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.
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.
SYNOPSIS: In the proposal the applicant will engineer neural progenitors to express photo-activatable ion channels or pumps and use them to stimulate signaling pathways with temporal precision in differentiating neuronal progenitors or hESC, in vitro as well as in vivo. The goal is to induce the generation of glutaminergic and dopaminergic neurons. The applicant proposes to develop, employ, and disseminate a noninvasive optical-engineering technology for long-term control of biochemical signaling using optically-activated molecular tools to regulate with a high-degree of temporal precision Ca++ and cAMP signaling pathways in vivo. The proposal has four aims. Aim 1 involves hardware and software development for an enclosed, gas and temperature controlled robotic system for high-throughput culture, optical stimulation, and microscopic monitoring of cultured stem cells. In Aim 2, the applicant will perform high-throughput mapping of the differentiation space of neural stem cells and mouse and human ES cells undergoing glutamatergic and dopaminergic differentiation. Aim 3 is to deliver optical stimulus protocols (i.e., drive specific patterns of gene expression or depolarization) 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 biochemical 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 grant is its novelty, and the fearlessness of combining engineering and stem cell biology. The applicant sees an opportunity to bring his optical technology to stem cell biology which is a positive. His stem cell experience is related to work on native adult neural/progenitor cells, and 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 grant 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 his choice of methods to differentiate human ES cells get the field past this hurdle? In fact a longer time frame for hES cell differentiation into neurons may be what is needed (patience) for efficient neuronal subtype differentiation, in which case the ability to provide quick pulses of expression pushes to cells may not offer a great advantage. All the biology that has detail in the grant sufficient to interpret involves neurogenesis, and there are not sufficient biologic detail in the methods regarding human ES cell differentiation (and dedifferentiation, which is also mentioned) to determine whether the biologic plan makes sense. Furthermore, all the genes discussed in any detail drive depolarization of the cells, and though there are populations in murine embryoid bodies (which have a lot of neurosphere potential) that have the Ca channels of interest, it is not clear from the grant proposal that these channels are present in undifferentiated hES cells. The PCR data does not cover these cells. Furthermore, though the message for the channel is expressed in the hES cells, the protein-level organization of functional channel may not occur in the undifferentiated cells, as is true for other channel types.
At some point the plan is to drive up to four different transgenes optically in the same cells, but the promoter 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 potential tumorigenesis tendencies of 4 lentiviral insertions cannot be overcome by driving all the genes with one promoter packaged in a single virus.
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, choosing control only over CO2 and temperature, and not oxygen, for example, as part of the set-up. Also, how is sterility maintained? In Aim 2, the PI will “begin by deriving glutamatergic neurons” from the Thomson ES cells, but no indication is given of how the differentiation will be achieved – which genes will be driven in what sequence? Presumably these are distinct from the genes chosen for the later stages of neurogenesis, but there simply was not enough detail to fully understand this set of experiments.
In Aim 3, Ca++ and cAMP are the biologic tools used in vivo for driving neuronal differentiation and integration (with or without transcranial magnetic stimulation). 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. Ca++ and cAMP (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 the PI acknowledges that “we may find it helpful to transduce ES cells with a second transgene” implying that all the differentiation will be accomplished by a single optically controlled transgene system. Since there is no biological discussion of the problems getting from undifferentiated human ES cells to neuroblasts, it is very hard to evaluate this Aim from the proposed design. Moreover, the preliminary data supporting this aim (Figure 5) do not mention transgenes at all, only pharmacologic manipulation of the cells.
Overall, the plan may be logical, but it is difficult to judge because of a lack of details and 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 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 of Bioengineering and Psychiatry at Stanford University since 2005. He did his MD/PhD at Stanford with R. Tsien, and then was a postdoc at Stanford with Robert C. Malenka (2001-2004). He has an impressive publication list, including seven in past the three years (in Nature, Science, J Neurosci, and PNAS). He has RO1 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. His research plan synergizes in an exciting way with his clinical work and he as demonstrated his 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. He has already established himself as a leading scientist with major research publications and multiple grant awards. He says the CIRM New Faculty program will allow him the time and resources to develop and validate his technology in the human ES cell system. In addition to developing this technology, this proposal also will lead to dissemination and exchange of information and tools. He 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 he is an Assistant Professor, he has been very successful in obtaining funding, and already has a directorship position in the psychiatry department, which he highlights in the grant. Furthermore within the confines of his current funding, it seems there would be room to explore the kinds of studies he proposes here. In fact, a concern is that he already has many large and small grants, plus spends a day in the clinic, thus can he commit enough time to the proposal? Exclusive of grants that will expire in 2008, the PI has an NIH grant ending in 2009, a McKnight grant ending in 2010, a second NIH grant ending in 2010, a third NIH grant ending in 2012, a Stanford BioX grant ending in 2009, an NSF grant ending in 2012, and he is co-PI on a CIRM grant ending in 2010.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Stanford 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. Dr. Deisseroth is jointly appointed in Bioengineering and Psychiatry in an independent, tenure-track position, and his independent laboratory includes all of the required space in the highly collaborative Clark Center for Science and Engineering at Stanford, with state-of-the-art- research facilities, situated between the medical school and the engineering school and administrative support. The proposal's interface with Stanford 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. Controlling the closed lab environment with CO2 and temperature only, 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 he is a pioneer in terms of tools development. A third reviewer felt that the application was innovative, but impossible to understand. This reviewer was not enthusiastic, particularly because the PI is far along his 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 he can activate with light pulses 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 as one needs to stick a probe into the brain of the test animal, 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 3 R01s that have to do with neural stem cells. One discussant pointed out that the PI has extensive time constraints already, and the real question is whether he could reasonably devote enough time to the research proposed here. If the point is to bring physician-scientists into the stem cell field, he's already there. The motion to recommend that this application be moved to Tier 1 failed.