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.
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.
This project is based on developing technologies to increase the information generated per mouse in order to reduce cohort sizes and also to include refinements of existing animal procedures to minimize stress to animals involved in the study.
During this project we will:
- Develop mouse induced Pluripotent Stem cell (iPSc) lines transgenic for transcription factor activated reporters (TFAR) that express both FLuc and eGFP (iPSc-TFAR) and constitutively expressing reporters (CER) that express VLuc and mCherry (iPSc-CER) from Gba1-/- and Gba1+/+ mouse embryonic fibroblasts.
iPSc-TFAR will be developed that are responsive to NFkB (inflammation), NRF2 (reactive oxygen), HIF (hypoxia), STAT3 (potency), FOXO (metabolism) and NFAT (Ca2+ signaling) transcription factors, all of which we hypothesise are involved in neural stem cell (NSC) differentiation and Gaucher's disease (GD) pathology.
- TFAR/CER-iPSc will be differentiated to NSC in vitro using established methodologies and compare Gba1-/- and Gba1+/+.
- Intracranially inject TFAR/CER-NSC into Gba1-/- and Gba1+/+ mouse fetuses and assay for FLuc versus VLuc expression as neonates develop and disease progresses over a 14-day period.
The data generated in this study will address the following sub-hypotheses:
- Gba1-/- and Gba1+/+ NSC differentiate readily to all neural cell-types in vitro.
- Gba1-/- and Gba1+/+ NSC employ the same cellular signaling pathways during neuronal differentiation in vitro.
- Gba1+/+ NSC successfully engraft in the fetal brains of Gba1+/+ mice.
- Gba1-/- NSC successfully engraft and signal normally when engrafted into the brains of Gba1+/+ mice.
- Gba1+/+ NSC successfully engraft and signal normally when engrafted into the brains of Gba1-/- mice.
For the first time this technology will allow us to see whether normal neurons are affected by a Gba1 knockout environment or vice versa whilst concurrently giving us new information as to how the cells react at the sub-cellular level.
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
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
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
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
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
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
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
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
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
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
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
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
Buckley SM et al (2015). In vivo bioimaging with tissue specific transcription factor activated luciferase reporters. Nature Scientific Reports.doi: 10.1038/srep11842
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
Principal investigatorDr Tristan McKay
InstitutionManchester Metropolitan University
Co-InvestigatorDr Simon Waddington
Dr Rahim Ahad