21 results on '"Orlova, V.V."'
Search Results
2. On-chip analysis of glycolysis and mitochondrial respiration in human induced pluripotent stem cells
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Fuchs, S., Helden, R.W.J. van, Wiendels, M., Graaf, M.N.S. de, Orlova, V.V., Mummery, C.L., Meer, B.J. van, and Mayr, T.
- Subjects
Biomaterials ,Oxygen ,Extracellular flux ,Metabolism ,Sensors ,pH ,Biomedical Engineering ,Bioengineering ,Human stem cells ,Cell Biology ,Molecular Biology ,Organ -on -Chip ,Biotechnology - Abstract
Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human -induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under con-ditions of fluidic flow with the same precision and relevance as current standard static cell cultures.
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- 2022
3. Perspectives for Future Use of Cardiac Microtissues from Human Pluripotent Stem Cells
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Arslan, U., Orlova, V.V., and Mummery, C.L.
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Biomaterials ,human induced pluripotent stem cells ,Tissue Engineering ,engineered heart tissue ,cardiac microtissues ,Induced Pluripotent Stem Cells ,Biomedical Engineering ,Animals ,Humans ,pluripotent stem cells - Abstract
Cardiovascular disorders remain a critical health issue worldwide. While animals have been used extensively as experimental models to investigate heart disease mechanisms and develop drugs, their inherent drawbacks have shifted focus to more human-relevant alternatives. Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, collectively called hPSCs) have been identified as a source of different cardiac cells, but to date, they have rarely offered functional and structural maturity of the adult human heart. However, the combination of patient derived hPSCs with microphysiological tissue engineering approaches has presented new opportunities to study heart development and disease and identify drug targets. These models often closely mimic specific aspects of the native heart tissue including intercellular crosstalk and microenvironmental cues such that maturation occurs and relevant disease phenotypes are revealed. Most recently, organ-on-chip technology based on microfluidic devices has been combined with stem cell derived organoids and microtissues to create vascularized structures that can be subjected to fluidic flow and to which immune cells can be added to mimic inflammation of tissue postinjury. Similarly, the integration of nerve cells in these models can provide insight into how the cardiac nervous system affects heart pathology, for example, after myocardial infarction. Here, we consider these models and approaches in the context of cardiovascular disease together with their applications and readouts. We reflect on perspectives for their future implementation in understanding disease mechanisms and the drug discovery pipeline.
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- 2022
4. Vascular defects associated with hereditary hemorrhagic telangiectasia revealed in patient-derived isogenic iPSCs in 3D vessels on chip
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Orlova, V.V., Nahon, D.M., Cochrane, A., Cao, X., Freund, C., Hil, F. van den, Westermann, C.J.J., Snijder, R.J., Amstel, J.K.P. van, Dijke, P. ten, Lebrin, F., Mager, H.J., and Mummery, C.L.
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Activin Receptors, Type II ,Induced Pluripotent Stem Cells ,Mutation ,Genetics ,Endoglin ,Endothelial Cells ,Humans ,Telangiectasia, Hereditary Hemorrhagic ,Cell Biology ,Biochemistry ,Developmental Biology - Abstract
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENG(c.)(1678C>)(T)) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1(c.)(1678C)(>T)-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays.
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- 2021
5. Introductions to the Community: Early-Career Researchers in the Time of COVID-19
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Ganesh, K., Patel, J., Orlova, V.V., Gifford, C., Elias, S., and Vaughan, A.
- Abstract
COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.
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- 2021
6. Prospects for production of smokeless fuel briquettes from coal from open pit mines in Kazakhstan
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Kim, S.V., primary, Bogoyavlenskaya, O.A., additional, Kudarinov, S.Kh., additional, Orlov, A.S., additional, and Orlova, V.V., additional
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- 2020
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7. Generation of 3 human induced pluripotent stem cell lines LUMCi005-A, B and C from a Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type patient
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Daoutsali, E. (Elena), Buijsen, R.A.M. (Ronald), Pas, S. (Simone) van de, Jong, A. (Anke 't), Mikkers, H. (H.), Brands, T. (Tom), Eussen, H.J.F.M.M. (Bert), Klein, A. (Annelies) de, van der Graaf, L.M. (Linda M.), Pepers, B.A. (Barry), Freund, C. (Christian), Terwindt, G.M. (Gisela), Orlova, V.V. (Valeria V.), Roon-Mom, W.M.C. (Willeke) van, Daoutsali, E. (Elena), Buijsen, R.A.M. (Ronald), Pas, S. (Simone) van de, Jong, A. (Anke 't), Mikkers, H. (H.), Brands, T. (Tom), Eussen, H.J.F.M.M. (Bert), Klein, A. (Annelies) de, van der Graaf, L.M. (Linda M.), Pepers, B.A. (Barry), Freund, C. (Christian), Terwindt, G.M. (Gisela), Orlova, V.V. (Valeria V.), and Roon-Mom, W.M.C. (Willeke) van
- Abstract
Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.
