal differentiation routes. Alternative differentiation routes in the mesodermal and endodermal germ lines have received less attention. This review provides an inventory of achievements in the latter areas of embryonic stem cell differentiation, with a view to possibilities for their use in non-animal test systems in developmental toxicology. This includes murine and human stem cell differentiation models, and also gains information from the field of stem cell use in regenerative medicine. Endodermal stem cell derivatives produced in vitro include hepatocytes, pancreatic cells, lung epithelium, and intestinal epithelium, and mesodermal derivatives include cardiac muscle, osteogenic, vascular and hemopoietic cells. This inventory provides an overview of studies on the different cell types together with biomarkers and culture conditions that stimulate these differentiation routes from embryonic stem cells. These models may be used to expand the spectrum of embryonic stem cell based new approach methodologies in non-animal developmental toxicity testing.
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Affiliation(s)
- R H Mennen
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, the Netherlands.
| | | | - A H Piersma
- Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, the Netherlands
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10
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Mostafavi S, Balafkan N, Pettersen IKN, Nido GS, Siller R, Tzoulis C, Sullivan GJ, Bindoff LA. Distinct Mitochondrial Remodeling During Mesoderm Differentiation in a Human-Based Stem Cell Model. Front Cell Dev Biol 2021; 9:744777. [PMID: 34722525 PMCID: PMC8553110 DOI: 10.3389/fcell.2021.744777] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 09/21/2021] [Indexed: 12/17/2022] Open
Abstract
Given the considerable interest in using stem cells for modeling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), plays a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that the metabolic “switch” from glycolysis to OXPHOS is germ layer-specific as glycolysis remains active during early ectoderm commitment but is downregulated during the transition to mesoderm and endoderm lineages. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remain unclear. Here, we address the question of mitochondrial adaptation by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to differentiated mesodermal derivatives, including functional cardiomyocytes. In contrast to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptations upon further differentiation. Our results improve the understanding of how mitochondrial remodeling and the metabolism interact during mesoderm differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content.
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Affiliation(s)
- Sepideh Mostafavi
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Novin Balafkan
- Department of Clinical Medicine, University of Bergen, Bergen, Norway.,Division of Psychiatry, Haukeland University Hospital, Bergen, Norway.,Norwegian Centre for Mental Disorders Research (NORMENT)-Centre of Excellence, Haukeland University Hospital, Bergen, Norway
| | | | - Gonzalo S Nido
- Department of Clinical Medicine, University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
| | - Richard Siller
- Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Charalampos Tzoulis
- Department of Clinical Medicine, University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
| | - Gareth J Sullivan
- Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Norwegian Center for Stem Cell Research, Oslo University Hospital and the University of Oslo, Oslo, Norway.,Institute of Immunology, Oslo University Hospital, Oslo, Norway.,Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Laurence A Bindoff
- Department of Clinical Medicine, University of Bergen, Bergen, Norway.,Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Bergen, Norway
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11
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Xu X, Nie Y, Wang W, Ullah I, Tung WT, Ma N, Lendlein A. Generation of 2.5D lung bud organoids from human induced pluripotent stem cells. Clin Hemorheol Microcirc 2021; 79:217-230. [PMID: 34487028 DOI: 10.3233/ch-219111] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70% confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90% confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.
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Affiliation(s)
- Xun Xu
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany
| | - Yan Nie
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany.,Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany
| | - Weiwei Wang
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany
| | - Imran Ullah
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany
| | - Wing Tai Tung
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany.,Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany
| | - Nan Ma
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany.,Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany
| | - Andreas Lendlein
- Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany.,Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.,Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany
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12
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Nowzari F, Wang H, Khoradmehr A, Baghban M, Baghban N, Arandian A, Muhaddesi M, Nabipour I, Zibaii MI, Najarasl M, Taheri P, Latifi H, Tamadon A. Three-Dimensional Imaging in Stem Cell-Based Researches. Front Vet Sci 2021; 8:657525. [PMID: 33937378 PMCID: PMC8079735 DOI: 10.3389/fvets.2021.657525] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2021] [Accepted: 03/19/2021] [Indexed: 12/14/2022] Open
Abstract
Stem cells have an important role in regenerative therapies, developmental biology studies and drug screening. Basic and translational research in stem cell technology needs more detailed imaging techniques. The possibility of cell-based therapeutic strategies has been validated in the stem cell field over recent years, a more detailed characterization of the properties of stem cells is needed for connectomics of large assemblies and structural analyses of these cells. The aim of stem cell imaging is the characterization of differentiation state, cellular function, purity and cell location. Recent progress in stem cell imaging field has included ultrasound-based technique to study living stem cells and florescence microscopy-based technique to investigate stem cell three-dimensional (3D) structures. Here, we summarized the fundamental characteristics of stem cells via 3D imaging methods and also discussed the emerging literatures on 3D imaging in stem cell research and the applications of both classical 2D imaging techniques and 3D methods on stem cells biology.
