Mitochondrial Stem cell research
The MMN group led by Laurence Bindoff is also part of the Bergen Stem Cell Consortium. The BSCC received strategic funding form the Regional Health Authority and is in the process of establishing new laboratories, including a GMP facility, in the hospital. It comprises groups working with different types of disease from cancer to diabetes. Our work with stem cells is done in collaboration with Gareth Sullivan, University of Oslo.
We are using stem-cells transformed from the patient’s own fibroblasts to study POLG related disease. These “induced pluripotent stem cells” (iPSC) offer a unique opportunity to model human disease in a renewable and tissue specific manner. Thus far we have successfully generated cardiomyocytes, hepatocytes and neurones from these iPS cells.
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 our own specialised laboratories located in the Department of Neurology, Haukeland University Hospital by postdoctoral fellow (Xiao Liang) and PhD student (Novin Balafkan) working on different aspects of the project. They are helped by Sepideh Mostafavi, M.Sc, who has been working with the group for the last 12 months. 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.
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/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.
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).