Research Projects on Mitochondrial Stem Cell Group
We have established the required competence and facilities for human iPSCs reprogramming and differentiation, enabling the investigation of neuronal cells from patients and healthy controls. Validated iPSCs have been differentiated to neural stem cells (NSCs) and regionalized neuronal subtypes, as well as astrocytes/ glial populations. We have also established a 3D spheroid system using iPSC-derived neurons, astrocytes, and oligodendrocytes, and successfully generated cortical region-specific brain organoids from human iPSCs (see the figure below for our in vitro iPSC-based systems).
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Project 1:Modelling diseases
By using iPSCs as a tool, we provided the first proof-of-principle for the usefulness of iPSC-derived cell models in the study of mitochondrial diseases due to defects of mtDNA homeostasis. Comparing patient fibroblasts, their undifferentiated iPSC and both neural stem cells (NSCs) and dopaminergic neurons and astrocytes derived from NSCs, we found that NSCs, neurons and manifested a cellular phenotype that mimicked precisely the changes found in postmortem neuron with mtDNA depletion, loss of complex I. In addition, we were able to show that the cellular phenotype included increased neuronal ROS and cellular senescence, and disturbed NAD+ metabolism. These findings have been published in EMBO Molecular Medicine and Experimental Neurology.
Our Ph.D. student Sepideh Mostafavi has differentiated iPSC to cardiomyocytes and investigated the levels of mitochondrial DNA as they mature. This is vital since we know that patients with POLG disease lose mtDNA in their cells. Interestingly, this “depletion” of mtDNA occurs mostly in neurons and liver cells while skeletal muscle shows other mtDNA defects. Our collaborator Dr. Sullivan in UiB has differentiated iPSCs into liver cells giving us a range of tissues to monitor tissue specificity in POLG disease.
To investigate mitochondrial function in living cells, we have established a flow-cytometry assay to monitor mitochondrial volume, membrane potential, ROS production, and other parameters.
Project 2: Developing high-throughput iPSC-based screening platform
We aim to develop a pharmacological screening platform using iPSC-derived NSCs. In this way, we do not have to expose the patient to any compounds that we have not already tested and found to be helpful. Further, using the technology (CRISPR/Cas9) that allows us to correct the genetic defect in living cells, the iPSC that are the patient’s own cells, will have the disease-causing corrected and this will open the way for potential treatment using stem cells differentiated to whichever tissue is required. Ph.D. student Cecilie Katrin Kristiansen is working on this project.
Project 3: Generating 3D human brain organoid system
Organoids generated from human pluripotent stem cells are a new and promising development that can potentially provide a rapid and cost-effective method for drug discovery. We aim to develop a 3D brain organoid system to study POLG related disorders and establish a high-throughput screening platform for both compounds. We have shown that it is possible to generate a 3D brain organoid from POLG patient iPSCs. We will then explore multidimensional phenotyping of brain organoids generated from POLG iPSCs by using single-cell transcriptomics methods and establish a high-throughput 3D brain organoid drug screening platform. Postdoc. Yu Hong is working on this project.
Project 4: Cardiomyocytes generated from iPSCs
While cardiomyocytes do demonstrate mtDNA abnormalities and biochemical abnormalities, cardiac involvement in POLG disease is very uncommon. We have generated cardiomyocytes from controls and POLG patient fibroblasts and are investigating how these cells survive despite have mutations that disturb POLG function and therefore mtDNA homeostasis. This work was started by Novin Balafkan and after he finished his Ph.D. carried on by Sepideh Mostafavi. These findings have been published in Scientific Report.