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RS1-00156-1: Designing monitoring systems for beta-cell differentiation from ES cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Type I diabetes (TID) patients suffer from insufficient or lack of insulin to regulate blood glucose levels. Due to loss of pancreatic beta-cells, the only treatment for TID patient is transplantation of donor islets. With the improved transplantation technique, it still requires 2-4 donor pancreas for each successful transplantation. The shortage of donor islets and inefficiency of beta-cell regeneration presented an urgent need for alternative sources of beta-cells. Embryonic stem (ES) cell-derived insulin producing cells are regarded as a possible means to overcome these limitations. However, difficulties in controlling the fate of ES cell differentiation have presented significant challenge for using ES cells to treat type I diabetes. Current protocols of ES cell differentiation to beta-cells rely on trying different combinations of medium factors. A lack of monitoring system has made the effort to improve the beta-cell proliferation protocol a blind search. In this grant proposal, we intend to design a monitoring system to monitor the differentiation process. This monitoring system is based on the physiological development of the pancreas. During pancreas development, several distinct stage with different cell markers have been identified to be critical. Using these markers as a monitoring guide will aid in our effort of improving beta-cell differentiation protocol. In this study, we propose to design fluorescent reporter markers to monitor the appearance and disappearance of these markers. The system designed here will be useful tools for future studies targeted at improving the current beta-cell differentiation protocol. A efficient and reliable beta-cell differentiation protocol is the critical step toward using ES cell based therapy for the treatment of type I diabtetes.

Statement of Benefit to California (provided by applicant)

The proposed study is an important step towards establishing a reliable, robust differentiation system for beta-cells. The percentage of population who has diabetes in California is among the top in the 50 states. Therefore, a treatment targeted at diabetes will be of significant benefit to the California citizens. For type I diabetes for whom transplantation is the only reliable treatment, an alternative treatment is urgently needed and is of significant benefit to the Californians.

Review

SYNOPSIS: The goal of the proposed research is to create a fluorescently tagged genetic reporter system in human embryonic stem cells (hESCs) that will be useful to monitor the directed differentiation of cultured hESCs toward the pancreatic beta-cell phenotype. Toward this goal, the Principal Investigator (PI) will derive separate hESC lines with GFP, YFP or RFP reporters for the expression of a number of genes which not only mark but also control pancreatic endocrine cell development. Three strategies for homologous recombination will be attempted in order until one is successful. The PI will test the veracity of expression of the reporters in a variety of controlled ways.

SIGNIFICANCE AND INNOVATION: The PI proposes to create and test reporter hESC lines for endodermal and pancreatic differentiation by creating several fluorescently tagged reporter gene constructs, introducing these into mouse and hESCs, and testing the constructs in vitro and in vivo. The approach is innovative and the research is potentially very significant. The generation of replacement beta-cells for transplantation therapy of diabetics is a major and highly worthy goal of stem cell technology. Progress has been limited for hESCs, in part due to lack of cell lines and reagents as are available for the equivalent mouse work. Engineered human stem cell lines with intrinsic reporter genes that can be used to monitor separate endocrine developmental stages during attempts to induce step-wise beta-cell differentiation in vitro would be an important technical advance.

STRENGTHS: A strength of this proposal is the plan to explore several recombination strategies to create intrinsic reporters in hESCs that would precisely mimic the expression of definitive markers for embryonic beta-cell development, from endoderm to the mature beta-cell. Although homologous recombination experiments with hESCs have been difficult, collaborators with extensive pioneering success with this strategy greatly enhance the chance for success.

A limitation of creating reporter lines in this manner is the lack of a direct assay for proper developmental expression of the engineered reporters. To overcome this, three different assays are proposed. In addition, assays other than using an intrinsic fluorescent reporter to follow the progress of hESC differentiation in vitro are described. But these do not have the advantage of being able to both assess the fraction of cells in the population at a particular developmental stage and use the potential for FACS enrichment of these, potentially minority cell populations, for next-step induction experiments.

The PI has good experience creating similar constructs to those proposed here and has worked with mouse ESCs. The preliminary data presented demonstrates that some of this work has begun in mouse ESCs. The PI recognizes the difference between mouse and human ESCs and realizes that there are substantial hurdles to be overcome in order to carry out the same work using hESC lines.

There are technical challenges. There are very few reports of successful attempts of homologous recombination in hESC lines and this proposal seems very ambitious in its aims. Although the PI will first test the constructs in mouse ESCs, there are a few significant differences between mouse and human ESCs and showing efficacy in mouse ESCs does not guarantee success in hESCs.

WEAKNESSES: Each of the assays proposed to verify correct reporting by the fluorescent proteins has intrinsic limitations. For example, creating equivalent reporters in mouse embryos is time consuming, involves re-creation of targeting vectors for homologous recombination with mouse gene sequences, is sensitive to any mouse-specific problems with the locus,is insufficient alone and would require analysis of the human reporters in a human background. Each problem needs careful consideration in advance.

Some of the markers that will be used to follow development have complexities that have not been considered and may confound some differentiation experiments. Other useful markers should be considered. Considering the complexities of homologous recombination in hESCs, the testing protocols and the pace of animal work, the timeline of 2 years is unrealistically short.

As written, this proposal is not eligible for NIH funding because of the specific hESC lines chosen. The only mention of why these lines were chosen is because the PI requires “hESCs that have not been grown on mouse feeders.” However, the lines chosen by the PI were derived and expanded on mouse feeders, very similar to the NIH-eligible hESC lines. A significant part of this work (that deals with mouse ESC lines) is eligible for NIH funding.

Feasibility: The PI only designates 5% of time to this project but is stated as being responsible for all experimental planning, data analysis and distribution, writing manuscripts, and training and directing technical staff. It is hard to imagine how all this will be accomplished with only a 5% commitment.

DISCUSSION: There was no further discussion beyond the comments by the reviewers.

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:

• None