The Koehler and Teitell labs are working hard on an interesting observation that could be important for bringing stem cell therapies to the clinic. The Koehler lab identified a small molecule called MitoBloCK-6 (MB6) that inhibits the function of a protein called ALR (also called Erv1) that resides within the mitochondria or all mammalian cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). ALR/Erv1 has been shown to be essential for the survival of mouse embryonic stem cells (mESCs) and in a study published in the journal Developmental Cell (Dabir, et al., Dev Cell 25:81-92, 2013), the Koehler and Teitell labs showed that exposure of hESCs to MB6 caused them to die, but differentiated progeny cells from hESCs exposed to MB6 were unaffected and functioned normally. This background work led to our current CIRM award, which is focused on determining whether MB6 can efficiently kill both hESCs and hiPSCs but leave unharmed progeny cells that are being differentiated along the main lineages that make all of the cells in our bodies called endoderm (e.g. blood vessels), mesoderm (e.g. muscles), and ectoderm (e.g. skin, brain). If this proves to be the case, then we suggest that MB6 could have clinical utility, by eliminating undifferentiated cells in tissue culture protocols that aim to produce replacement cells for cell therapy usage. By eliminating those cells that ‘fail to differentiate’, MB6 will make cell therapies safer for patients by reducing or, hopefully, eliminating the possibility of transferring cells that could cause benign and sometimes malignant tumors in recipient patients.
To evaluate MB6 for potential clinical use, we have two lines of studies. In the first area (Aim 1 of the parent grant), we have begun determining the MB6 killing spectrum. We have efficiently killed multiple human pluripotent stem cell (HPSC) lines including H9 and HSF1 hESCs and UCLA1 hiPSCs with MB6 in culture. We have successfully generated endoderm, mesoderm, and ectoderm cells from these hPSC lines with varying efficiencies. We have begun testing the effects of MB6 killing on H9 hESCs induced to become mesoderm and determined that these cells are resistant to MB6-induced cell death. We are continuing this Aim 1 work with additional lines differentiated into all 3 lineages to obtain a more complete picture of the range of MB6 killing activities and generality of the process.
In the second area (Aim 2) of studies, we are working toward defining the mechanism(s) by which MB6 preferentially kills hPSCs. We have generated two hPSC lines, H9 and UCLA1, that overexpress a tagged version of ALR/Erv1 in an attempt to increase the level of ALR/Erv1 present in mitochondria to ask whether an increased level of the protein is cell protective from MB6. Preliminary results suggest that there is less cell death from added MB6 from increased ALR/Erv1 expression, which is good because it suggests that the MB6 effect is on-target for its known interacting protein and not an off-target drug effect that can often occur. We are in the process of confirming tagged ALR/Erv1 expression in the mitochondria, quantifying the steady state level of reactive oxygen species (a by product of mitochondrial respiration affected by ALR/Erv1 function and a trigger for inducing cell death), and pull-down of ALR/Erv1 to identify bound proteins in the MB6 sensitive state (pluripotency) versus the MB6 resistant state (differentiated). Thus far, our studies remain highly promising and on track with our original hypotheses, aims, and goals.