Basic Biology I
$1 812 267
Neurodegenerative diseases comprise a heterogeneous spectrum of neural disorders that cause severe and progressive cognitive and motor deficits. A histological hallmark of these disorders is the occurrence of disease-specific cell death in specific regional subpopulations of neurons, such as the loss of cholinergic neurons in Alzheimer’s disease, gaba-ergic interneurons in various forms of Batten’s disease, spiny interneurons of the basal ganglia in some forms of metabolic disease, etc. Neurodegenerative disease can also possibly occur from the loss or dysfunction of selected glial cell subsets, such as the dysfunction of supportive glial cells around somatic motor neurons in amyotrophic lateral sclerosis. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among the problems associated with this are the wide variability of neural differentiation potential of different PSC lines and the lack of comparison of the resulting neural cells to those derived from the brain itself. PSCs, because of their broad neuro-developmental potential, are expected to help provide a therapeutic cure for a wide variety of neurodegenerative diseases. To achieve this expectation, we need to identify all the factors involved in neural differentiation such that our understanding of the mechanisms involved becomes more complete. Moreover, we need to be capable of manipulating differentiation pathways such that desired subtypes of neuronal progenitors can be selected that will provide functional phenotypes upon transplantation. We are taking a whole genome expression analysis approach to help identify markers and networks of gene associated with distinct stages of neural differentiation and also inhibition of neural differentiation. We hypothesize that once such factors are identified, we may be able to more readily generate and isolate a transplantable population of neural cells.
Statement of Benefit to California:
Current conservative estimates indicate that at least 16 million individuals in the US (2 million in California alone) are afflicted and currently living with a brain disease. This incidence may be higher as the estimates exclude rare disorders and childhood neurological disorders such as neuro-metabolic diseases and autism. An estimated 16% of California households may be dealing with the care of a loved one with brain disease. Many of the diseases (Alzheimer’s, Stroke and Parkinson’s) that affect the brain are progressive and their incidence and prevalence increase with age. By 2020, it is estimated that almost 1 million people will be aged 85+ in California alone, with a high proportion (36%) having moderate or severe neurological function. This represents an immense challenge to California’s health care system. Neural stem cell (NSC) populations have great potential for revolutionizing medicine by providing successful neuroprotective or regenerative therapy for brain disease following transplantation. The use of neural stem cells in the clinical therapy of brain disease and injury continues to remain an area of intense focus. The recent groundbreaking work of the derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has additionally created the reality of deriving immune matched NSCs from adult cells such as skin. However, difficulties in the development of these potential therapies relate to insufficient tools to isolate, identify and characterize NSC populations. We propose to further develop existing molecular pathway analysis tools to identify a "NeuroNet" or molecular fingerprint (s) specific for NSC and neural induction/differentiation pathways from embryonic - derived NSCs, brain - derived NSCs and iPSC - derived NSCs. Realizing the full potential of all such NSCs as a source of defined cells for cell based neurological therapies will ultimately require a critical in - depth knowledge of factors present within these cells that are responsible for inducing an early neural phenotype and for orchestrating differentiation down specific neural lineage(s). Defining the NeuroNet will be instrumental in facilitating both new discoveries in neural development and providing a means of simplifying characterization and quality control of these cells and, most importantly, guiding neural differentiation into clinically useful cell types.
The proposal is designed to reveal similarities and differences between neural stem cells (NSCs) isolated from human brain (bNSCs), NSCs generated from human embryonic stem cells (hESC-NSCs) and NSCs derived from induced pluripotent stem cells (iPSC-NSCs). Specifically, the applicant proposes to examine gene expression in these cells with the goal of identifying molecules that regulate neural differentiation. In Aim 1, the applicant proposes to characterize global gene expression in donor-matched pairs of bNSC and iPSC-NSC at two developmental time points in vitro. The applicant will also assess the proliferation, migration and neurogenic potential of these cells. In Aim 2, bioinformatic technologies will be used to identify a network of protein-protein interactions that may be shared between NSCs derived from all three sources. The applicant hypothesizes that the information derived from these aims will allow them to identify pluripotent stem cells with robust neural potential and therefore be able to more readily direct neural differentiation and isolate transplantable NSCs for clinical applications. Although reviewers appreciated that this proposal addresses a significant scientific problem, they were not convinced that it would have a major impact in the field. While the proposal has the potential to generate a huge amount of data describing different types of NSCs, reviewers were unsure how useful the data would be, especially since gene expression profiling is unable to discriminate between “drivers” and “passengers” in the neural differentiation process. Reviewers did not find the proposal to be innovative, nor focused on molecular mechanisms. They felt that a major strength of the proposal is the applicant’s unique resource that enables the comparison of bNSCs and iPSC-NSCs derived from the same donors. Reviewers found the research plan to be straightforward and generally feasible. Feasibility relies on the applicant’s ability to generate NSCs from hESCs and iPSCs, which is supported by some preliminary data and proposed alternative strategies. A reviewer disagreed with the applicant’s hypothesis that iPSC-NSC populations are equivalent to bNSC populations and would actually assume the opposite and focus experiments on how to make them equivalent. The reviewer suggested using the tools at the applicant’s disposal to evaluate protocols for generating iPSCs and inducing neural differentiation in order to yield NSCs that most closely resemble bNSCs. Reviewers raised a few concerns about the research team. They noted that the applicant is a junior investigator with a modest publication record and only recent experience in stem cell biology. However, reviewers praised the co-investigator’s expertise working with human stem cells and appreciated the 40% effort s/he devotes to the project. One reviewer felt that the proposal would be strengthened if the primary investigator and co-investigator switched roles. Reviewers were also unclear about another key collaborator’s role in the project. They noted that much of the foundational work was performed in this collaborator’s lab and from his/her letter of support it appears that much of the proposed work will be performed there as well. Yet this collaborator is only devoting 5% effort to the project and receiving a minimal subcontract. Overall, while reviewers found the research plan to be logical and feasible, they raised concerns about the research team and were not convinced the project would make a significant impact in the field.