Funding opportunities

Engineering Bioactive Hydrogels for Neuronal Differentiation of hESCs

Funding Type: 
SEED Grant
Grant Number: 
RS1-00300
Funds requested: 
$399 999
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells (hESCs) have strong potential as sources of cells for the treatment for disease and injury (e.g. Parkinson’s Disease, amyotrophic lateral sclerosis, spinal cord injury, diabetes, congestive heart failure, etc.). The successful integration of hESC into such therapies will hinge upon three critical steps: their expansion without differentiation, their differentiation into a specific cell type or collection of cell types, and the promotion of their survival and functional integration at the site of disease or injury. Precisely controlling each of these steps will be essential to maximize hESC’s therapeutic efficacy, as well as to minimize potential side effects that can occur when the cells numbers and types are not properly controlled. However, hESCs are typically grown on murine or human feeder cells, in conditioned media derived from these cells, and/or within complex mixtures of animal or human proteins. Such growth conditions present major problems: there is a possibility of pathogen transmission from feeder cells or proteins, hESCs can acquire non-human antigens that will lead to immune rejection following implantation into a patient, and these growth conditions are difficult to precisely control and reproducibly scale up to a clinical process for the treatment of large patient populations. To achieve the intended goals of regenerative medicine, methods for the precise control of the proliferation, differentiation, and survival of stem cell populations in cell culture and in the body after cell implantation are necessary. We have made significant progress in developing a novel technology platform consisting of completely synthetic polymer-based synthetic matrices to support hESC proliferation and self-renewal. We now propose to create synthetic microenvironments to support hESC differentiation into two important neuronal lineages: dopaminergic neurons with potential for Parkinson’s Disease therapy and motor neurons with potential for Lou Gehrig’s Disease. Previous protocols have been developed for controlled differentiation into these lineages; however, they have typically involved culture conditions with animal and human proteins and ECM. Furthermore, after implantation into the site of injury or disease, the majority of neurons typically die. We hypothesize that implanting neurons differentiated from hESCs along with a supporting, bioactive matrix will enhance cell survival and therefore future efforts to utilize grafts for tissue engineering and repair. The result will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying cell differentiation. Therefore, this novel integration of stem cell biology, neurobiology, bioengineering, and materials science has the potential to overcome a major challenge in regenerative medicine.
Statement of Benefit to California: 
Stem cell research in general, and this proposed research in particular, have great potential for enhancing the scientific and economic development of the state of California. First, this project is highly integrative in that it melds expertise and investigators from a number of scientific fields including tissue engineering, materials science, chemical engineering, stem cell biology, neurobiology, electrophysiology, and genomics. It therefore represents a model project for the development of interdisciplinary research teams, since success in research increasingly relies upon taking the initiative to draw from numerous fields of science and engineering. Furthermore, this project will represent a highly valuable and unique interdisciplinary training environment. Two trainees will be able to draw from leading scientific expertise in five research groups in five departments and two institutes to make progress in this high impact work. Finally, the collaborative expertise that this group develops as a result of this funding will be in place to continue this and other research areas, with the aid of numerous additional trainees, in the future. In addition, the field of stem cells represents a unique economic opportunity for the state of California. Both Northern and Southern California are dominant areas for biotechnology research and companies. We anticipate that the products of this research will be of interest to numerous sectors of biotech, not only for its potential in neuronal differentiation but in its generality for both embryonic and adult stem cell culture and differentiation into numerous lineages. First, the use of stem cells for in vitro pharmacology and toxicology screening will rely upon the development of scaleable and reproducible systems for stem cell expansion and differentiation, which this work can provide. Second, research product companies may be interested in providing reproducible, synthetic culture systems for stem cell experimentalists. Finally, this work potentially has its largest promise in the development of scaleable systems to support stem cell differentiation in vitro and cell transplantation in vivo for therapeutic application in tissue engineering and repair.
Review Summary: 
SYNOPSIS: Dr. Schaffer from UC Berkeley proposes to develop and test synthetic polymer-based matrices to support human embryonic stem cell (hESC) proliferation and self-renewal, and differentation to dopaminergic neurons and motoneurons. He will be working with others at Berkeley, including Kevin Healy (Bioengineering and Materials Science), John Ngai (head of Neuroscience Graduate Program), Brian Kaspar at OSU, an expert on gene therapy, and Mu-Ming Poo, a well-known neuroscientist. He presents signficant preliminary data indicating that a synthetic microenvironment can support hESC differentiation into neurons and proposes to determine whether a synthetic biodegradable hydrogel can enhance the viability of differentiated neurons following engraftment into the central nervous system. SIGNIFICANCE AND INNOVATION: The idea of creating a 3-dimensional synthetic hydrogel environment in which to grow and differentiate hESC is not particularly innovative or original. Nonetheless, the research is important because it may provide a wholly synthetic and controlled environment for cell growth, expansion, and differentiation. For clinical applications of hESC it will be highly desirable to cultivate and differentiate them in a completely chemically defined environment that includes both the soluble factors, nutrients and physical surface. It is even more desirable if the chemical environment is free of animal derived components which may harbor adventitious agents (either known but undetected, or agents that are not yet known such as adventitious agents). The proposed work to develop a polymer based material (not quite extracellular matrix, although the proposal terms it “ECM”) for supporting hESC, if fulfilled will be a major step forward in stem cell technology. The work is based on highly successful prior biomaterial research of Dr. Healy. The materials proposed have been shown previously to support various behaviors of neuronal cells. The applications to hESC are therefore rather innovative. STRENGTHS: This is an important area of investigation. As the applicant pointed out, current methods of growing hESC are still relatively primitive and use organic materials and even feeder cells that may introduce complications for transplanting the cells. The investigator is experienced and productive and should be able to deliver on this research. This is a strong team of highly reputable scientists each with unique expertise complementing each other. The bioreactor peptides have all been shown to promote cell adhesion and trigger the corresponding receptor mediated response. The likelihood of success in supporting cell proliferation is high. Whether the growth rate, proliferation potential will be the same or not is yet to be investigated. The experimental procedure for both material synthesis and hESC characterization are well laid out. WEAKNESSES: The proposal is not particularly original and there is not much discussion of high throughput methods that would be necessary to evaluate the large number of synthetic materials. The work appears to be more on material research than truly focusing on stem cells. A number of questions were raised. Will the work be compared to hECS cultivated on feeder layer or in serum-free media (SFM) on plastic surface? Is it critical not only to evaluate survival, but also the population behavior? Much biomeaterial related work test small samples, relying overly on microscopic observation of local cell behavior. The percentage of cells differentiated, survived vs. degenerated is also critical. The relationship between cells and SIPN is less than clear. How is cell distribution controlled? It would be good to provide either a more hypothesis-driven approach (i.e. whether particular classes of molecules are better than others) or a high-throughput screening method for identifying and optimizing the hydrogels. DISCUSSION: This proposal is from an experienced and productive investigator who has assembled a team of well-known collaborators with a proven track record. The proposal aims to test synthetic, polymer-based matrices for hESC proliferation and differentiation to dopaminergic neurons and motoneurons. This proposal lacks a discussion of the high throughput method for optimizing hydrogel and one reviewer is uncertain as to whether there is a sufficient plan to cover 3 years' time. Both reviewers commented that developing 3-D synthetic hydrogels is not particularly innovative nor original. The plan for generating useful and important data is insufficient. Many variables in the high-throughput screen are not specified, and the reviewers noted that NIH would not be interested in such a "fishing expedition".
Conflicts: 

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