Development of multiparameter based flow cytometry technology for characterization and isolation of purified population of hESC derived differentiated cells for use in cell therapy - pilot system with cholinergic neurons.
Tools and Technologies I
A key problem in using stem cells to better understand human disease and to develop new therapies is the need to generate large quantities of highly purified, rigorously characterized, functionally robust and viable cells of various types. Here we propose to develop the tools and flow cytometry technology needed to solve this problem in the specific case of neurons in the human brain that are damaged in Parkinson's Disease and in Alzheimer's Disease. Our goal is to develop tools to generate and purify these special neurons from human ES and iPS cells. The lessons we learn in the solution of these cases will then be extended to the generation of a general technological approach to solving in vitro differentiation problems in any cellular system using flow cytometry and surface markers. We seek to define systems that enable and permit safe and effective utilization of the many approaches in regenerative medicine that are being developed from hESC or iPS cell sources, to be safely used in Phase I and subsequent trials to establish their utility in clinical therapy. Such purified cells will also be crucial reagents for finding and testing new drugs for the treatment of AD, PD, and other diseases.
Statement of Benefit to California:
Developing better understanding and treatments for a variety of diseases using human stem cell technology requires not only inducing differentiation of the required cell types, but also requires purifying them sufficiently that they would be useful for research or safe for transplantation. Our proposal is to develop methods to generate and purify two defined types of useful neurons for AD and PD, which has the long-term benefit on contributing to therapy development for very costly and devastating diseases. In addition, the methods and tools we develop may be commercializable and stimulate new business and beneficial economic activity in California. It is our expectation that this technological approach to greatly reducing one of the key identified risks in the potential therapeutic use of hESC derived differentiated cells, may thus more rapidly enable the initiation of Phase I and further cllnical trials of various cell types being developed in California under the CIRM program.
This proposal focuses on the development of flow cytometry methods to generate purified populations of neurons differentiated from human embryonic stem cells (hESCs). The Principal Investigator (PI) proposes to first define cell surface markers and protocols for the differentiation and positive selection of basal forebrain cholinergic neurons (BFCNs) derived from human pluripotent stem cells with a goal of isolating at least one million neurons at greater than 99% purity. The second aim is to optimize their flow cytometric methods to enable scale-up and isolation of GLP quality cells. Finally, in their third aim, the group proposes to apply these optimized methods to dopaminergic neuron differentiation and purification. Reviewers agreed that this proposal addresses a significant technological roadblock in stem cell research. They praised the expertise of the PI and his/her co-investigators. However, they strongly questioned the project’s feasibility based on the preliminary data and research design. Reviewers commented that the potential impact of this proposal is high. Current methods for differentiation of hESCs are highly variable and give rise to mixed populations of neurons. While much work is being done to improve differentiation protocols, this proposal pursues a solution based on the development of methods for selection and purification of desired cell types from a mixed population. The potential result of this proposal, a high-throughput, scalable method of differentiating neuronal cell types and selecting to high purity, would be a significant advance to moving stem cell research toward to the clinic. The reviewers’ main problems with this proposal were with its feasibility. They pointed out that the cell surface marker the applicant emphasizes for its utility in selecting BFCNs, is expressed on a range of neural sub types, including motor and sensory neurons as well as mesenchymal stem cells. Thus, the reviewers would not predict that antibodies to this surface marker would be specific for BFCNs. This is a major problem with the proposal, as the efficacy of cell selection by flow cytometry will depend on the specificity of the cell surface marker(s) for the desired cell population. The applicant also proposes to screen a monoclonal antibody library for novel cell surface markers for BFCNs. However, reviewers noted that few details were provided about this library (i.e. how it was derived or what types of protein targets were represented); therefore it was difficult to determine how useful this resource might be. Reviewers also raised concerns about the quality of the preliminary data, noting that it was not quantitated and was presented in small panels of low resolution that precluded assessment. One reviewer would have liked to see data addressing the viability of cells following sorting. Finally, reviewers commented that aim 3 which extended the work to dopaminergic neurons, was extremely vague and appeared to be tacked onto the proposal. This aim contained little experimental detail and insufficient explanation of how the PI would find and develop dopaminergic cell markers. The reviewers praised the expertise of the three team leaders and the strength of their public/private collaboration. However they were disappointed by their listed efforts (10, 10, and 5%) and would have liked to see greater commitments. The reviewers also noted that much of the work would be supervised by a postdoctoral clinical fellow with a sparse publication record and very little demonstrable stem cell culture or supervisory experience. Overall, while this proposal addresses an important roadblock in stem cell research, the reviewers had serious doubts about its feasibility based on the preliminary data and research design.