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Affiliation(s)
- Fariborz Nowzari
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Huimei Wang
- Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, Shanghai, China
| | - Arezoo Khoradmehr
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Mandana Baghban
- Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Neda Baghban
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Alireza Arandian
- Laser and Plasma Research Institute, Shahid Beheshti University, Tehran, Iran
| | - Mahdi Muhaddesi
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Iraj Nabipour
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
| | - Mohammad I. Zibaii
- Laser and Plasma Research Institute, Shahid Beheshti University, Tehran, Iran
| | - Mostafa Najarasl
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Payam Taheri
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Hamid Latifi
- Laser and Plasma Research Institute, Shahid Beheshti University, Tehran, Iran
- Department of Physics, Shahid Beheshti University, Tehran, Iran
| | - Amin Tamadon
- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
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13
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Aprigliano R, Aksu ME, Bradamante S, Mihaljevic B, Wang W, Rian K, Montaldo NP, Grooms KM, Fordyce Martin SL, Bordin DL, Bosshard M, Peng Y, Alexov E, Skinner C, Liabakk NB, Sullivan GJ, Bjørås M, Schwartz CE, van Loon B. Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities. Cell Rep Med 2021; 2:100240. [PMID: 33948573 PMCID: PMC8080178 DOI: 10.1016/j.xcrm.2021.100240] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Revised: 01/18/2021] [Accepted: 03/16/2021] [Indexed: 02/06/2023]
Abstract
Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.
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Affiliation(s)
- Rossana Aprigliano
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
- Department of Molecular Mechanisms of Disease, University of Zurich, 8057 Zürich, Switzerland
| | - Merdane Ezgi Aksu
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Stefano Bradamante
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
- Department of Pathology and Medical Genetics, St. Olavs University Hospital, 7049 Trondheim, Norway
| | - Boris Mihaljevic
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Wei Wang
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Kristin Rian
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Nicola P. Montaldo
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Kayla Mae Grooms
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Sarah L. Fordyce Martin
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Diana L. Bordin
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Matthias Bosshard
- Department of Molecular Mechanisms of Disease, University of Zurich, 8057 Zürich, Switzerland
| | - Yunhui Peng
- Computational Biophysics and Bioinformatics, Department of Physics and Astronomy, Clemson University, Clemson, SC 29631, USA
| | - Emil Alexov
- Computational Biophysics and Bioinformatics, Department of Physics and Astronomy, Clemson University, Clemson, SC 29631, USA
| | | | - Nina-Beate Liabakk
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
| | - Gareth J. Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0315 Oslo, Norway
- Hybrid Technology Hub, Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, 0315 Oslo, Norway
| | - Magnar Bjørås
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
- Department of Pathology and Medical Genetics, St. Olavs University Hospital, 7049 Trondheim, Norway
- Department of Microbiology, Oslo University Hospital, Department of Medical Biochemistry, Oslo University Hospital and University of Oslo, 0372 Oslo, Norway
| | | | - Barbara van Loon
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), 7049 Trondheim, Norway
- Department of Molecular Mechanisms of Disease, University of Zurich, 8057 Zürich, Switzerland
- Department of Pathology and Medical Genetics, St. Olavs University Hospital, 7049 Trondheim, Norway
- Corresponding author
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14
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Goliusova DV, Klementieva NV, Panova AV, Mokrysheva NG, Kiselev SL. The Role of Genetic Factors in Endocrine Tissues Development and Its Regulation In Vivo and In Vitro. RUSS J GENET+ 2021; 57:273-281. [DOI: 10.1134/s102279542103008x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/13/2020] [Accepted: 06/16/2020] [Indexed: 02/05/2023]
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15
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Skottvoll F, Hansen FA, Harrison S, Boger IS, Mrsa A, Restan MS, Stein M, Lundanes E, Pedersen-Bjergaard S, Aizenshtadt A, Krauss S, Sullivan G, Bogen IL, Wilson SR. Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies. Anal Chem 2021; 93:3576-3585. [PMID: 33534551 PMCID: PMC8023518 DOI: 10.1021/acs.analchem.0c05082] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Accepted: 01/25/2021] [Indexed: 12/20/2022]
Abstract
Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 μL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.
