Human embryonic stem cells (hESCs) hold enormous promise for providing novel cellular therapies for neurological disorders such as Parkinson’s Disease and spinal cord injuries, for blood disorders such as leukemia, aplastic anemia, and sickle cell disease, for metabolic disorders such as diabetes, and for a long list of other diseases for which we have suboptimal or no therapies. A somewhat less appreciated fact is that hESCs can be used as experimental model systems to better understand normal human development and the mechanisms underlying various human diseases. Through such model systems, hESCs provide novel avenues to identify and develop non-cellular therapies. Given all this, it is easy to understand why there is so much excitement surrounding these cells.
Central to the ultimate success of cell-based therapies using hESCs is the ability to generate a specific cell of interest from hESCs, and to do so in a regulated, predictable, and reproducible fashion. Many of the methods currently employed to generate specific cell types use poorly defined conditions that often include the use of ‘other’ cells in what is called a ‘co-culture’ system. This is certainly true for developing blood cells from hESCs. To systematically screen well-controlled conditions to identify specific methods that will direct the desired cellular development, we need to have what are called reporter cell lines that identify themselves once they have achieved the desired developmental destination. For example, if we had a cell that would express a fluorescent protein only if it developed into a hematopoietic stem cell, then we could perform massive screening of culture conditions, including high-throughput screens of existing compound libraries, to identify molecules that direct hESCs to generate hematopoietic cells. Such lead compounds could then be used to study the cellular mechanisms that control the desired development, and be used to establish the desired goal of regulated, predictable, and reproducible development of therapeutic cells.
In this proposal we seek to develop technologies that will be globally useful for generating such reporter cell lines for any cell type of interest. The technologies will also be directly applicable for establishing hESC lines that can be used to generate model systems for various human diseases, significantly advancing the ability to develop non-cellular therapies for human disease and to test for possible human toxicity prior to the introduction of drugs currently under development. The technology we wish to develop is called gene targeting and it has proven invaluable over the past 20 years in studies involving mouse embryonic stem cells. We have every reason to believe that we can develop approaches using hESCs and achieve a similarly robust benefit.
Statement of Benefit to California:
This proposal seeks to develop efficient and broadly useful methodologies for performing gene targeting in hESCs with an emphasis on applying this technology to the development of a variety of lineage-specific reporter cell lines that will serves as the critical reagent needed to advance the development of hESC-based cellular therapeutics. Benefits to the State of California include:
1. The methodologies developed will be made immediately available through online protocols to all stem cell scientists in California.
2. Developmentally-specific reporter hESC lines generated by our technologies will significantly advance the effort of many California investigators as they seek to develop safe cell-based therapies for a myriad of medical disorders.
3. Developmentally-specific reporter hESC lines generated by our technologies will be available to other investigators to develop non cell-based therapeutic agents to treat a variety of disorders.
4. This work will provide new jobs at [REDACTED] and hopefully will spawn additional research activities in the California academic and private sectors, all of which promotes the stateís economy.
5. To the extent that patents or licensing agreements can be developed from the technology we advance, citizens of California will see a direct financial return on their investment in stem cell research.
The applicant proposes to improve methods for gene targeting (homologous recombination) in human embryonic stem cells (hESC). In aim 1, targeting vectors will be generated using bacterial artificial chromosomes (BAC) and targeting efficiency will be optimized. The applicant proposes to insert reporter genes into 5 loci, whose expression patterns are relevant for hematopoietic development. In aim 2, the applicant will establish the recombinase-mediated cassette exchange (RMCE) method to enable a second modification of a previously targeted locus. Finally, the resulting cell lines will be analyzed to evaluate the efficiency of the targeting methodology and to monitor the expression of the reporters during hESC differentiation into blood cells.
The proposed technology addresses a significant roadblock in stem cell science: the lack of robust and efficient methods for gene modification. As a result of this deficiency, it has not been possible to investigate many fundamental aspects of hESC biology. If successful, the proposed technology would represent an extremely valuable and important tool for generating modified hESCs including reporter cell lines, homozygous loss-of-function lines and cell lines that could serve as models for specific diseases. The mechanistic understandings that could emerge from such tools would be of substantial utility for regenerative medicine.
Reviewers judged the research plan to be systematic, realistic, and well-written and preliminary studies to be encouraging, but they identified several issues that diminished their enthusiasm. In particular, a reviewer expressed concern about required controls for aberrant recombination events, and felt that the limitation of the proposed studies to NIH registered hESC lines was inadequate. Another reviewer cautioned that the use of BACs for homologous recombination can lead to artifacts. Additionally, reviewers criticized that the applicants did not provide a compelling reason for their focus on hematopoietic differentiation, and did not indicate how the proposed strategy would significantly move the field forward, either for driving hESC differentiation into specific cell types or for elucidating the principles that underlie differentiation processes.
The research team was viewed as highly qualified for the proposed study. The principal investigator (PI) has expertise in areas of gene targeting and immunology. The PI presents a good publication record, however he/she is not the senior author on many of the listed papers. The co-investigator has extensive experience in studying blood cell differentiation. Despite these credentials, the reviewers expressed some concern that the 10% commitment of the PI might not be sufficient for an effort of the proposed scope and magnitude.