Non-invasive real-time bioluminescence imaging in living mice to interrogate transcription factor activity and fate of engrafted stem cells
Lead Research Organisation:
Manchester Metropolitan University
Department Name: School of Healthcare Science
Abstract
Lay Summary
Understanding the genetic basis of disease involves ascribing gene mutations directly to a deleterious effect. Technological advances in whole genome sequencing now provide us with a plethora of information linking genetics to disease. An emerging bottleneck in translating that information to curing disease is a more complete understanding of how cells affected by disease interact in the body. Broadly, genetic disease can be caused at the level of the cell by gene mutation(s) preventing a certain cell-type from doing its job correctly. In the body, cells interact in 3 dimensions in a dynamic way within tissues and organs but also potentially send signals throughout the body. For example, neurons in the brain pass signals through complex local circuits that are initiated remotely and have consequences remotely. This circuitry can be damaged by neurological disease that can result in wide-ranging affects such as in Huntington's disease. However, the brain is not made up wholly of neurons and many neurological diseases are caused by dysfunction in other cells such as microglia, astrocytes and oligodendrocytes. This is exemplified by the now clear understanding that Multiple Sclerosis (MS) is caused by defects in oligodendrocyte function. Great strides have been made recently in the understanding of the cellular events that underlie MS resulting in novel drugs being trialed to treat the disease.
We have developed new technologies and techniques to unravel the way that cells communicate with each other in living animals as disease develops. This study is focused on investigating type 2 Gaucher's disease (GD) that severely affects the brain but the tools and technologies involved would be broadly applicable to the study of virtually any type of cell in any tissue of the body. For the first time, we will combine the ability to generate neurons (but we can generate virtually any cell type) from mouse or human stem cells in the lab and genetically manipulate them so they emit quantifiable light when stimulated. Once these cells have been proven to respond to such stimuli we will engraft these normal reporter neurons into the brains of mice affected by GD before they are born. These experiments have been designed so as disease starts to affect the mice, the injected normal neurons will send signals by emitting light that will tell us how the cells are behaving and how they are interacting with cells affected by disease.
Scientific Abstract
We aim to combine for the first time induced pluripotent stem cell (iPSc) technologies with next generation light-emitting reporters to gain new insights into the cell:cell interactions that underlie disease progression in living animals. The use of luciferase luminescence for continued bioimaging of engrafted cells has already resulted in increased data output in substantially reduced cohorts of animals to track cell fate and distribution. This has so far been largely restricted to models of tumorigenesis and stem cell tracking. Here, we propose to use dual reporter cell lines generated from mouse iPSc to not only track cell fate but also transcription factor activity within these cells to provide new insights into how the engrafted cells interact with the local environment.
For this focused study we will intracranially inject normal dual reporter neural stem cells (NSC) into fetal Gba1 knockout mice; a model of type 2 neuronopathic Gaucher's Disease and serially image for both reporters within the 14 day window after which most GD mice are sacrificed at a humane endpoint. We will also compare the reverse i.e. Gba1 knockout NSC into normal mice to determine if it is primarily the neural cells or resident activated microglia that are primarily responsible for neural degeneration. The technology being developed is not brain specific but is broadly applicable to many cell-types in many disease settings.
Understanding the genetic basis of disease involves ascribing gene mutations directly to a deleterious effect. Technological advances in whole genome sequencing now provide us with a plethora of information linking genetics to disease. An emerging bottleneck in translating that information to curing disease is a more complete understanding of how cells affected by disease interact in the body. Broadly, genetic disease can be caused at the level of the cell by gene mutation(s) preventing a certain cell-type from doing its job correctly. In the body, cells interact in 3 dimensions in a dynamic way within tissues and organs but also potentially send signals throughout the body. For example, neurons in the brain pass signals through complex local circuits that are initiated remotely and have consequences remotely. This circuitry can be damaged by neurological disease that can result in wide-ranging affects such as in Huntington's disease. However, the brain is not made up wholly of neurons and many neurological diseases are caused by dysfunction in other cells such as microglia, astrocytes and oligodendrocytes. This is exemplified by the now clear understanding that Multiple Sclerosis (MS) is caused by defects in oligodendrocyte function. Great strides have been made recently in the understanding of the cellular events that underlie MS resulting in novel drugs being trialed to treat the disease.
