Funding opportunities

Molecular and Cellular Transitions from ES Cells to Mature Functioning Human Neurons

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00115
Principle Investigator: 
Funds requested: 
$2 879 210
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
SYNOPSIS OF PROPOSAL: This proposal will look at three important “transition points” during the differentiation of hES (human embryonic stem cells) into human neural stem cells (hNS), and then from hNS to committed lineage restricted neuronal progenitors (hNP). The final transition point is from hNP to mature functioning hN of specific neuronal lineages, and the studies proposed here will focus on dopaminergic (hNda) and cholinergic (hNchat) neurons that are clinically significant for Parkinson’s Disease and ALS. Mechanisms associated with cellular changes at these transition points will be studied at the protein and small non-coding RNAS level. IMPACT AND SIGNIFICANCE: This proposal has impact and significance to the field in several areas. There is a need to develop better cell culture models of human diseases such as PD and ALS, and there is also a need to generate large numbers of transplantable cells that possess the appropriate phenotype (e.g. DA and ChAT); ES cells are appreciated as offering these outcomes become of their extensive proliferative potential and their “blank slate” making them suitable for controlled differentiation and specific fate choice-inducing protocols. The choice of looking at protein expression and small non-coding RNAS at specific checkpoints of commitment to ultimately DA and ChAT precursor cells is a good one. Once the specific molecular mechanisms and pathways are gleaned to direct these cells to these phenotypes, they can then be used not only for transplantation in animal models of disease, but they can be used for high throughput screening of drugs and factors that underlie or reverse disease processes. In doing this, the cells can be studied for how mutant genes wreak havoc in these and other types of CNS cells. This would be an extremely significant advance to the field of restorative neurology. The purpose of this project is to unravel the molecular basis underlying the generation of neuronal diversity using human embryonic stem cells. Just as any differentiated cells, the formation of the subtype of neurons involves a hierarchy of decisions that allow embryonic cells to transit from one to the other. The PI proposes the effort on 2 types of neuronal lineages: dopaminergic neurons from the midbrain and cholinergic motor neurons from the spinal cord. The final objective is to produce homogeneous populations of these two types of neurons for in vivo studies in mouse with chimeric brains. One area of focus is the generation of homogeneous hESC-derived cell populations to examine the developmental transition points from hESCs to specialized neurons. Another is to define the molecular signatures of developmental transition points from hESC to functional hN. The PI also proposes to investigate the differential molecular profile in cellular models for PD and ALS. Finally, there is intent to execute in vivo modeling for PD and ALS. The basic understanding of neuronal subtype specification presents one of the highest priorities in hESC work, and it is very clear that the basic molecular knowledge of these early events will have a tremendous impact on our understanding of the normal state of differentiation, as well as the diseased state. In addition, the success of this project will establish powerful models to understand human disease, such as Parkinsons and ALS. These models are going to be different than the ones currently available, as they represent mouse with human neurons in vivo providing the platform for direct study of the state of the disease in human cells. This work proposes a very general and broad approach to study neural development and neuronal differentiation in hESCs, and to develop hES derived hNS models relevant to disease. The use of a number of innovative techniques is proposed including high throughput sequencing of sm non-coding RNAs and exon arrays (both commercially available). QUALITY OF THE RESEARCH PLAN: Different ES and more committed precursor cell types (all five of them) will be generated, and there is a focus on (hESC, hNS, hNP,hNda, and hNchat). Specifically, the applicant proposes to use high-density Affymetrix exon arrays for gene identification and Deep Sequencing for characterization of small non-coding RNAs. Validation of molecular targets will be performed in tissue culture. Differential molecular profiles in cellular models for PD and ALS will be assessed via overexpressing alpha-synuclein and SOD1 human mutant isoforms. The molecular phenotype of mutant and wild type cells will also be compared. Chimeras will be generated, following grafting of the cells from the other aims, into mouse embryos, for the study of cell-autonomous versus cell-non-autonomous effects of the disease genes, alpha-synuclein and SOD1. This is a very reasonable interconnected series of goals that will provide novel and important findings on ES cell manipulation for studying and potentially treating PD and ALS. The timetable for performing these studies is appropriate. This is a proposal of very high quality written by a world specialist in the field. The PI has pioneered the use of in vivo mouse chimeras harboring human cells within the central nervous system, and has proven successfully that human neurons can home, integrate, differentiate, and contribute to the circuitry in the context of the mouse CNS. The research plan is logical and well lined out – though in very big strokes. While there is a considerable lack of detail and preliminary data, the outstanding record of the PI in the field can overcome many of these concerns. For example there is no data on motoneuron derivation (no HB9+ cells), and the data on dopamine neuron derivation is largely limited to TH expression. Comparison of hES derived NS data with the wealth of data available in the Gage lab from primary hNS of various brain regions is an interesting idea. However, there is no discussion about the potential limitations of this approach (i.e. hES derived NS will likely represent an earlier developmental stage compared with primary hNS). There is a clear acknowledgement that cell purification may be required for some of these studies. However, no details are provided on which markers are going to be the few key markers selected for this study (catalogue of marker provided). The use of promoter-specific lentiviral vectors requires extensive optimization for any given construct as stably integrated vectors often get misregulated depending on integration site(s) and the nature of the promoter/enhancer elements. Many of the proposed experiments are interdependent, and it would have been valuable to provide a marker scheme