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CL1-00502-1: Center for hESC Research

Laboratory Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

The therapeutic use of stem cells in regenerative medicine will require the ability to control stem cell expansion and differentiation into specific tissue types, such as pancreatic ?-cells, heart tissue, bone or specific neuronal lineages. We have taken a chemical approach toward this problem in which large collections of synthetic small molecules are being screened in cell-based assays to identify drug-like molecules that control stem cell processes. Preliminary experiments in our institute have demonstrated that we can identify molecules that control the self-renewal and directed differentiation of murine embryonic stem cells. The characterization of the biological mechanisms of the molecules has also provided new insights into the underlying biology of stem cells. We now propose to extend these studies to hESC lines not eligible for federal funding, for which our research activities have been restricted to date. In addition, such lines may be better suited for specific applications, including the use of small molecules to derive specific cell lineages and investigate ES derived cell-based models of genetic disease. To this end, we would like to establish a human embryonic stem cell core facility. This facility will house the necessary equipment to genetically manipulate and culture hESCs on a large scale for a variety of studies including cell-based screens of small molecule libraries, as well as screens of arrayed genomic cDNA and siRNA libraries. We anticipate that this facility will serve our faculty as well as other labs that would like to collaboratively exploit this chemical approach to the study and manipulation of stem cells.

Statement of Benefit to California (provided by applicant)

Historically, small molecules have been more useful than genetic approaches in the treatment of human disease. However, much of our ability to control embryonic stem cell self-renewal and directed differentiation currently involves genetic manipulation of these cells or complex mixtures of protein factors. The demonstration that one can systematically identify, optimize and characterize the mechanism of action of small drug-like molecules that selectively control stem cell biology both in vitro and in vivo will: (1) provide important tools to manipulate stem cells in the lab; (2) provide new insights into the complex biology that regulates stem cell differentiation; and (3) provide an important first step which may ultimately lead to drugs that facilitate the clinical application of stem cells.

Scientific Review Summary of Part One Application

SHARED LABORATORY

SYNOPSIS OF PROPOSAL: This is an application for a Shared Research Laboratory (SRL)focused on the identification of synthetic small molecules that regulate the self-renewal and lineage-specific differentiation of human embryonic stem cells (hESCs). The plan is that the laboratory will include equipment needed to culture non-presidential hESC lines on a large scale for cell-based high throughput screening (HTS) of small molecules and for screens of arrayed genomic cDNA and siRNA libraries. No “safe haven” (e.g., free of federal funds) facility currently exists at this institution for non-presidential hESC lines. The 1000 sq ft facility will be housed at the institution and will be directed by a program director (PD)who has an established track record in HTS. The shared use laboratory will serve as a core for 18 principal investigators (PIs) from the host institution and 12 PIs from other investigators in the region as part of a consortium.

QUALITY AND IMPACT OF THE SCIENCE: The PD is clearly a leader in the area of HTS and was one of the first to propose applications in stem cells including hESCs. Many of these studies were carried out in collaboration with a former student of the PD who is now an independent investigator at the institution. This individual has been the leading person advancing the application of HTS to hESC and s/he has developed novel culture protocols for the maintenance of hESCs. The principal strength of this application is the unique expertise in HTS of the PD and the outstanding set of ~30 potential users who could benefit from the facility from the other institutions in the area. These investigators include many leaders in the field of stem cell/hESC biology. Another strong aspect of the application is the integration of the facility within the community where a consortium for regenerative medicine has been established.

There is a very strong focus on HTS assays. While this is an important research area, the laboratory proposed seems to be small and too specialized to serve the community as a shared hESC resource. A lab with a single hood and single centrifuge will not allow sufficient access to the 30 investigators listed on the application * (see STAFF NOTE below). Running a single HTS assay for 100,000 compounds can easily fill an entire incubator with hESC culture plates. Clearly this facility will not suffice as a core for all the various hESC HTS assays from the regional community. It seems likely that the use of the SRL will be limited primarily to the PD’s laboratory groups. The cell lines proposed for use in the facility are several lines from WiCell and the HUES lines 1-13 from Doug Melton’s group; a rationale for selecting these specific lines was not provided in the application.

A reviewer points out that although the PD is a respected scientist, neither the quality nor quantity of hESC work to be conducted in the SRL is made clear in the application. In addition, the host institution is proposing a new hESC facility and has not invested in building one previously.

APPROPRIATENESS OF SPACE AND EQUIPMENT TO SCOPE OF PLAN: A laboratory of 1000 sq ft is proposed. No dedicated hESC laboratory is currently available at this institution that would allow work with non-NIH approved cell lines. Nearly all the costs for equipment will go into a high content confocal spinning disk plate scanner that allows 4-color imaging and automation. The application includes purchase of only a single hood and centrifuge *(see STAFF NOTE below) which, according to the reviewers, would be highly inadequate for the proposed user base. The listed personnel also seem inadequate as there is no PhD-level manager who would run the facility, and the PD will contribute only 5% effort; this is considered inadequate. The PD has little direct hESC experience, although s/he is clearly a world leader in the area of HTS assays. The technical staff working in the facility is listed as “to-be-named”. Overall, this plan is viewed as inadequate for the proposed large user base.

