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.
Reporting Period:
Year 3
Mitochondria are the powerhouse of the cell, providing energy in the form of ATP and NADH for cellular activities. In the pluripotent state, mitochondrial activity is relatively limited, but mitochondrial energy production increases as a cell differentiates and typically moves from a hypoxic to normoxic environment. Initial studies showed that a small molecule probe that interfered with mitochondrial function selective killed pluripotent cells but not the differentiated lineages. With the goal of stem cell therapeutics, transplanting stem cells to patients to correct a disease could be potentially dangerous if a population of stem cells that fail to differentiate moved to an environment that could induce teratomas. The probe was tested to determine if it could be useful for characterizing the differentiation process, with the goal of making stem cell therapies safer. Mechanistic studies were also completed to understand important aspects of the mitochondrial function in stem cell pluripotency and differentiation pathways. A novel signaling pathway was identified that is required for activating mitochondrial function as the stem cells differentiate and move from a hypoxic to normoxic environment. Future studies to further these outcomes will focus on optimizing the probe for stem cell differentiation studies and identifying key steps in the signaling pathway that are required to induce mitochondrial function during differentiation.
Reporting Period:
Year 4 (NCE 6-mo)
Mitochondria are the power of the cell, providing energy in the form of ATP and NADH for cellular activities. In the pluripotent state, mitochondrial activity is relatively limited. However, as a pluripotent stem cell differentiates into a determined lineage, mitochondrial energy production increases as the cell moves from a typically hypoxic to normoxic environment. Initial studies showed that a small molecule probe that interfered with mitochondrial function selectively killed pluripotent cells but not differentiated lineages. With the ultimate goal of developing stem cell therapies, transplanting stem cells into patients to correct a disease is a potentially important application. However, transplanting stem cells can have a potential danger if the cells fail to differentiate into the specified lineage and instead move into an environment that could induce teratomas. This probe was tested to determine if it could be useful for characterizing the differentiation process with the ultimate goal of making stem cell therapies safer. Mechanistic studies were also completed to understand important aspects of mitochondrial function as the stem cell differentiates and changes environments. Future studies to further these outcomes will focus on optimizing the probe for stem cell differentiation studies and identifying key steps in mitochondrial physiology that are important in the stem cell differentiation process. Studies with these probes highlighted the importance of mitochondrial physiology in stem cell maintenance and differentiation pathways.
Grant Application Details
Application Title:
A small molecule tool for reducing the malignant potential in reprogramming human iPSCs and ESCs
Public Abstract:
This research project aims to solve a key bottleneck in the use of differentiated human embryonic stem cells and induced pluripotent stem cells for the regeneration and replacement of diseased or damaged tissues. This bottleneck is the potential of unintended transplants containing failed-to-differentiate stem cells developing into benign growths called teratomas, or worse, malignant teratocarcinomas. It is essential to overcome this safety concern before stem cell-derived therapies can become acceptable for human use. Stem cells and cancer cells have many common properties. Both can replenish themselves indefinitely, and can potentially grow in different parts of the body. Before they are administered to patients, stem cells must be forced in the laboratory to turn into more mature cells that are programmed to become neurons, heart cells, beta cells of the pancreas, and other differentiated cell types. The mature cells, unlike the stem cells, do not grow indefinitely, but rather can replace a specific function that is defective in disease. We have identified a specific small molecule tool that selectively kills pluripotent stem cells but does not damage differentiated lineage cells. We will investigate the mechanism of action of the tool and test the tool for specificity in a variety of pluripotent stem cells and their differentiated lineages. The end goal is to develop a technology that will minimize the potential of developing unexpected tumors from stem cell therapies.
Statement of Benefit to California:
Our proposal benefits California by adding new essential knowledge on mitochondrial mechanisms that control human pluripotent stem cell (hPSC) function to support the taxpayers' commitment to personalized cell therapies. This work builds on highly successful CIRM Seed & Basic Biology I awards. CIRM funds to date resulted in 20+ publications and training of 14 individuals including post-docs, graduate students, undergraduates, and CIRM Bridges to Stem Cell Biology program trainees, some of whom have entered the California workforce. Here we have identified a small molecule modulator of a mitochondrial redox protein that selectively kills pluripotent stem cells but not their differentiated lineages. Because contamination by hPSCs in transplanted donor cell pools is a key concern for regenerative cell therapies, there is a critical need to develop methods for reproducibly eliminating potentially cancerous cells. Our small molecule is an exciting candidate tool and will be characterized extensively. Our ongoing work underpins therapy development in California’s major academic centers and will provide data for many of California's biotechnology companies in the growing stem cell industry, whose success will propel hiring and increased economic prosperity for the state. With success, tangible health and economic impact on California, its academic institutions and companies, and the rest of the nation will be achieved as California leads the way forward with personalized medicine.