We have developed new technologies and techniques to unravel the way that cells communicate with each other in living animals as disease develops. This study is focused on investigating type 2 Gaucher's disease (GD) that severely affects the brain but the tools and technologies involved would be broadly applicable to the study of virtually any type of cell in any tissue of the body. For the first time, we will combine the ability to generate neurons (but we can generate virtually any cell type) from mouse or human stem cells in the lab and genetically manipulate them so they emit quantifiable light when stimulated. Once these cells have been proven to respond to such stimuli we will engraft these normal reporter neurons into the brains of mice affected by GD before they are born. These experiments have been designed so as disease starts to affect the mice, the injected normal neurons will send signals by emitting light that will tell us how the cells are behaving and how they are interacting with cells affected by disease.
Scientific Abstract
We aim to combine for the first time induced pluripotent stem cell (iPSc) technologies with next generation light-emitting reporters to gain new insights into the cell:cell interactions that underlie disease progression in living animals. The use of luciferase luminescence for continued bioimaging of engrafted cells has already resulted in increased data output in substantially reduced cohorts of animals to track cell fate and distribution. This has so far been largely restricted to models of tumorigenesis and stem cell tracking. Here, we propose to use dual reporter cell lines generated from mouse iPSc to not only track cell fate but also transcription factor activity within these cells to provide new insights into how the engrafted cells interact with the local environment.
For this focused study we will intracranially inject normal dual reporter neural stem cells (NSC) into fetal Gba1 knockout mice; a model of type 2 neuronopathic Gaucher's Disease and serially image for both reporters within the 14 day window after which most GD mice are sacrificed at a humane endpoint. We will also compare the reverse i.e. Gba1 knockout NSC into normal mice to determine if it is primarily the neural cells or resident activated microglia that are primarily responsible for neural degeneration. The technology being developed is not brain specific but is broadly applicable to many cell-types in many disease settings.
Technical Summary
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.
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.
Planned Impact
We propose to develop substantial advances using the latest technology in the field of in vivo bioimaging. Luciferase bioimaging in rodents has already substantially reduced the number of animals required for assessing continued gene expression or tracking engrafted cells. Furthermore, the advent of light-emitting transgenics has enabled the continued monitoring of gene activation in response to disease or drug induction. Our ethos is anchored by a commitment to developing innovations to increase data output whilst minimizing the number of animals used in our and others experiments. Our previous work has been focused on developing tissue specific, somatic, light-emitting transgenics for modeling disease. This is achieved by administering tissue-targeted lentiviruses expressing a transcription factor activated reporter (TFAR), in this case firefly luciferase (FLuc) to rodents during the fetal/neonatal period which retains fidelity throughout the lifetime of the animal. This technology was developed to further reduce the numbers of animals currently used to generate and maintain light-emitting transgenic mouse colonies.
In parallel experiments we have developed reliable and reproducible protocols for reprogramming mouse and human fibroblasts to induced Pluripotent Stem cells (iPSc) as in vitro disease models. We are able to differentiate iPSc to many diverse cell types including neural, hepatic and cardiac lineages. This provides a powerful tool in interrogating the underlying mechanisms of development and disease.
Here we are proposing to expand on our existing expertise in terms of quantifying dual luciferase produced light emission from fetally engrafted stem cells as opposed to endogenous tissues. The challenges are overlapping but distinct. We will combine a constitutively expressing reporter (CER), vargula luciferase (VLuc), with our recently developed TFAR library to effectively double the information we can obtain from continued bioimaging of a single animal.