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Affiliation(s)
- Frøydis
Sved Skottvoll
- Department
of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
| | - Frederik André Hansen
- Department
of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, NO-0316 Oslo, Norway
| | - Sean Harrison
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
| | - Ida Sneis Boger
- Department
of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
| | - Ago Mrsa
- Department
of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
| | - Magnus Saed Restan
- Department
of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, NO-0316 Oslo, Norway
| | - Matthias Stein
- Institute
of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr.
55, DE-38106 Braunschweig, Germany
| | - Elsa Lundanes
- Department
of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway
| | - Stig Pedersen-Bjergaard
- Department
of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, NO-0316 Oslo, Norway
- Department
of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
| | - Aleksandra Aizenshtadt
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
| | - Stefan Krauss
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
- Department
of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 1110, Blindern, 0317, Oslo, Norway
| | - Gareth Sullivan
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
- Department
of Pediatric Research, Oslo University Hospital
and University of Oslo, P.O. Box 1112,
Blindern, 0317 Oslo, Norway
| | - Inger Lise Bogen
- Section
for Drug Abuse Research, Department of Forensic Sciences, Oslo University Hospital, P.O. Box 4950, Nydalen, NO-0424 Oslo, Norway
- Institute
of Basic Medical Sciences, Faculty of Medicine, University of Oslo, P.O. Box 1103,
Blindern, NO-0317 Oslo, Norway
| | - Steven Ray Wilson
- Department
of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway
- Hybrid
Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, NO-0317 Oslo, Norway
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16
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Jacobson EF, Chen Z, Stoukides DM, Nair GG, Hebrok M, Tzanakakis ES. Non-xenogeneic expansion and definitive endoderm differentiation of human pluripotent stem cells in an automated bioreactor. Biotechnol Bioeng 2020; 118:979-991. [PMID: 33205831 DOI: 10.1002/bit.27629] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2020] [Revised: 10/13/2020] [Accepted: 11/08/2020] [Indexed: 12/15/2022]
Abstract
Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with the maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.
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Affiliation(s)
- Elena F Jacobson
- Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA
| | - Zijing Chen
- Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA
| | - Demetrios M Stoukides
- Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA
| | - Gopika G Nair
- Department of Medicine, Diabetes Center, University of California - San Francisco, San Francisco, California, USA
| | - Matthias Hebrok
- Department of Medicine, Diabetes Center, University of California - San Francisco, San Francisco, California, USA
| | - Emmanuel S Tzanakakis
- Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA
- Clinical and Translational Science Institute, Tufts Medical Center, Boston, Massachusetts, USA
- Department of Developmental, Molecular and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts, USA
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17
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Liang KX, Vatne GH, Kristiansen CK, Ievglevskyi O, Kondratskaya E, Glover JC, Chen A, Sullivan GJ, Bindoff LA. N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation. Exp Neurol 2020; 337:113536. [PMID: 33264635 DOI: 10.1016/j.expneurol.2020.113536] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2020] [Revised: 11/22/2020] [Accepted: 11/22/2020] [Indexed: 01/03/2023]
Abstract
The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms in neurons remains difficult due to the limited access to human tissue. Using human induced pluripotent stem cells (hiPSCs), we generated functional dopaminergic (DA) neurons showing positive expression of dopaminergic markers TH and DAT, mature neuronal marker MAP2 and functional synaptic markers synaptophysin and PSD-95. These DA neurons were electrophysiologically characterized, and exhibited inward Na + currents, overshooting action potentials and spontaneous postsynaptic currents (sPSCs). POLG patient-specific DA neurons (POLG-DA neurons) manifested a phenotype that replicated the molecular and biochemical changes found in patient post-mortem brain samples namely loss of complex I and depletion of mtDNA. Compared to disease-free hiPSC-derived DA neurons, POLG-DA neurons exhibited loss of mitochondrial membrane potential, loss of complex I and loss of mtDNA and TFAM expression. POLG driven mitochondrial dysfunction also led to neuronal ROS overproduction and increased cellular senescence. This deficit was selectively rescued by treatment with N-acetylcysteine amide (NACA). In conclusion, our study illustrates the promise of hiPSC technology for assessing pathogenetic mechanisms associated with POLG disease, and that NACA can be a promising potential therapy for mitochondrial diseases such as those caused by POLG mutation.
