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.
Statement of Benefit to California:
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.
Year 1The potential of stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), has been widely recognized for cell replacement therapy, modeling human diseases and serving as a platform for drug screening and validation. In this grant, we proposed to use Rett syndrome as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat embryos) and perform in vivo analyses to study the neurotransmission characteristics of normal and diseased human neurons. We initially determined that it was feasible to use the lentiviral CamKII-ChR2 construct to drive excitatory neuronal-specific expression of ChR2 in mouse hippocampal pyramidal neurons as well as human embryonic stem cell derived neurons. Importantly, we have found that both ChR2 expressing mouse hippocampal neurons and human neurons derived from embryonic stem cells can spike action potentials when stimulated in vitro, indicating that exogenously expressed ChR2 is functional. Furthermore, we successfully transplanted human embryonic stem cell derived neural stem/progenitor cells into fetal rat forebrain at embryonic day 17. Our analysis of the recipient animals at postnatal day 21 showed that approximately 40-50% of the cells survived and began to express neuronal markers, such as NeuN, indicating the neuronal differentiation, as well as the long-term survival, of transplanted human cells in the recipient animals. As originally proposed, we will proceed with the documentation of the in vivo phenotype of Rett syndrome diseased neurons. Our approach will be particularly crucial to not only validate candidate drugs or other therapeutic interventions to treat Rett syndrome using xeno-transplanted human Rett neurons, but also to study the in vivo behavior of those neurons with and without the therapeutic intervention.
Year 2Stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), carry great potentials for cell replacement therapy, human diseases modeling and drug screenings. We proposed to use Rett syndrome (RTT) as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. During the 2nd year of funding, we gained new insights into the electrophysiological characteristics of RTT neurons. Specifically, we found that the neurotransmission phenotype of neurons derived from RTT patient-specific iPSCs was highly circuitry-dependent. On the other hand, when cell-intrinsic electrophysiological properties were measured, extremely stable abnormalities in action potential profiles, resting membrane potentials, etc. were observed, indicative of the validity of the culture system. Given that currently scientists have very limited control over the features of neuronal connections formed in culture conditions, our findings make the in vivo assessment of RTT neuronal properties even more desirable, because the circuitry features are more amenable in vivo, with anatomical cues. In light of aforementioned in vitro findings, we focused our attention to both cell-intrinsic electrophysiological characteristics of RTT neurons, as well as their connectivity or neural network properties, after neurons were integrated into host circuits in vivo following xenotransplantation. Our preliminary data demonstrated that the action-potential abnormalities of RTT neurons are preserved in vivo after xenotransplantation. So far we have established a relatively optimized system for studying human iPSC-derived RTT neurons integrated into mouse brains. We are poised to uncover not only the neuronal intrinsic electrophysiological properties but also the connectivity of wild type and RTT neurons with host circuits. Moreover, we have made substantial progress with regards to a novel technology, i.e., single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Up to now, 8 RTT iPSC-derived neurons were profiled via RNA sequencing following electrophysiological recordings, and some interesting clues have already been revealed. Currently we are collecting more neurons and we expect to make unprecedented discoveries with mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. This paradigm is also generally applicable for studying other neurological disorders.