NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research
Strategic grant

Non-invasive real-time bioluminescence imaging in living mice to interrogate transcription factor activity and fate of engrafted stem cells

A stock image of round glass dishes containing blue and green liquid arranged closely together.

At a glance

Completed
Award date
October 2014 - September 2016
Grant amount
£165,884
Principal investigator
Dr Tristan McKay

Co-investigator(s)

Institute
Manchester Metropolitan University

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Contents

Overview

Aims

The aim of this project is to develop and demonstrate that a novel dual luciferase reporter technology can be employed to minimise the number of animals used in research on stem cell engraftment and differentiation.

Background

Tracking stem cell fate in vivo has become a focus in multiple fields, including regenerative medicine where stem cells can be employed to repopulate or repair damaged tissues. Current approaches to study cell fate in vivo often require large cohorts of animals to be killed at multiple time points followed by histological and molecular analysis of excised tissue. A number of established imaging modalities exist for tracking engrafted cells, but these are limited (i) to short term studies because of the rapid degradation of the reporter probes available, (ii) by the short depth of effective light emission, and (iii) because they fail to quantify the activity of the cells.

This project will overcome these challenges by developing new reporter probes capable of tracking stem cells and their daughter cells and quantifying their activity over long term studies. The utility of the system will be demonstrated using neural stem cells in a mouse model of type 2 Gaucher’s Disease, an infantile genetic disease in which lipids accumulate in cells and certain organs. This approach will increase the amount of data generated in smaller cohorts of animals. During the course of this project a total of 72 mice will be used compared with 520 mice using a more traditional serial sacrifice model.

Research details and methods

For the first time induced pluripotent stem (iPS) cell technologies will be combined with next generation light-emitting reporters to gain new insights into the cell:cell interactions that underlie disease progression in living animals. Using dual reporter cell lines generated from mouse iPS cells will enable not only cell fate to be tracked, but also transcription factor activity within these cells to provide new insights into how the engrafted cells interact with the local environment. The dual reporter iPS cell lines will be generated from embryonic fibroblasts harvested from Gba1 knockout mice; a model of type 2 neuropathic Gaucher's Disease. iPS cells will be differentiated to neurons and the neuronal stem cells will be utilised in both an in vitro and in vivo model of Gaucher’s Disease for comparison. 

Publications

  1. Karda R et al. (2020). Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors. Scientific Reports 10: e2121. doi: 10.1038/s41598-020-59075-3
  2. FitzPatrick LM et al. (2018). NF-κB Activity Initiates Human ESC-Derived Neural Progenitor Cell Differentiation by Inducing a Metabolic Maturation Program. Stem Cell Reports 10(6):1766-1781. doi: 10.1016/j.stemcr.2018.03.015
  3. Delhove JMKM et al. (2017). Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo. In: Gould D. (eds) Mammalian Synthetic Promoters. Methods in Molecular Biology, vol 1651. Humana, New York, NY doi: 10.1007/978-1-4939-7223-4_5
  4. Delhove JMKM et al. (2017). Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice. Scientific Reports 7:41874. doi: 10.1038/srep41874
  5. Karda R et al. (2017). Continual conscious bioluminescent imaging in freely moving somatotransgenic mice. Scientific Reports 7(1):6374. doi: 10.1038/s41598-017-06696-w
  6. Mattar CNZ et al. (2017). In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model. Molecular Therapy 25(8):1843-1852. doi: 10.1016/j.ymthe.2017.04.003
  7. Miller DC et al. (2017). Ajmaline blocks I and I without eliciting differences between Brugada syndrome patient and control human pluripotent stem cell-derived cardiac clusters. Stem Cell Research 25:233-244. doi: 10.1016/j.scr.2017.11.003
  8. Munye MM et al. (2017). BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity. American journal of physiology. Lung cellular and molecular physiology 312(2):L258-L267. doi: 10.1152/ajplung.00471.2016
  9. Poliandri A et al. (2017). Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells. Molecular and cellular endocrinology 447(12-22). doi: 10.1016/j.mce.2017.02.030
  10. Teasdale JE et al. (2017). Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells. Scientific Reports 7:39945. doi: 10.1038/srep39945
  11. Vink CA et al. (2017). Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy. Molecular Therapy 25(8):1790-1804. doi: 10.1016/j.ymthe.2017.04.028
  12. Hawkins KE et al. (2016). NRF2 orchestrates the metabolic shift during induced pluripotent stem cell reprogramming. Cell Rep. 14(8):1883-91. doi: 10.1016/j.celrep.2016.02.003 
  13. Buckley SM et al (2015). In vivo bioimaging with tissue specific transcription factor activated luciferase reporters. Nature Scientific Reports.doi: 10.1038/srep11842 
  14. Nivsarkar MS et al. (2015). Evidence for contribution of CD4+ CD25+ regulatory T cells in maintaining immune tolerance to human factor IX following perinatal adenovirus vector delivery. Journal of Imunology Research 397879. doi: 10.1155/2015/397879