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Affiliation(s)
- Kristina Xiao Liang
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway; Department of Clinical Medicine (K1), University of Bergen, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway.
| | - Guro Helén Vatne
- Department of Clinical Medicine (K1), University of Bergen, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway
| | - Cecilie Katrin Kristiansen
- Department of Clinical Medicine (K1), University of Bergen, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway
| | - Oleksandr Ievglevskyi
- The Intervention Centre, Oslo University Hospital, P. O. Box 4950, Nydalen, 0424 Oslo, Norway; Laboratory of Neural Development and Optical Recording (NDEVOR), Division of Physiology, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1103, Blindern, 0317 Oslo, Norway
| | - Elena Kondratskaya
- Laboratory of Neural Development and Optical Recording (NDEVOR), Division of Physiology, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1103, Blindern, 0317 Oslo, Norway
| | - Joel C Glover
- Laboratory of Neural Development and Optical Recording (NDEVOR), Division of Physiology, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1103, Blindern, 0317 Oslo, Norway; Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, P. O. Box 4950, Nydalen, 0424 Oslo, Norway
| | - Anbin Chen
- Department of Clinical Medicine (K1), University of Bergen, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway; Department of Neurosurgery, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012, Shandong Province, China; Shandong Key Laboratory of Brain Function Remodeling, Shandong University, 107 Wenhua Xi Road, Jinan 250012, Shandong Province, China
| | - Gareth John Sullivan
- Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, P. O. Box 4950, Nydalen, 0424 Oslo, Norway; Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1105, Blindern, 0317 Oslo, Norway; Institute of Immunology, Oslo University Hospital, PO Box 4950, 0424 Oslo, Norway; Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1110, Blindern, 0317 Oslo, Norway; Department of Pediatric Research, Oslo University Hospital, P. O. Box 4950, Nydalen, 0424 Oslo, Norway
| | - Laurence A Bindoff
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Department of Neurology, Haukeland University Hospital, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway; Department of Clinical Medicine (K1), University of Bergen, Jonas Lies vei 87, P. O. Box 7804, 5021 Bergen, Norway.
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18
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Balafkan N, Mostafavi S, Schubert M, Siller R, Liang KX, Sullivan G, Bindoff LA. A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates. Sci Rep 2020; 10:18498. [PMID: 33116175 PMCID: PMC7595118 DOI: 10.1038/s41598-020-73656-2] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Accepted: 09/21/2020] [Indexed: 12/11/2022] Open
Abstract
The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.
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Affiliation(s)
- Novin Balafkan
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway
| | - Sepideh Mostafavi
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway
| | - Manja Schubert
- Department of Neurology, Haukeland University Hospital, 5021, Bergen, Norway
| | - Richard Siller
- Norwegian Center for Stem Cell Research, Oslo University Hospital, Domus Medica, Oslo, Norway.,Institute of Immunology, Oslo University Hospital-Rikshospitalet, P.O. Box 4950, 0424, Nydalen, Oslo, Norway
| | - Kristina Xiao Liang
- Department of Neurology, Haukeland University Hospital, 5021, Bergen, Norway
| | - Gareth Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Norwegian Center for Stem Cell Research, Oslo University Hospital, Domus Medica, Oslo, Norway.,Institute of Immunology, Oslo University Hospital-Rikshospitalet, P.O. Box 4950, 0424, Nydalen, Oslo, Norway.,Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Blindern, Oslo, Norway
| | - Laurence A Bindoff
- Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway. .,Department of Neurology, Haukeland University Hospital, 5021, Bergen, Norway. .,Neuro-SysMed, Department of Neurology, Haukeland University Hospital, Bergen, Norway.
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19
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Liang KX, Kristiansen CK, Mostafavi S, Vatne GH, Zantingh GA, Kianian A, Tzoulis C, Høyland LE, Ziegler M, Perez RM, Furriol J, Zhang Z, Balafkan N, Hong Y, Siller R, Sullivan GJ, Bindoff LA. Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations. EMBO Mol Med 2020; 12:e12146. [PMID: 32840960 PMCID: PMC7539330 DOI: 10.15252/emmm.202012146] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 07/30/2020] [Accepted: 07/31/2020] [Indexed: 12/12/2022] Open
Abstract
Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post‐mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG‐driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD+ metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.
