Stem cells, including human embryonic stem cells, provide extraordinary new opportunities to model human diseases and may serve as platforms for drug screening and validation. Especially with the ever-improving effective and safe methodologies to produce genetically identical human induced pluripotent stem cells (iPSCs), increasing number of patient-specific iPSCs will be generated, which will enormously facilitate the disease modeling process. Also given the advancement in human genetics in defining human genetic mutations for various disorders, it is becoming possible that one can quickly start with discovery of disease-related genetic mutations to produce patient-specific iPSCs, which can then be differentiated into the right cell type to model for the disease in vitro, followed by setting up the drug screening paradigms using such disease highly relevant cells. In the context of neurological disorders, both synaptic transmission and gene expression can be combined for phenotyping and phenotypic reversal screening and in vitro functional (synaptic transmission) reversal validation. The missing gap for starting with the genetic mutation to pave the way to drug discovery and development is in vivo validation-related preclinical studies. In order to fill this gap, in this application we are proposing to use Rett syndrome as a proof of principle, to establish human cell xenografting paradigm and perform optogenetics and in vivo recording or functional MRI (fMRI), to study the neurotransmission/connectivity characteristics of normal and diseased human neurons. Our approach will be applicable to many other human neurological disease models and will allow for a combination of pharmacokinetic, and in vivo toxicology work together with the in vivo disease phenotypic reversal studies, bridging the gap between cell culture based disease modeling and drug screening to in vivo validation of drug candidates to complete the cycle of preclinical studies, paving the way to clinical trials. A success of this proposed study will have enormous implications to complete the path of using human pluripotent stem cells to build novel paradigms for a complete drug development process.
Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by primarily loss-of-function mutations in the X-linked MeCP2 gene. It mainly affects females with an incidence of about 1 in 10,000 births. After up to 18 months of apparently normal development, children with RTT develop severe neurological symptoms including motor defects, mental retardation, autistic traits, seizures and anxiety. RTT is one of the Autism Spectrum Disorders (ASDs) that affects many children in California. In this application, we propose to use our hESC-based Rett syndrome (RTT) model as a proof-of-principle case to define a set of core transcriptome that can be used for drug screenings. Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Unfortunately, most of the characterization of hESCs are performed in vitro when disease models are generated using hESC-derived neurons. In this application, using RTT as a proof of principle study, we will bridge the gap and perform in vivo characterization of transplanted normal and RTT human neurons. Our findings will not only benefit RTT and other ASD patients, but also subsequently enable broad applications of this approach in drug discovery using human pluripotent stem cell-based disease models to benefit the citizens of California in a broader spectrum.
This basic biology application proposes to develop a new way to monitor connectivity and function of transplanted neurons in vivo. The applicants will engineer glutaminergic and gabaergic neurons from normal human embryonic stem cell (hESC) and an hESC model of Rett Syndrome, an autism spectrum disorder, such that they can be modulated by a light dependent switch. The cells will then be study the fate of these cells in vivo using a small animal model. The group will utilize the switch, combined with neurophysiological and imaging methods, to assess functional integration of transplanted normal and disease neurons into host cortical circuitry.
Significance and Innovation:
- Reviewers agreed that the proposed work could have a powerful impact upon both basic and translational aspects of stem cell research. If successful, this work could improve the ability to model neurological diseases in vivo with stem cells, enable validation of results from in vitro studies, and allow study of drug effects upon specific neurons. However, they cautioned that the proposed studies themselves do not address specific biological mechanisms.
- Reviewers unanimously praised the use of innovative techniques such as optical brain imaging and neurophysiology in xenotransplanted neurons.
- One reviewer questioned the selection of knockdown hESC over patient iPSC for these studies, as this choice will not allow modeling of the random X-inactivation associated with Rett Syndrome.
Feasibility and Experimental Design:
- Reviewers agreed that preliminary data supporting feasibility of the project are quite strong overall with many important tools already developed such as Rett Syndrome model hESC lines that demonstrate an appropriate in vitro phenotype, neuronal differentiation expertise and the ability to achieve switch-modulated neuronal function.
- The proposal would benefit from demonstration of successful switch activation and recording data from transplanted cells and evidence of selectivity with the selected neuronal subtype markers.
- There was some concern that expertise in the in utero assays appears limited and this element of the preliminary data is not very robust.
Principal Investigator (PI) and Research Team:
- Reviewers felt that the PI has excellent training, productivity and relevant track record of high profile publications.
- Overall, the team is strong and possesses the majority of the required expertise to perform the proposed studies. Reviewers did suggest the team would be further strengthened by the inclusion of in utero transplantation specialist.
Responsiveness to RFA:
- In general, the panel agreed that this proposal is a good fit for the RFA considering it’s use of human embryonic cells and the potential for this work to ultimately enable better understanding of human disease. However, there was a general responsiveness concern regarding the lack of mechanistic focus that lowered scores.
- A motion was made to move this application into Tier 1, Recommended for funding. The reviewers were all in agreement that although the project does not have a direct mechanistic focus this grant will develop a valuable new stem cell based disease model that could benefit the field. The motion carried.