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- 2019
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8. Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells
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Halaidych, O.V., Freund, C., Hil, F. van den, Salvatori, D.C.F., Riminucci, M., Mummery, C.L., and Orlova, V.V.
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- 2018
9. Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo
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Sala, L., primary, van Meer, B.J., additional, Tertoolen, L.G.J., additional, Bakkers, J., additional, Bellin, M., additional, Davis, R.P., additional, Denning, C., additional, Dieben, M.A.E., additional, Eschenhagen, T., additional, Giacomelli, E., additional, Grandela, C., additional, Hansen, A., additional, Holman, E.R., additional, Jongbloed, M.R. M., additional, Kamel, S.M., additional, Koopman, C.D., additional, Lachaud, Q., additional, Mannhardt, I., additional, Mol, M.P.H., additional, Orlova, V.V., additional, Passier, R., additional, Ribeiro, M.C., additional, Saleem, U., additional, Smith, G.L., additional, Mummery, C.L., additional, and Burton, F.L., additional
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- 2017
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10. Endoglin haploinsufficency in endothelial cells from Rendu-Osler-Weber patient derived induced pluripotent stem cells leads to upregulation of MTUS1 gene expression
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Mummery, C.L., Gkatzis, K., Freund, C., Lebrin, F., Dijke, P. ten, Passier, R., Westermann, C.J.J., Snijder, R., Mager, H.J., and Orlova, V.V.
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- 2015
11. Differentiation and functionality of endothelial cells and pericytes from human induced pluripotent stem cells
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Orlova, V.V., Freund, C., Petrus-Reurer, S., Hil, F.E. van den, Drabsch, Y., Dijke, P. ten, and Mummery, C.L.
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- 2014
12. Controlling angiogenesis by two unique TGF-β type I receptor signaling pathways
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Orlova, V.V., Liu, Z., Goumans, M.J., and Dijke, P. ten
- Abstract
Genetic studies in mice and humans have revealed a pivotal function for transforming growth factor-beta (TGF-β) in vascular development and maintenance of vascular homeostasis. Mice deficient for various TGF-β signaling components develop an embryonic lethality due to vascular defects. In patients, mutations in TGF-β receptors have been linked to vascular dysplasia like Hereditary Hemorrhagic Telangiectasia (HHT) and pulmonary arterial hypertension (PAH). Besides indirect effects by regulating the expression of angiogenic regulators, TGF-β also has potent direct effects on endothelial cell growth and migration, and we have proposed that TGF-β regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways, activin receptor-like kinase (ALK)1 and ALK5. TGF-β is also critical for the differentiation of mural precursors into pericytes and smooth muscle cells. Furthermore, defective paracrine TGF-β signaling between endothelial and neighboring mural cells may be responsible for a leaky vessel phenotype that is characteristic of HHT. In this review, we discuss our current understanding of the TGF-β signaling pathway and its regulation of endothelial and vascular smooth muscle cell function.
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- 2011
13. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo
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Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., Burton, F.L., Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., and Burton, F.L.
- Abstract
Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of contractility from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models
- Full Text
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14. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo
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Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., Burton, F.L., Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., and Burton, F.L.
- Abstract
Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of contractility from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models
- Full Text
- View/download PDF
15. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo
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Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., Burton, F.L., Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., and Burton, F.L.
- Abstract
Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of contractility from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models
- Full Text
- View/download PDF
16. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo
- Author
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Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., Burton, F.L., Sala, L., van Meer, B.J., Tertoolen, L.G.J., Bakkers, J., Bellin, M., Davis, R.P., Denning, Chris, Dieben, M.A.E., Eschenhagen, T., Giacomelli, E., Grandela, C., Hansen, A., Holman, E.R., Jongbloed, M.R.M., Kamel, S.M., Koopman, C.D., Lachaud, Q., Mannhardt, I., Mol, M.P.H., Mosqueira, D., Orlova, V.V., Passier, R., Ribeiro, M.C., Saleem, U., Smith, G.L., Mummery, C.L., and Burton, F.L.
