Basic Biology V
$1 152 668
The goal of this proposal is to elucidate a regulatory RNA program underlying trans-differentiation of adult human fibroblasts to functional neurons. We recently discovered that down-regulation of a critical RNA binding protein called PTB is sufficient to trans-differentiate mouse cells to functional neurons. It is well known that human cells are harder to convert. Strikingly, we are now able to convert adult human fibroblasts into complex neurons with nearly 100% efficiency using a newly improved protocol. However, such neurons are arrested at a premature stage. This is likely due to the induction of a neuronal homologue of PTB called nPTB in premature neurons, which is then switched off in mature neurons. We hypothesize that this sequential switch defines critical phases during neural induction and maturation, which may be more stringently regulated in humans than mice. We propose to test this hypothesis by sequential removal of PTB and nPTB to characterize induced neurons and define the RNA programs differentially regulated by the RNA binding proteins. We will also attempt to use this new approach to induce nascent neurons on a rat model for Parkinson’s disease. We believe that the proposed project has the potential to reveal new regulatory principles during neuronal differentiation, define critical checkpoints for neuronal development in human cells, and provide the theoretical basis for developing new therapeutic strategies against neurodegenerative diseases.
Statement of Benefit to California:
Stem cell research promises to provide resources to dissect molecular mechanisms underlying various human diseases, including a range of neurodegenerative diseases, and to develop effective therapies against those diseases, which is the central goal of the California stem cell initiative. However, stem cell-based replacement strategy is not without problems because of the potential to induce oncogenic mutations and/or trigger immune rejection after the long conversion process in vitro. Direct conversion may overcome these problems, but it has been a bottleneck in converting adult human fibroblasts to defined cell lineages, such as neurons. Furthermore, most popular approaches in this direction rely on overexpressing lineage-specific transcription factors, which may also induce mutational side effects. Our approach is fundamentally different from those overexpression-based approaches, as we are removing key regulators that are naturally down regulated during cell lineage conversion. Thus, our approach offers a new strategy to change cell fate without using any genetic material. If successfully developed with detailed understanding of the molecular mechanism behind the approach, we will have a new way to intervene with critical diseases. The proposed research will thus have the potential to transform stem cell research, which will allow California to remain at the competitive edge for development of new biotechnologies and new disease treatment strategies.
The proposal aims to define the effects of recently discovered RNA binding protein know as PTB and a neuronal variant of this protein on RNAs that contribute to neuronal differentiation, and to elucidate the mechanism by which suppression of the gene encoding the RNA binding protein enhances reprogramming. They will test the hypothesis and check whether or not the RNA binding protein acts as a differentiation checkpoint. The proposed project has the potential to reveal new regulatory principles during neuronal differentiation, define critical checkpoints for neuronal development in human cells, and provide the theoretical basis for developing new therapeutic strategies against neurodegenerative diseases. Significance and Innovation - Significance is quite modest, because it is not at all clear if the transdifferentiated cells are in fact neurons. -While direct trans-differentiation has potentially translational interest, current iPSCs, hESCs, and SCNTs techniques actually generate fully mature neurons that are of higher quality than those that are being generated here - Combination of shRNA to knock down the RNA binding protein-encoding gene, coupled to small molecule inhibitors of several relevant factors, could produce a highly homogeneous population of cells. - The developmental and functional potential of these "neuron-like" trans-differentiated cells has yet to be rigorously demonstrated. Feasibility and Experimental Design - Preliminary data is unpersuasive. No potential difficulties or alternative approaches are described. - Differences in neurogenesis in humans vs. rodents undermine the significance of the proposed animal studies. - Hypothesis that the neuronal variant of this protein blocks maturation of neurons by imposing a differentiation checkpoint is interesting, and if proven, would greatly enhance development of induced neurons. Principal Investigator (PI) and Research Team - Well established and productive investigator, who has recruited an internationally regarded expert in neurogenesis to support this relatively new direction for the PI's lab. - Unclear what the consultant’s role or input will be given that he is not receiving salary support. Responsiveness to the RFA - All reviewers agree that the proposal is highly responsive to the RFA.