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

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. 

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:

  1. 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.
  2. TFAR/CER-iPSc will be differentiated to NSC in vitro using established methodologies and compare Gba1-/- and Gba1+/+.
  3. 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.

Hawkins KE, Joy S, Delhove JM, Kotiadis VN, Fernandez E, Fitzpatrick LM, Whiteford JR, King PJ, Bolanos JP, Duchen MR, Waddington SN, McKay TR (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, Delhove JM, Perocheau DP, Karda R, Rahim AA, Howe SJ, Ward NJ, Birrell MA, Belvisi MG, Arbuthnot P, Johnson MR, Waddington SN, McKay TR (2015). In vivo bioimaging with tissue specific transcription factor activated luciferase reporters. Nature Scientific Reports.doi: 10.1038/srep11842 

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