to be used for selection prior to using exon arrays and sm non-coding RNA analysis. The selection criteria for the identification of candidates from these arrays and the tools for GOF and LOF are also not described in any detail. However, the extensive expertise of the PI in comparable studies mitigates some of these concerns. The biggest concern however relates to grafting hESCs (wt or mutant) into the E14.5 ventricle. This would most certainly not lead to the robust integration of such cells in the spinal cord as motoneurons to model of ALS, or in the midbrain as dopamine neurons to model of PD. In both cases all host motoneurons or dopamine neurons have been already generated (postmitotic), and it seems very unlikely that the signals would be still available to instruct undifferentiated hES rapidly into the appropriate phenotype. In addition there are obviously a lot of other issues regarding experimental variability and differentiation into unexpected phenotypes that will make it very unlikely to yield a robust human disease model. It would be extremely surprising if these experiments works as proposed. There is also no discussion of alternative strategies in the event that the proposed experiments are unsuccessful. STRENGTHS: Small non-coding microRNAs and other approaches used here to discover ways to control the fate of ES and adult neural stem cells into dopaminergic and cholinergic neuronal phenotypes will without question provide invaluable information to the field. This is a very strong investigator and the experiments are logical, well-described, and with the proper insights for translating these new findings to reagents for the field as well as for therapeutic applications (a major goal of CIRM) in motor neuron disease and Parkinson’s. The experiments are complex and time consuming, but there is a great deal of enthusiasm for this investigator who continually provides the field with high quality science in the field of stem cells. Appreciation of the state of transitions during cell fate specification is a strength. This is an embryological concept mostly neglected by non-developmental biologists approaching the hESC field. Another strength of this proposal is that it combines basic research with direct potential clinical applications, not only unraveling the molecular aspect of differentiation, but also establishing a platform for modeling disease. Finally, and perhaps more importantly, the use of in vivo assays in chimeric mouse to study the behavior of the cell types in their natural in vivo milieu, again an area neglected in the hESC field. A series of very innovative techniques are proposed as an integral part of this proposal and the experiments are proposed in a logical progression. Also a strength is the acknowledgment that cell sorting may be necessary for these studies. WEAKNESSES: There are really very few if any weaknesses with this very nice proposal. The Deep Sequence studies are certainly ambitious, considering the massive number of small RNAs that will present themselves; the productivity and expertise of this PI and his group reduces concerns of the amount of work involved, and the potential payoff in new insights and reagents allay any concern. The PI will focus on determining “… the consequences of overexpressing mutant variants of alpha-synuclein (the gene mutated in PD) at different transition points as cells are induced to express the dopaminergic neuronal phenotype…” Certainly synuclein is not the only “gene mutated in PD”, and even though it is reasonable to focus on this mutation for the current model studies, there are other genes and mutations that might be more interesting and potentially important in the hES cells models. The PI checks the box that these hES cells studies have clinical relevance to Parkinson’s and “Alzheimer’s”. Certainly cholinergic cells do have relevance to AD, with regard to at-risk ACh cells in the cholinergic basal forebrain, however, this reviewer thought that most of this grant is directed to motor neuron production for ALS. This grant can be qualified as overly ambitious by regular funding agencies. However the track record and productivity of the PI is tremendously high. Therefore, this should not impact this grant negatively. Scarce preliminary data is available: no HB9 data, no differentiation, characterization and promoter driven isolation of phenotypes, and the experiments are interdependent. If the first ones fail, little useful information will be gained. The description of promoters to be used for purification is vague. There is little discussion of potential pitfalls throughout the proposal. Limited information is offered on how the PI will progress from screening to identification of candidates, to functional loss-of-function and gain-of-function screens. The generation of disease specific models seems problematic (motoneurons and dopamine neurons are born at E10 – 12 and it would be very surprising to observe significant numbers of properly integrated MN or DA neurons when grafting at E14.5 into the lateral ventricle). Such a study may require early intraparenchymal grafts and should be supported by preliminary data in the spinal cord and midbrain (e.g. checking for MN and DA integration in the embryos from the recent PNAS paper). DISCUSSION: The proposal offers a compelling approach to using non-coding RNAs to probe the transition points of differentiation of hESC into mature functioning dopaminergic and cholinergic neurons. There was agreement that the proposal is ambitious, and concern about the workload required to sequence small RNAs as proposed, but it was generally agreed that based on the track record of the PI, the research team would be likely to handle the challenges. There was also concern that the proposal is vague on specific details relating to the genetic screen proposed, on how the applicant will use the data from the screen and about how candidates will be selected for validation. The discussants were troubled by the transplantation experiments as proposed. There was agreement that the chimera experiments may be unlikely to work given that precise timing is needed in integration of appropriate local signals for differentiation of neurons. The reviewers felt that transplantation may need to be done at a much earlier stage than that proposed in order to achieve the proposed goal. To improve the proposal, there were several suggestions. Additional preliminary data for Aim1 would strengthen the proposal, as would a better description of the promoters & GFP constructs to be used for selection, and selection criteria for candidates to be used for GOF and LOF efforts. Discussion of alternative strategies should be included throughout the manuscript. The chimera experiments should be re-designed and appropriate preliminary data should be provided.
Conflicts: 

© 2013 California Institute for Regenerative Medicine