In this instance we will exemplify the efficacy of this platform technology by tracking the engraftment, cell fate and intra-cellular signaling of iPSc-derived neural stem cells (NSC) in a mouse model of neuronopathic Gaucher's disease (GD). Our initial analyses of the differentiation potential of CER-TFAR-iPSc will all be carried out in vitro to minimize the risk of failure in vivo. It is difficult to directly compare what animal numbers this could reduce as failed in vivo experiments are rarely detailed in the literature.
Our team has previous success in intracranially engrafting stem cells into fetal mice (Kennea et al. 2009). In that study a minimum of 6 mice were sacrificed at 6 time points and compared to controls totaling a minimum of 42 mice per experiment. A comparable analysis using our continued bioimaging would involve 6 mice, a 7-fold reduction. However, we will not only be tracking engrafted NSC; we will be gaining valuable information of their cell signaling as GD progresses, thus the data we generate is substantially more informative. During the development of this model we have made further refinements in our methodologies that now allow us to image pups without anesthesia thus minimizing stress during experiments.
Fetal stem cell therapy is unlikely to become clinically translatable and we must clarify this is a model for studying disease as well as learning more about stem cell engraftment. This neurological model is designed to exemplify the technology which is broadly applicable to most cell-types in many organs. It would be feasible to quantify engraftment, differentiation and signaling of cells in principle regenerative models where it is critical to learn more about not only cell fate but how those cells signal. Examples could include cardiac injection of mesenchymal stem cells (already in clinical trials but the therapeutic mechanism is unclear) or intrahepatic injection of hepatic stellate cells in a fibrotic liver model.
In parallel experiments we have developed reliable and reproducible protocols for reprogramming mouse and human fibroblasts to induced Pluripotent Stem cells (iPSc) as in vitro disease models. We are able to differentiate iPSc to many diverse cell types including neural, hepatic and cardiac lineages. This provides a powerful tool in interrogating the underlying mechanisms of development and disease.
Here we are proposing to expand on our existing expertise in terms of quantifying dual luciferase produced light emission from fetally engrafted stem cells as opposed to endogenous tissues. The challenges are overlapping but distinct. We will combine a constitutively expressing reporter (CER), vargula luciferase (VLuc), with our recently developed TFAR library to effectively double the information we can obtain from continued bioimaging of a single animal.
In this instance we will exemplify the efficacy of this platform technology by tracking the engraftment, cell fate and intra-cellular signaling of iPSc-derived neural stem cells (NSC) in a mouse model of neuronopathic Gaucher's disease (GD). Our initial analyses of the differentiation potential of CER-TFAR-iPSc will all be carried out in vitro to minimize the risk of failure in vivo. It is difficult to directly compare what animal numbers this could reduce as failed in vivo experiments are rarely detailed in the literature.
Our team has previous success in intracranially engrafting stem cells into fetal mice (Kennea et al. 2009). In that study a minimum of 6 mice were sacrificed at 6 time points and compared to controls totaling a minimum of 42 mice per experiment. A comparable analysis using our continued bioimaging would involve 6 mice, a 7-fold reduction. However, we will not only be tracking engrafted NSC; we will be gaining valuable information of their cell signaling as GD progresses, thus the data we generate is substantially more informative. During the development of this model we have made further refinements in our methodologies that now allow us to image pups without anesthesia thus minimizing stress during experiments.
Fetal stem cell therapy is unlikely to become clinically translatable and we must clarify this is a model for studying disease as well as learning more about stem cell engraftment. This neurological model is designed to exemplify the technology which is broadly applicable to most cell-types in many organs. It would be feasible to quantify engraftment, differentiation and signaling of cells in principle regenerative models where it is critical to learn more about not only cell fate but how those cells signal. Examples could include cardiac injection of mesenchymal stem cells (already in clinical trials but the therapeutic mechanism is unclear) or intrahepatic injection of hepatic stellate cells in a fibrotic liver model.