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RN1-00535-1: Bioengineering technology for fast optical control of differentiation and function in stem cells and stem cell progeny
Recommendation: Recommended if funds available
Scientific Score: 61
First Year Funds Requested: $582,098
Total Funds Requested: $2,979,719
Public Abstract (provided by applicant)
Embryonic stem (ES) cells potentially could provide clinically important replacement tissue for central nervous system (CNS) disease treatment, and regenerative medicine approaches involving ES cells have been suggested for common CNS disorders. But it has been difficult to produce the right kind of replacement tissues from ES cells because the “differentiation”, or cell-type specification process, takes many days to weeks, during which time many different stimuli and signaling molecules need to be physically applied to the stem cells. This process of “stem cell differentiation” is slow, costly, laborious, variable, prone to error and contamination, and ultimately rate-limiting in the long road leading to clinical translation. We propose to develop and apply fast, inexpensive, and robust optical technologies to the fundamental problem of stem cell differentiation and regenerative medicine, with particular focus on CNS disease.
Statement of Benefit to California (provided by applicant)
Neuropsychiatric diseases like Parkinson’s disease and major depression are leading causes of disability and death in California and worldwide. They are difficult to treat, poorly understood, and devastating for patients, families, and society as a whole. Our proposed fusion of engineering technology with clinically-inspired stem cell technology represents a unique opportunity, which we anticipate will lead not only to fundamentally new, potent, and specific therapies for diseases representing major burdens for the state, but also to engineering and medical commercial ventures that will add resources, money, and skilled jobs to the robust and growing state economy.
Review
SYNOPSIS: In the proposal the applicant will engineer embryonic stem cells (ESC) or neural progenitors to express photo-activatable cellular factors. The Principal Investigator (PI) will use these factors to stimulate signaling pathways with temporal precision to effect differentiation, in vitro as well as in vivo. The goal is to induce the generation of specific neuronal types. The applicant proposes to develop, employ, and disseminate a noninvasive optical-engineering technology for long-term control of signaling with a high-degree of temporal precision. The proposal has four aims. Aim 1 involves hardware and software development for a robotic system for high-throughput culture, optical stimulation, and microscopic monitoring of cultured stem cells. In Aim 2, the applicant will examine the differentiation of neural stem cells and mouse and human ES cells undergoing differentiation to specific neuronal types. Aim 3 is to deliver optical stimulus protocols to promote the terminal stages of neuronal differentiation in vivo after transplantation of cells into the brain. Finally, Aim 4 is to develop precise control of new tissues in order to selectively drive the function of stem cell-derived progeny within intact tissue.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a very ambitious research plan. The PI is pioneering technology that would control multiple intracellular signaling or plasma membrane events in stem/progenitor cells, without need for solution changes. These optical-molecular technologies may have many uses for noninvasive stimulation of differentiating stem cells with superb resolution. This is bold and imaginative technology that would represent a new means of spatially and temporally controlling the generation of particular differentiated cells from stem cells in vivo for therapeutic purposes. If successful, it would be revolutionary in its impact.
The strength of the proposal is its novelty, and the fearlessness of combining engineering and stem cell biology. The applicant sees an opportunity to bring his/her optical technology to stem cell biology which is a positive. His/her stem cell experience is related to work on native adult neural/progenitor cells, and s/he proposes to make a concerted effort to move into stem cell technology development and distribution.
Although the technological aspect of the plan is innovative and sufficiently detailed the biological underpinnings are weak, making the likelihood of success uncertain. The weaknesses of the application all center around the lack of acknowledgement of the biological pitfalls (the technical pitfalls are fairly well acknowledged). As an example, the PI does not acknowledge that the transfer of differentiation protocols from mouse ES cell to human ES cells for neurogenesis has not really proved fruitful. How will the choice of methods to differentiate human ES cells get the field past this hurdle? A long time frame for hES cell differentiation into neurons may be what is needed for efficient neuronal subtype differentiation, in which case the ability to provide quick pulses of activation pushes to cells may not offer a great advantage. All the biology that has detail in the proposal sufficient to interpret involves neurogenesis, but there is not sufficient detail in the methods regarding human ES cell differentiation to determine whether the biologic plan makes sense. Furthermore, it is not clear from the grant proposal that the cellular factors used in the photo-activation are present in undifferentiated hES cells. The preliminary data does not cover these cells. Also, the message for the cellular factors may be expressed in the hES cells, but may not be functional as is true for other types of these factors.
At some point the plan is to drive up to four different factors optically in the same cells, but the expression considerations are glossed over. Furthermore, the induced pluripotent cell experience with four lentiviral vectors is cautionary (offspring with tumors), and in the case of this proposed research, the proposed expression approach may not totally mitigate the potential for tumorigenicity.
Regarding the specific aims, in many ways Aim 1 to develop the robotic stem cell differentiation setup is the most exciting since the physical structure of traditional laboratories clearly needs a re-think for all of stem cell biology and tissue culture models of disease. A criticism of this aim is that it does not take advantage of the bioreactor literature in choosing the parameters to control as part of the set-up. Also, how is sterility maintained? In Aim 2, no indication is given of how neuronal differentiation will be achieved – which genes will be driven in what sequence? There simply was not enough detail to fully understand this set of experiments.