- Abstract
Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of contractility from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models
- Full Text
- View/download PDF
17. Unravelling cell fate decisions through single cell methods and mathematical models
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Mircea, M., Garlaschelli, D., Schmidt, T., Semrau, S., Mahfouz, A., Orlova, V.V., Aarts, J., Merks, R.M.H., Noort, S.J.T. van, and Leiden University
- Subjects
Computational biology ,In vitro ,Stem cells ,Single-cell RNA-seq - Abstract
Despite being the object of intense study, embryonic development has been difficult to model due to a number of reasons. First, complex tissues can be comprised of many cell types, of which we probably only know a subset. Therefore, we first focused on the discovery of cell types by single-cell RNA-sequencing (scRNA-seq). Cell types are routinely identified by clustering scRNA-seq data, however, there was no principled way to determine the right number of clusters. To improve cell type classification, we developed phiclust, a clusterability measure for scRNA-seq. Another challenge in a developing tissue is that many signaling processes and morphogenic events occur simultaneously, which makes it hard to isolate the individual contributions. For this purpose, I looked at stem cell derived in vitro systems, in which a small number of specific cell types can be combined deliberately and studied in isolation. My analysis of different model systems shows that cellular communication causes structural and transcriptional changes in the developing cells. Finally, while tissue organization has been characterized extensively, we lack generative models that can relate specific patterns to the underlying gene regulatory mechanisms. Therefore, I later focused on deep learning-based approaches to infer gene regulatory networks from observed spatial patterns.
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- 2022
18. Metabolic regulation of differentiation and maturation
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Es - Tiemeier, G.L. van, Rabelink, T.J., Berg, B.M. van den, Berg, C.W. van den, Zonneveld, A.J. van, Kooten, C. van, Eikenboom, J., Drukker, M.E., Orlova, V.V., Levi, M., and Leiden University
- Subjects
PGC1-alpha ,iPSC-EC differentiation ,iPSC-derived endothelial cells ,mitochondrial permeability transition pore ,Weibel-Palade bodies ,von Willebrand factor ,cyclosporine A ,glycocalyx ,shear stress ,metabolic switch - Abstract
In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging.
- Published
- 2021
19. [Untitled]
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Rabelink, T.J., Berg, B.M. van den, Berg, C.W. van den, Zonneveld, A.J. van, Kooten, C. van, Eikenboom, J., Drukker, M.E., Orlova, V.V., Levi, M., and Leiden University
- Subjects
PGC1-alpha ,iPSC-EC differentiation ,iPSC-derived endothelial cells ,mitochondrial permeability transition pore ,Weibel-Palade bodies ,von Willebrand factor ,cyclosporine A ,glycocalyx ,shear stress ,metabolic switch - Abstract
In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging.
- Published
- 2021
20. Gene regulation in embryonic development
- Author
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Berg, P.R. van den, Schmidt, T., Semrau, S., Orlova, V.V., Zon, J. van, Eliel, E.R., Noort, S.J.T. van, Drukker, M.E., and Leiden University
- Subjects
Proteomics ,DNA methylation ,Embryonic development ,Micro-RNA (miRNA) ,Single cell transcriptomics ,Gene regulation - Abstract
The human body consists of hundreds, perhaps thousands of different types of cells, each with different morphologies and functions, despite having the same genome. This diversity is created by gene regulation, a set of mechanisms that determine, which genes are used to make proteins and which genes are kept silent. During embryonic development, gene are turned on and off in a tightly orchestrated manner, to make sure that the right cell type is created at the right time and place.In this thesis we report several studies pertaining to gene regulation in embryonic development. Each of the four chapters will cover a different layer of the gene regulation toolbox: gene inactivation by DNA methylation, transcriptional regulation in the developing kidney, regulation of protein turnover and translational regulation through micro-RNAs. Together, these studies provide a refined understanding of the crucial role of gene regulation for embryonic development.
- Published
- 2021
21. Towards functional analysis of cerebrovascular cell types derived from human induced pluripotent stem cells
- Author
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Halaidych, O.V., Mummery, C.L., Orlova, V.V., Zonneveld, A.J. van, Lebrin, F.P.G., Maassen van den Brink, A., Hordijk, P.L., and Leiden University
- Subjects
junctional integrity ,impedance spectroscopy ,Ca2+ release ,hiPSC-derived smooth muscle cells ,inflammatory responses ,endothelial cell barrier function ,automated image analysis ,cell contraction ,Human Induced Pluripotent Stem Cells ,hiPSC-derived endothelial cells - Abstract
The work presented in this thesis provides novel contributions to advanced quantitative functional assessment of vascular endothelial and smooth muscle cells differentiated from hiPSCs. The automated assays described provide an unbiased and quantitative assessment of the functionality of hiPSC derivatives which are extremely needed for disease modeling and drug screening. Also, we have demonstrated the consistency of results obtained from cells differentiated from multiple healthy hiPSC lines which makes them good candidates for realistic in vitro models of human vasculature.
- Published
- 2019
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