The goal of this CIRM seed grant is to extend the potential of a new technology for stem cell studies by bringing to bear state-of-the-art microengineering techniques to the challenges of stem cell screening and selection. The completed system will provide a practical and flexible device with far-ranging applications. The capability to sort cells after experimental manipulation will fuel basic research by providing a means to establish new cell lines for stem cell study. The capabilities of the new instrumentation will enable greater precision and flexibility in the exposure of cells to growth factors to maintain cells in a sem-cell-like state of to differentiate cells into desired tissue types for regenerative medicine. Experiments are envisioned to more accurately recreate developmental events or the functions of stem cell in the living organism. Precise control of the cellular environment along with isolation of precursor cells for the treatment of a particular disorder would have a dramatic impact for medical applications.
Statement of Benefit to California:
The research to be funded by this CIRM Seed Grant has to potential to directly benefit the citizens of California. The funds will be used to develop a new instrument that stem cell researchers will utilize to test and select unique stem cells for either further study or to grow and apply to medical therapies. The technology has practical and widely applicable uses, so that many researchers in the academic and industrial laboratories of the state will receive benefit through increased efficiency of their studies and the ability to perform new types of experiments to better understand the biology of stem cells and how to use them for regenerative medicine. The research will also stimulate economic development through creation of intellectual property which will be owned by citizens of the California through the state university where the research will take place. This research and that of other investigators funded by these grants will enhance the state’s competitiveness in biotechnology research and development, thus furthering California’s lead in this important area of economic growth.
SYNOPSIS: In this proposal, the investigators will develop a microplatform wherein single ES cells can be manipulated using different adherence substrate and media to evaluate in a high throughput fashion the influence of these factors on hESC fate. The method allows for changing culture conditions on an individual cell basis as well as harvesting of single colonies for subsequent FACS and / or RT-PCR analysis of the status of the cells. ES cells that will be used are H1 and H9. SIGNIFICANCE AND INNOVATION: The investigators propose to combine two innovative technologies: pallet-based adherent-cell sorting and microfluidic gradient generators. The pallet technology, recently developed in the PI's lab, is an impressive new tool for sorting of adherent cells and is worthy of further development. This proposal is innovative in that it will apply the strengths from bioengineering, i.e., microfabrication of culture plates merged with microfluidics that allows manipulating large numbers of single cells or colonies simultaneously, with hESC technology. The approach if successful should allow high(er) throughput screening of specific conditions for differentiation or conditions that support ESC expansion without differentiation. STRENGTHS: The main strengths of this proposal are the team, including the extensive experience of the PI and the long-standing collaboration of the investigators, and the technology, particularly the pallet-based sorting. This merger between bioengineers knowledgeable about micropatterning combined with microfluidics and investigators in ES cell biology is a significant strength of the proposal. If the proposal succeeds, a high throughput system to test multiple cytokine cocktails, alone or sequentially or combined with exposure to different adherence substrates should become possible. This should significantly speed up the ability of testing conditions that support maintenance of the undifferentiated state of hESCs or induces differentiation towards a specific lineage. WEAKNESSES: There are two main weaknesses of this proposal due to an apparent mismatch between what the sorting technology offers and the needs of hESC research. First, the investigators propose to test factors and conditions on single ESCs, yet culture of human ESC at a clonal level is still very difficult. Advances in single-cell plating using neurotrophic factors is recent and has not been substantially validated by others, making this a risky route. The more conventional route is to plate hESCs as clumps, and the investigators will also test small clumps of cells, but it is surprising that the investigators do not plan to use ES cells that may be more easily grown at the clonal level than H1 and H9 cells. If the cells must be plated as clumps, then the concept of sorting clonally derived colonies is not applicable, as the colonies will result from multiple cells. Second, there is typically as much heterogeneity across a single colony as there is across multiple colonies, and so the idea of using single colonies to decrease variability is unlikely to work. The studies proposed in Aim 3 are to test the effect of different concentrations of BMP4 on the differentiation of single ESCs. Although these studies will provide proof of concept that the system allows high throughput screening for the effect of factors on the fate of ESCs, the readout proposed, i.e., to perform gene array analysis on the progeny of single ESCs cultured with BMP4 is unlikely to yield very useful information. Since the investigators will be analyzing expression of many pooled cells, it is unclear that sorting and analyzing one colony will be an improvement over analyzing a whole chip. In addition, it appears that the investigators suggest that as few as several thousand cells are sufficient to perform gene array analysis without the need for amplification of the target RNA, which will not be possible. A more minor issue is in overselling of microfluidics and of the pallet technology in the Rationale section. Microfluidics always has the "potential" for massively parallel assays and all sorts of wonderful functionality, but in practice the only group that even comes close to this ideal is the Quake group and their technology. Most microfluidics perform one axis of variation on a device and that's it, which is what the team indeed proposes to do. Additionally, the motivation would be stronger if the investigators were more reasonable in their claimed advantages. For example, suggesting that 10,000+ pallets are possible on a slide does not allow for spacing between pallets and assumes one can use the whole slide surface, which is never the case. Finally, there is no strong rationale for why this work should be funded by CIRM. Nothing proposed here would be unsuitable for the NIH, and the risk level, which is used to justify CIRM funding, is not measurably higher than any given NIH R21 proposal. DISCUSSION: This proposal aims to build micropallet arrays to screen and select stem cells. The effects of microfluidics and shear force on differentiation will be assessed. The applicant will work with Dr. Donovan, who is an expert in hESCs. A BMP gradient will be applied to the plate look at gene arrays. Reviewers felt that the technology is fine, but the proposed ESC cell lines H1 and H9 are difficult to culture as single cells, and even if the applicant could get single cells reviewers anticipate there will be heterogeneity in the resulting population which will be a major concern. They suggest that the applicant look at other cell lines. One reviewer noted that there was nothing here that NIH couldn't fund. The proposed technology for cytokine/growth factor gradient was regarded as a good idea, feasible and useful; however, one panel member felt that testing multiple combinations of growth factors would be quite hard.