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Affiliation(s)
- Kristina Xiao Liang
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, Bergen, Norway.,Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | | | - Sepideh Mostafavi
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Guro Helén Vatne
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Gina Alien Zantingh
- Leiden University Medical Centre, Leiden University, Leiden, The Netherlands
| | - Atefeh Kianian
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Charalampos Tzoulis
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, Bergen, Norway.,Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | | | - Mathias Ziegler
- Department of Biomedicine, University of Bergen, Bergen, Norway
| | | | - Jessica Furriol
- Department of Clinical Medicine, University of Bergen, Bergen, Norway.,Department of Medicine, Haukeland University Hospital, Bergen, Norway
| | - Zhuoyuan Zhang
- State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China.,Department of Head and Neck Cancer Surgery, West China School of Stomatology, Sichuan University, Chengdu, China
| | - Novin Balafkan
- Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Yu Hong
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, Bergen, Norway.,Department of Clinical Medicine, University of Bergen, Bergen, Norway
| | - Richard Siller
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Norwegian Center for Stem Cell Research, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Gareth John Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Norwegian Center for Stem Cell Research, Oslo University Hospital and University of Oslo, Oslo, Norway.,Institute of Immunology, Oslo University Hospital, Oslo, Norway.,Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
| | - Laurence A Bindoff
- Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, Bergen, Norway.,Department of Clinical Medicine, University of Bergen, Bergen, Norway
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20
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Naumovska E, Aalderink G, Wong Valencia C, Kosim K, Nicolas A, Brown S, Vulto P, Erdmann KS, Kurek D. Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells. Int J Mol Sci 2020; 21:ijms21144964. [PMID: 32674311 PMCID: PMC7404294 DOI: 10.3390/ijms21144964] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 07/03/2020] [Accepted: 07/10/2020] [Indexed: 12/15/2022] Open
Abstract
Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.
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Affiliation(s)
- Elena Naumovska
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
- Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (C.W.V.); (S.B.)
| | - Germaine Aalderink
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
| | - Christian Wong Valencia
- Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (C.W.V.); (S.B.)
| | - Kinga Kosim
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
- Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (C.W.V.); (S.B.)
| | - Arnaud Nicolas
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
| | - Stephen Brown
- Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (C.W.V.); (S.B.)
| | - Paul Vulto
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
| | - Kai S. Erdmann
- Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; (C.W.V.); (S.B.)
- Correspondence: (K.S.E.); (D.K.)
| | - Dorota Kurek
- Mimetas BV, Model Development, J.H. Oortweg 16, 2333 CH Leiden, The Netherlands; (E.N.); (G.A.); (K.K.); (A.N.); (P.V.)
- Correspondence: (K.S.E.); (D.K.)
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21
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Strano A, Tuck E, Stubbs VE, Livesey FJ. Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways. Cell Rep 2020; 31:107732. [PMID: 32521257 PMCID: PMC7296348 DOI: 10.1016/j.celrep.2020.107732] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Revised: 03/09/2020] [Accepted: 05/14/2020] [Indexed: 12/12/2022] Open
Abstract
Directed differentiation of human pluripotent stem cells varies in specificity and efficiency. Stochastic, genetic, intracellular, and environmental factors affect maintenance of pluripotency and differentiation into early embryonic lineages. However, factors affecting variation in in vitro differentiation to defined cell types are not well understood. To address this, we focused on a well-established differentiation process to cerebral cortex neural progenitor cells and their neuronal progeny from human pluripotent stem cells. Analysis of 162 differentiation outcomes of 61 stem cell lines derived from 37 individuals showed that most variation occurs along gene expression axes reflecting dorsoventral and rostrocaudal spatial expression during in vivo brain development. Line-independent and line-dependent variations occur, with the latter driven largely by differences in endogenous Wnt signaling activity. Tuning Wnt signaling during a specific phase early in the differentiation process reduces variability, demonstrating that cell-line/genome-specific differentiation outcome biases can be corrected by controlling extracellular signaling.
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Affiliation(s)
- Alessio Strano
- The Wellcome Trust/Cancer Research UK Gurdon Institute & Department of Biochemistry, University of Cambridge, Cambridge, UK; UCL Great Ormond Street Institute of Child Health, Zayed Centre for Research into Rare Disease in Children, University College London, 20 Guilford Street, London WC1N 1DZ, UK
| | - Eleanor Tuck
- UCL Great Ormond Street Institute of Child Health, Zayed Centre for Research into Rare Disease in Children, University College London, 20 Guilford Street, London WC1N 1DZ, UK
| | - Victoria E Stubbs
- The Wellcome Trust/Cancer Research UK Gurdon Institute & Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Frederick J Livesey
- UCL Great Ormond Street Institute of Child Health, Zayed Centre for Research into Rare Disease in Children, University College London, 20 Guilford Street, London WC1N 1DZ, UK.