QUALITY OF MANAGEMENT PLAN: A reviewer believes that the research and management plans are not well-developed and that the procedures and goals are not described in sufficient detail. It is unclear from the proposal who will be running the facility as there is no technical staff listed beyond the two technician positions that are listed as to-be-named and the 5% effort of the PD. The research associates will “routinely maintain hESC cultures and grow hESCs on a large scale for chemical and genomic screens and molecular profiling. They will also assist in carrying out the screens (plating compounds, imaging, etc.), as well as carry out functional studies on hESCs”. The reviewer did not consider this a sufficiently detailed management plan.

Another reviewer advises that it would be critical to identify a PhD level person with hESC experience to run the day-to-day operations of the facility. Developing HTS assays for hESCs is rather complex and time consuming and a PhD level scientist should be involved as an interface between the user and SRL leadership to help in assay development. One solution may be to have a stronger contribution from staff trained in the laboratory of the PD’s former student, as s/he seems to have the most hESC expertise within the users group at this institution.

The PD is a molecular biologist, not a stem cell biologist steeped in the latest technology of growing and manipulating hESCs. S/he is director of a research foundation and has founded a number of companies; however, it is not clear that these experiences necessarily support the ability to direct a shared laboratory facility dedicated to serving multiple scientists. The proposed shared research laboratory plan seems, according to one reviewer, to be designed * (see STAFF NOTE below) primarily to serve a few core users rather than providing a starting point for other researchers at the host institution and surrounding institutions who want to develop hESC technology in the context of HTS.

There also are no plans described for how HTS costs will be managed. The costs for running multiple high throughput chemical and genetic screens as proposed will clearly run beyond the listed budget. Will these costs be directly charged to users? Which screens will be subsidized by the supplies budget and – if so – how much?

No support letters are provided from the potential user base but a generic letter from the regional consortium is attached. Access to the facility will require a formal proposal from the host institution and members of neighboring institutions. There is no discussion about hESC training beyond fulfilling the regulatory requirements (biosafety etc.).

DISCUSSION: The discussion acknowledged the strength of the PD as a leader in his/her field and the uniqueness of the high throughput chemical and functional genomics screening approach amongst the members of the regional consortium and the value of this approach for stem cell research. A reviewer pointed out that it would be a tremendous advantage to develop these tools to drive hESCs along specific lineages. Thus far, their work has only been done using mouse ESCs and adult stem cells; there currently is not such a resource as an hESC laboratory available although there is a need. A discussion centered on the weakness of the management plan as presented in the application, as well as the perception by the Grants Working Group that this proposal seemed intended to fund a core lab for screening, not for hESC work as a shared research facility because funds were requested for only a single hood and centrifuge * (see STAFF NOTE below). The lack of hESC experience for the PD was considered a real negative especially since the technical staff included was to-be-named. A discussant pointed out that working with hESC and developing high throughput screens are not trivial and likely will require PhD level expertise for implementation, yet that is not the level of personnel contemplated and the PD’s 5% time is not likely to be adequate to provide support to the ~ 30 investigators. According to another discussant, their plan is to set up an infrastructure which is needed for hESC HTS approaches. The Grants Working Group generally agreed that the final score should reflect the relatively weak management plan for a shared hESC laboratory.

PROGRAMMATIC DISCUSSION: A motion was made to recommend this Shared Research Laboratory application for funding. From the perspective of science quality, the PD is extremely qualified in HTS. There is no overlap with collaborating institutions on the proposed SRL core platform. The Grants Working Group agreed that the PD is a world class expert in her/his field and that this facility would provide a unique resource but there was not full agreement during discussion about whether or not the application was suitable for funding given the purpose of the RFA to provide a SRL. One discussant did not view the value of the facility in the context of immediate institutional environment, rather as an opportunity to fund a potential state-wide resource headed by one of the top experts in the world. This advantage was counterbalanced by the inadequate size of the cell culture facility *(see STAFF NOTE below) and the inadequate management plan, limiting its utility as a shared resource.

Three conditions were added as a friendly amendment to a motion to fund:

1. hire 1-2 management people with proper SRL management experience to meet the demand;
2. require a management plan that assures access to multiple investigators and sets priorities, and
3. perform an annual review to confirm that the facility is serving the region with this shared resource. An additional comment was made that because the facility as proposed is inadequate to support such a broad use, the facility itself would have to be re-designed * (see STAFF NOTE below). The motion to recommend this Shared Research Laboratory application for funding, as amended with conditions, failed.

*STAFF NOTE: The equipment list on Part Two of this application was not the same as the equipment list on Part One. The reviewers on the Grants Review Working Group did not have access to the equipment list on Part Two of the application at the time of the review.

The following Grants 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:

• Lansing, Sherry