Mitochondria are key players in cellular physiology, not only are they the major producers of cellular energy (ATP), but they also play a role in many vital processes that affect the cell: these include processes such as apoptosis, cancer development, neurodegeneration and even ageing. Mitochondrial energy failure produces a wide range of devastating diseases that affect both adults and children: these diseases can affect any tissue, since all tissues rely on energy they produce, and they are among the commonest inborn errors of metabolism known. Indeed, evidence suggests that even the current prevalence figure of 1:3500 underestimates the problem. Mitochondrial disease phenotypes vary dramatically, even when caused by the same genetic defect; e.g. in diseases caused by mutations in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase (POLG), some patients show involvement of the brain and liver while others show only skeletal muscle disease. We have been studying diseases caused by mutations in this protein and find that it is the commonest cause of recessively inherited ataxia (unsteadiness) in Norway with two founder mutations each having a prevalence of 1:100.
We are studying POLG disease using stem-cell like cells transformed from the patient’s own fibroblasts. These “induced pluripotent stem cells” (iPSC) offer a unique opportunity to model human disease in a renewable and tissue specific manner. We also plan to use iPS cells to perform large scale screening of potential therapeutic agents. In this way, we do to expose the patient to any compounds that we have not already tested and found to be helpful. Further, using new technology (CRISPR-cas) 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.
Figure 1. The journey from a fibroblast to a functional cardiomyocytes
Reprograming starts with exposing primary cultured fibroblasts (A) to Yamanaka factor’s (Oct4, Sox2, Kalf4, c-Myc) for 48 hours. The cultured fibroblasts will go through dramatic changes and from week 2 begin to form colonies of “induced pluripotent cells” ( iPSC)s. These colonies are transferred to new dishes in order to expand and develop a mature iPSC clone (b). High level expression of pluripotency markers indicate the quality of iPSC colonies: Oct4 is a known marker of pluripotency (green) and cells are clearly expressing this protein (C). IPSC’s have the potential to form all three germ layers (endoderm, mesoderm, ectoderm). In the experiment shown here, we have differentiated iPSC into cardiomyocytes. This takes ~2 weeks and it starts by forming mesoderm progenitors on day 1 of differentiation (D) and developing into cardiac mesoderm after exposing to different growth factors (E) and the final results are functional, beating cardiomyocytes (F).
The work on iPSC is performed in specialised laboratories located in the Department of Neurology, Haukeland University Hospital. Currently, there are a postdoctoral fellow (Xiao Liang) and one PhD student (Novin Balafkan) working on different aspects of the project: Novin Balafkan is differentiating cells to cardiomyocytes and hepatocytes (Figure 1) and looking at the levels of mitochondrial DNA as they mature. This is vital since we know that patients with POLG disease loose mtDNA in their cells. Interestingly, this is mostly in neurons and liver cells while skeletal muscle shows other mtDNA defects. Xiao Liang is preparing to differentiate iPSC into neurons and at the same time establishing methods to enable us to screen cells quickly so that we can begin screening compounds as potential therapies (Figure 2, 3). To do this we are investigating the use of flow-cytometry to see if we can develop methods to screen for mitochondria membrane potential.
Figure 2. Immunofluorescence staining with OCT4 in human iPSC lines.
Oct4 is one of the factors that we use to stimulate cells to become stem cell-like. Continued expression of Oct4 defines the cells as maintaining the undifferentiated state. Here we see that both the patient and control iPSC lines are expressing Oct4.
Collaboration is established with Gareth Sullivan from the Stem Cell Centre in Oslo. Gareth heads the work on iPSC in Oslo and is a major collaborator for this project. Funding is provided by NFR (to X. Liang) and UiB (N. Balafkan).
Figure 3. Images of mitochondrial membrane potential using TMRE staining in fibroblasts and human iPSC lines.
Upper panel: negative control was measured by exposure to 100 µM FCCP for 10 min, when the dye is released from mitochondria after depolarization (“fluorescence unquenching”) prior to incubation of TMRE. Lower panel: After the cells were incubated with 200 nM TMRE for 20 min and fluorescence pictures were acquired using digital fluorescence microscopy. Mag x20