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22
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Chen YF, Li YSJ, Chou CH, Chiew MY, Huang HD, Ho JHC, Chien S, Lee OK. Control of matrix stiffness promotes endodermal lineage specification by regulating SMAD2/3 via lncRNA LINC00458. SCIENCE ADVANCES 2020; 6:eaay0264. [PMID: 32076643 PMCID: PMC7002135 DOI: 10.1126/sciadv.aay0264] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 11/22/2019] [Indexed: 05/07/2023]
Abstract
During endoderm formation, cell identity and tissue morphogenesis are tightly controlled by cell-intrinsic and cell-extrinsic factors such as biochemical and physical inputs. While the effects of biochemical factors are well studied, the physical cues that regulate cell division and differentiation are poorly understood. RNA sequencing analysis demonstrated increases of endoderm-specific gene expression in hPSCs cultured on soft substrate (Young's modulus, 3 ± 0.45 kPa) in comparison with hard substrate (Young's modulus, 165 ± 6.39 kPa). Further analyses revealed that multiple long noncoding RNAs (lncRNAs) were up-regulated on soft substrate; among them, LINC00458 was identified as a stiffness-dependent lncRNA specifically required for hPSC differentiation toward an early endodermal lineage. Gain- and loss-of-function experiments confirmed that LINC00458 is functionally required for hPSC endodermal lineage specification induced by soft substrates. Our study provides evidence that mechanical cues regulate the expression of LINC00458 and induce differentiation of hPSC into hepatic lineage progenitors.
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Affiliation(s)
- Yu-Fan Chen
- Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Yi-Shuan J. Li
- Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA, USA
- Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA
| | - Chih-Hung Chou
- Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
- Department of Biological Science and Technology, Center for Intelligent Drug Systems and Smart Bio-devices (IDSB), National Chiao Tung University, Hsinchu, Taiwan
| | - Men Yee Chiew
- Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
| | - Hsien-Da Huang
- School of Life and Health Sciences, Warshel Institute for Computational Biology, The Chinese University of Hong Kong, Shenzhen, Guangdong, China
| | - Jennifer Hui-Chun Ho
- Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
- Corresponding author. (J.H.-C.H.); (S.C.); (O.K.L.)
| | - Shu Chien
- Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA, USA
- Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA
- Corresponding author. (J.H.-C.H.); (S.C.); (O.K.L.)
| | - Oscar K. Lee
- Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
- Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong
- Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong
- Corresponding author. (J.H.-C.H.); (S.C.); (O.K.L.)
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23
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Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids. J Hepatol 2019; 71:970-985. [PMID: 31299272 DOI: 10.1016/j.jhep.2019.06.030] [Citation(s) in RCA: 204] [Impact Index Per Article: 34.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2018] [Revised: 06/19/2019] [Accepted: 06/21/2019] [Indexed: 12/27/2022]
Abstract
BACKGROUND & AIMS The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed. METHODS We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses. RESULTS Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis. CONCLUSIONS Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine. LAY SUMMARY A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.
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24
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Liu LP, Zheng YW. Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges. World J Stem Cells 2019; 11:375-382. [PMID: 31396366 PMCID: PMC6682503 DOI: 10.4252/wjsc.v11.i7.375] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2019] [Revised: 06/12/2019] [Accepted: 06/20/2019] [Indexed: 02/06/2023] Open
Abstract
The capability of human pluripotent stem cell (hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by high-throughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo. Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation. For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost, restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.
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Affiliation(s)
- Li-Ping Liu
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Yun-Wen Zheng
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China.
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25
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Jacobson EF, Tzanakakis ES. Who Will Win: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells for β Cell Replacement and Diabetes Disease Modeling? Curr Diab Rep 2018; 18:133. [PMID: 30343423 DOI: 10.1007/s11892-018-1109-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
PURPOSE OF REVIEW Ever since the reprogramming of human fibroblasts to induced pluripotent stem cells (hiPSCs), scientists have been trying to determine if hiPSCs can give rise to progeny akin to native terminally differentiated cells as human embryonic stem cells (hESCs) do. Many different somatic cell types have been successfully reprogrammed via a variety of methods. In this review, we will discuss recent studies comparing hiPSCs and hESCs and their ability to differentiate to desired cell types as well as explore diabetes disease models. RECENT FINDINGS Both somatic cell origin and the reprogramming method are important to the epigenetic state of the hiPSCs; however, genetic background contributes the most to differences seen between hiPSCs and hESCs. Based on our review of the relevant literature, hiPSCs display differences compared to hESCs, including a higher propensity for specification toward particular cell types based on memory retained from the somatic cell of origin. Moreover, hiPSCs provide a unique opportunity for creating diabetes disease models.
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Affiliation(s)
- Elena F Jacobson
- Department of Chemical and Biological Engineering, Tufts University, Science and Technology Center, Room 276A, Medford, MA, 02155, USA
| | - Emmanuel S Tzanakakis
- Department of Chemical and Biological Engineering, Tufts University, Science and Technology Center, Room 276A, Medford, MA, 02155, USA.