In Aim 3, specific factors involved in cellular signaling are the biologic tools to be used in vivo for driving neuronal differentiation and integration. Once again, the technical feasibility issues are acknowledged, but not the biologic considerations – it is unlikely that this approach alone will be sufficient in the clinical situation. Stimulation of these factors (for when and how long?) won’t be sufficient for all the necessary machinery to drive integration. The variety of temporal regimens that will have to be tried to determine an optimal signaling are not acknowledged as potential feasibility issues.
In Aim 4 there is no biological discussion of the problems getting from undifferentiated human ES cells to neuroblasts, thus it is very hard to evaluate this aim from the proposed design. Moreover, the preliminary data supporting this aim do not mention the particular method used to express the photo-activatable cellular factors.
Overall, the plan may be logical, but it is difficult to judge because of the lack of both details and a discussion of biology. Stronger rationale needs to be presented to argue that the particular signaling pathways being manipulated, and the timing, duration and combinations of signals to be used are likely to achieve the desired cellular differentiation outcomes. As proposed, it is not clear how effective the approach will be in the long term in its application to hESCs.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is an outstanding physician-scientist who has been an Assistant Professor since 2005. S/he did an MD/PhD and was a post-doc at a top-tier university. The PI has an impressive publication list and has NIH funding support for similar research in adult neural stem cells.
Obviously this is a super-star physician-scientist investigator who is interested in neurogenesis and neural circuit control with a strong clinical translational context. The research plan synergizes in an exciting way with the PI’s clinical work, and the PI has demonstrated the ability to follow through on a major undertakings like this. One reviewer is concerned that this investigator does not fit the spirit of the New Faculty Award, which is designed to support good research in stem cell biology for investigators who “face tremendous pressure to obtain results, publish,…and acquire grants quickly” and for investigators who find it “difficult to obtain financial support, especially…in the early stages of their career.” In terms of career development, this investigator’s career is well underway. S/he is already established as a leading scientist with major research publications and multiple grant awards. The PI says the CIRM New Faculty program will allow the time and resources to develop and validate his/her technology in the human ES cell system. In addition to developing this technology, this proposal will lead to the dissemination and exchange of information and tools. This PI does not need a CIRM award, but the question is whether CIRM can do without having in its portfolio one of the best and brightest young investigators in California and the country.
Though the PI is an Assistant Professor, s/he has been very successful in obtaining funding, and already has a directorship position in his/her department, which is highlighted in the grant. Within the confines of the PI’s current funding, it seems there would be room to explore the kinds of studies proposed here; in fact, a concern is that the applicant already has many large and small grants, plus spends a day in the clinic, thus can s/he commit enough time to all of the work described this proposal?
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution has one of the strongest stem cell programs in the country and an unwavering commitment in developing new biomedical research faculty in this field, making this an exceptional and pre-eminent environment for proposed research. The PI is jointly appointed in two departments in an independent, tenure-track position, and his/her independent laboratory includes all of the required space in a highly collaborative science and engineering environment with state-of-the-art research facilities and administrative support. The proposal’s interface with the applicant institution’s engineering programs is a strength, and the PI’s department chair states that the PI will only have a one-day-a-week clinical commitment. Considering how spread out the applicant appears to be, one day a week may be too much. Nonetheless the environment and institutional track record are outstanding.
DISCUSSION: Reviewers agreed that this is a technologically innovative proposal which could have a huge impact on biology as a whole, but differed in their overall enthusiasm. One reviewer was very concerned that the PI is over-extended. Limiting the control parameters for the closed lab environment, and not describing when and how long the temporal stimulation would be administered diminished this reviewer’s enthusiasm. In contrast, another reviewer was extremely enthusiastic about the tools the PI is bringing to bear in this unique experimental cell environment, and notes that while the developmental biology was not sufficiently considered, the PI does seem to have credibility in the neural field and should not be naive to complexities of nervous system. Clearly the PI is a pioneer in terms of tools development. A third reviewer felt that the application was innovative, but difficult to understand. This reviewer was not enthusiastic, particularly because the PI is far along his/her career.
PROGRAMMATIC DISCUSSION: A motion was made to recommend that this application be moved to Tier 1 – Recommended for Funding. A discussant reiterated that this is a very innovative proposal from an exceptional and creative scientist with dazzling technology, but no biology. This exciting technology uses light-activated compounds to initiate signaling pathways. In this case, the PI is developing tools that will not only impact stem cell biology, but many fields because the light pulses can activate multiple signaling pathways in cells, using non-invasive tools that do not require genetic modification. There was disagreement on this latter issue, as another discussant pointed out that this technique is not quite non-invasive, which prompted another panel member to agree, saying that what the applicant is doing is not clinically translational, but in fact very technical. Panelists also discussed the fact that the PI has won many awards, and is amply funded with grants having to do with neural stem cells. One discussant pointed out that the PI has extensive time constraints already, and the real question is whether s/he could reasonably devote enough time to the research proposed here. If the point is to bring physician-scientists into the stem cell field, this person is already there. The motion to recommend that this application be moved to Tier 1 failed.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- Wagers, Professor Amy