Funding opportunities

Mechanisms of Lineage Commitment in Human Neural Crest Stem Cells

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01398
Funds requested: 
$1 593 000
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Diseases arising from defects in NC specification, migration and differentiation referred to as neurocristopathies (total ~74) include various skeletal dysmorphology syndromes (e.g. Apert and Beare-Stevenson cutis gyrate syndromes), diseases of the nervous system (neurofibromatosis and Hirschsprung’s disease, peripheral neuropathies such as Familial Dysautonomia) and pigment disorders (Waardenburg syndrome). The defect is often confined to one or two NC lineages, as in pigment abnormalities and the treatment of PNS disorders will require a uniform population of lineage-committed progenitors. In addition, in vitro applications such as initial screening for drug toxicity will require large quantities of pure NC progenitors. In particular, derivation of functional chondrocytes may provide a much-needed alternative for cartilage regeneration. Recently, by means of the support from a CIRM Seed grant, we generated a uniform population of neural crest stem cells (NCSC). These human NCSC can differentiate in vitro producing all neural crest lineages including sensory and autonomic neurons, Schwann cells, smooth muscle cells, melanocytes, adipocytes and chondrocytes. We propose comprehensive in vitro analysis of differentiation in NC for the following two reasons: 1. All previous evidence was obtained in the model organisms. No studies investigating the role of transcription factors or extracellular matrix (ECM) in human neural crest self-renewal and differentiation were reported to date. 2. It is possible that unsuspected conditions will promote directed differentiation of human NCSC compared to physiological stimuli. Specifically we will: 1. Identify transcription factors regulating human NCSC differentiation using a functional genomic approach; 2. Investigate the role of various matrix components and their elasticity in human NCSC differentiation. The deliverables will include: first, a verified set of TFs that regulate human NCSC lineage commitment and differentiation; second, a knowledge of how the nature and stiffness of various ECM matrices affects differentiation into neural crest lineages. The knowledge, tools and reagents will be available to other academic researchers and commercial entities. If successful, the proposed approach will help us to understand mechanisms of NC differentiation along specific NC lineages. This knowledge will allow the development of tools and reagents for diagnostic and therapeutic applications such as screening assays for drugs affecting human sensory neurons, neuronal replacement for peripheral neuropathies, and the generation of functional Schwann cells.
Statement of Benefit to California: 
Diseases arising from defects in NC specification, migration and differentiation referred to as neurocristopathies (total ~74) include various skeletal dysmorphology syndromes (e.g. Apert and Beare-Stevenson cutis gyrate syndromes), diseases of the nervous system (neurofibromatosis and Hirschsprung’s disease, peripheral neuropathies such as Familial Dysautonomia) and pigment disorders (Waardenburg syndrome). The defect is often confined to one or two NC lineages, as in pigment abnormalities and the treatment of PNS disorders will require a uniform population of lineage-committed progenitors. We propose comprehensive in vitro analysis of differentiation in NC for the following two reasons: 1. All previous evidence was obtained in the model organisms. No studies investigating the role of transcription factors or extracellular matrix (ECM) in human neural crest self-renewal and differentiation were reported to date. 2. It is possible that unsuspected conditions will promote directed differentiation of human NCSC compared to physiological stimuli. The deliverables will include: first, a verified set of TFs that regulate human NCSC lineage commitment and differentiation; second, a knowledge of how the nature and stiffness of various ECM matrices affects differentiation into neural crest lineages. The knowledge, tools and reagents will be available to other academic researchers and commercial entities. If successful, the proposed approach will help us to understand mechanisms of NC differentiation along specific NC lineages. This knowledge will allow the development of tools and reagents for diagnostic and therapeutic applications such as screening assays for drugs affecting human sensory neurons, neuronal replacement for peripheral neuropathies, and the generation of functional Schwann cells. An effective, straightforward, and understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. In addition, this program and its many complementary programs will generate potentially very large, tangible monetary benefits to the citizens of California. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by the CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.
Review Summary: 
This proposal is focused on human embryonic stem (hES) cell-derived neural crest stem cells (NCSC) and examines the molecular control of their fate specification. The goal of Aim 1 is to identify transcription factors regulating human NCSC commitment and differentiation using a siRNA library in a functional genomic approach, and Aim 2 investigates the role of various extracellular matrix (ECM) components and their elasticity in this process. Since NCSC produce a wide array of cell types of clinical relevance, reviewers felt that a better understanding of the molecular and cellular basis of NCSC differentiation may impact future therapies. However, they identified several major problems with this proposal. Overall, the proposal is poorly written and contains numerous mistakes. Reviewers questioned the feasibility of the functional screen, since siRNA-mediated knock down of gene function is relatively transient and may not be able to impact human NCSC differentiation which occurs over 1-3 weeks in culture. Reviewers suggested that lentiviral-mediated shRNA delivery would be a more promising approach for the initial screen. Preliminary data did not provide much support toward the feasibility of the screen, since only a single gene had been used to validate the system, and actual levels and duration of knock down were not presented. Since this gene is a master regulator of neural crest formation, this does not demonstrate that the assay can identify factors that influence a single cell fate. Further criticism was leveraged against the assays used to assess differentiation of NCSC, as they are based on detection of endogenous proteins, a cumbersome process. Reviewers suggested this analysis would be better accomplished using reporter constructs. Lack of rescue experiments to control for siRNA off-target effects was cited as a further weakness of the proposal. The proposed analysis of ECM effects on NCSC differentiation is based on preliminary data that 2 ECM molecules differently affect NCSC differentiation. Reviewers felt that this is not adequate to justify a full program in ECM biology, especially with regard to effects based on elasticity. Furthermore, reviewers were concerned about the large number of conditions being examined without clear rationale for their selection, and about the fact that in the preliminary analyses, synthetic matrix stiffness was assumed to be that of published preparations, rather than actually measured and confirmed. Subtle differences in the formulation of synthetic matrices, including the thickness of the gel, can influence mechanical properties. Thus, any study attempting to ascribe biological results to matrix properties requires rigorous analysis of this parameter. The principal investigator has adequate expertise to conduct the proposed cellular and molecular biology studies with demonstrated experience working with hES cells and their differentiation toward neural crest. The publication track record is good but does not yet include papers in a significant number of high impact journals. Although a well-known neural crest cell biologist is included in the research team, it lacks significant inclusion of a co-investigator with experience in characterizing and formulating matrices with defined mechanical properties. A letter of support from an appropriate investigator is included, but that investigator is not listed as part of the formal team. In summary, reviewers expressed many reservations regarding the feasibility and design of both aims of this research proposal and thus did not recommend it for funding.
Conflicts: 

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