- Clinical and Translational Science Institute, Tufts Medical Center, Boston, MA, 02111, USA.
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26
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Gaspari E, Franke A, Robles-Diaz D, Zweigerdt R, Roeder I, Zerjatke T, Kempf H. Paracrine mechanisms in early differentiation of human pluripotent stem cells: Insights from a mathematical model. Stem Cell Res 2018; 32:1-7. [PMID: 30145492 DOI: 10.1016/j.scr.2018.07.025] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 07/13/2018] [Accepted: 07/24/2018] [Indexed: 02/01/2023] Open
Abstract
With their capability to self-renew and differentiate into derivatives of all three germ layers, human pluripotent stem cells (hPSCs) offer a unique model to study aspects of human development in vitro. Directed differentiation towards mesendodermal lineages is a complex process, involving transition through a primitive streak (PS)-like stage. We have recently shown PS-like patterning from hPSCs into definitive endoderm, cardiac as well as presomitic mesoderm by only modulating the bulk cell density and the concentration of the GSK3 inhibitor CHIR99021, a potent activator of the WNT pathway. The patterning process is modulated by a complex paracrine network, whose identity and mechanistic consequences are poorly understood. To study the underlying dynamics, we here applied mathematical modeling based on ordinary differential equations. We compared time-course data of early hPSC differentiation to increasingly complex model structures with incremental numbers of paracrine factors. Model simulations suggest at least three paracrine factors being required to recapitulate the experimentally observed differentiation kinetics. Feedback mechanisms from both undifferentiated and differentiated cells turned out to be crucial. Evidence from double knock-down experiments and secreted protein enrichment allowed us to hypothesize on the identity of two of the three predicted factors. From a practical perspective, the mathematical model predicts optimal settings for directing lineage-specific differentiation. This opens new avenues for rational stem cell bioprocessing in more advanced culture systems, e.g. in perfusion-fed bioreactors enabling cell therapies.
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Affiliation(s)
- Erika Gaspari
- Institute for Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, TU Dresden, Dresden, Germany; Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Wageningen, the Netherlands
| | - Annika Franke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH-Cluster of Excellence, Hannover Medical School, Germany
| | - Diana Robles-Diaz
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH-Cluster of Excellence, Hannover Medical School, Germany
| | - Robert Zweigerdt
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH-Cluster of Excellence, Hannover Medical School, Germany
| | - Ingo Roeder
- Institute for Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Thomas Zerjatke
- Institute for Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, TU Dresden, Dresden, Germany.
| | - Henning Kempf
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH-Cluster of Excellence, Hannover Medical School, Germany.
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27
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The potential of human induced pluripotent stem cells for modelling diabetic wound healing in vitro. Clin Sci (Lond) 2018; 132:1629-1643. [PMID: 30108152 DOI: 10.1042/cs20171483] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Revised: 06/28/2018] [Accepted: 07/23/2018] [Indexed: 12/11/2022]
Abstract
Impaired wound healing and ulceration caused by diabetes mellitus, is a significant healthcare burden, markedly impairs quality of life for patients, and is the major cause of amputation worldwide. Current experimental approaches used to investigate the complex wound healing process often involve cultures of fibroblasts and/or keratinocytes in vitro, which can be limited in terms of complexity and capacity, or utilisation of rodent models in which the mechanisms of wound repair differ substantively from that in humans. However, advances in tissue engineering, and the discovery of strategies to reprogramme adult somatic cells to pluripotency, has led to the possibility of developing models of human skin on a large scale. Generation of induced pluripotent stem cells (iPSCs) from tissues donated by diabetic patients allows the (epi)genetic background of this disease to be studied, and the ability to differentiate iPSCs to multiple cell types found within skin may facilitate the development of more complex skin models; these advances offer key opportunities for improving modelling of wound healing in diabetes, and the development of effective therapeutics for treatment of chronic wounds.
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28
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Asumda FZ, Hatzistergos KE, Dykxhoorn DM, Jakubski S, Edwards J, Thomas E, Schiff ER. Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Differentiation 2018; 101:16-24. [PMID: 29626713 PMCID: PMC6055513 DOI: 10.1016/j.diff.2018.03.002] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2017] [Revised: 03/18/2018] [Accepted: 03/21/2018] [Indexed: 12/19/2022]
Abstract
A variety of approaches have been developed for the derivation of hepatocyte-like cells from pluripotent stem cells. Currently, most of these strategies employ step-wise differentiation approaches with recombinant growth-factors or small-molecule analogs to recapitulate developmental signaling pathways. Here, we tested the efficacy of a small-molecule based differentiation protocol for the generation of hepatocyte-like cells from human pluripotent stem cells. Quantitative gene-expression, immunohistochemical, and western blot analyses for SOX17, FOXA2, CXCR4, HNF4A, AFP, indicated the stage-specific differentiation into definitive endoderm, hepatoblast and hepatocyte-like derivatives. Furthermore, hepatocyte-like cells displayed morphological and functional features characteristic of primary hepatocytes, as indicated by the production of ALB (albumin) and α-1-antitrypsin (A1AT), as well as glycogen storage capacity by periodic acid-Schiff staining. Together, these data support that the small-molecule based hepatic differentiation protocol is a simple, reproducible, and inexpensive method to efficiently drive the differentiation of human pluripotent stem cells towards a hepatocyte-like phenotype, for downstream pharmacogenomic and regenerative medicine applications.
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Affiliation(s)
- Faizal Z Asumda
- Schiff Center for Liver Diseases, University of Miami Miller School of Medicine, Miami, FL 33136, United States.
| | - Konstantinos E Hatzistergos
- Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Derek M Dykxhoorn
- John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Silvia Jakubski
- Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Jasmine Edwards
- Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Emmanuel Thomas
- Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Eugene R Schiff
- Schiff Center for Liver Diseases, University of Miami Miller School of Medicine, Miami, FL 33136, United States
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Pisal RV, Suchanek J, Siller R, Soukup T, Hrebikova H, Bezrouk A, Kunke D, Micuda S, Filip S, Sullivan G, Mokry J. Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth. Sci Rep 2018; 8:6168. [PMID: 29670257 PMCID: PMC5906561 DOI: 10.1038/s41598-018-24421-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 04/03/2018] [Indexed: 02/07/2023] Open
Abstract
The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.
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Affiliation(s)
- Rishikaysh V Pisal
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Jakub Suchanek
- Department of Dentistry, Faculty Hospital in Hradec Kralove and Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Richard Siller
- Norwegian Center for Stem Cell Research, University of Oslo, 0317, Oslo, Norway.,Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0317, Oslo, Norway
| | - Tomas Soukup
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Hana Hrebikova
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Ales Bezrouk
- Department of Biophysics, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - David Kunke
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Stanislav Micuda
- Department of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Stanislav Filip
- Department of Oncology and Radiotherapy, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic
| | - Gareth Sullivan
- Norwegian Center for Stem Cell Research, University of Oslo, 0317, Oslo, Norway.,Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0317, Oslo, Norway.,Institute of Immunology, Oslo University Hospital-Rikshospitalet, PO Box 4950 Nydalen, Oslo, 0424, Norway.,Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Blindern, 0317, Oslo, Norway.,Department of Pediatric Research, Oslo University Hospital, 0424, Nydalen, Norway
| | - Jaroslav Mokry
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 03, Hradec Kralove, Czech Republic.
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Siller R, Sullivan GJ. Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells. ACTA ACUST UNITED AC 2018; 43:1G.7.1-1G.7.23. [DOI: 10.1002/cpsc.36] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Affiliation(s)
- Richard Siller
- Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo Blindern Oslo Norway
- Norwegian Center for Stem Cell Research Blindern Oslo Norway
| | - Gareth J. Sullivan
- Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo Blindern Oslo Norway
- Norwegian Center for Stem Cell Research Blindern Oslo Norway
- Institute of Immunology, Oslo University Hospital Nydalen Oslo Norway
- Department of Pediatric Research, Oslo University Hospital Nydalen Norway
- Hybrid Technology Hub–Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo Blindern Oslo Norway
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31
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Keller A, Dziedzicka D, Zambelli F, Markouli C, Sermon K, Spits C, Geens M. Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells. Hum Reprod Update 2018; 24:162-175. [PMID: 29377992 DOI: 10.1093/humupd/dmx042] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Revised: 11/23/2017] [Accepted: 12/22/2017] [Indexed: 01/19/2023] Open
Abstract
BACKGROUND Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. OBJECTIVE AND RATIONALE Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. SEARCH METHODS Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. OUTCOMES Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. WIDER IMPLICATIONS As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.
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Affiliation(s)
- Alexander Keller
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Dominika Dziedzicka
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Filippo Zambelli
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Christina Markouli
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Karen Sermon
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Claudia Spits
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
| | - Mieke Geens
- Research group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Jette, Belgium
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Future Challenges in the Generation of Hepatocyte-Like Cells From Human Pluripotent Stem Cells. CURRENT PATHOBIOLOGY REPORTS 2017. [DOI: 10.1007/s40139-017-0150-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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