To use human embryonic stem cells (hESC) for regenerative medicine, it is essential to develop methods to direct them to develop into specific cell types for clinical transplantation, such as pancreatic beta cells, nerve cells, hematopoietic stem cells, etc. Experimental research with hESC will seek to understand the mechanisms that control the processes by which hESC can be turned into specific cell types, and this knowledge can then be translated for the development of stem cell therapeutics. Techniques that allow new genes to be transferred into hESC will have many important experimental applications in studies of stem cell biology that will increase our knowledge of how stem cells develop into mature tissue cells. Gene transfer techniques may also have important clinical uses for the development of stem cell-based therapies. Stem cell therapies for patients with inherited disorders (e.g. sickle cell disease, cystic fibrosis, muscular dystrophy, immune deficiencies) will likely need a gene therapy component, so that the inherited gene defect can be corrected in the context of the patient's own genes (by moving the nucleus from the cell of a patient into hESC and correcting their genetic defect to produce “matching” and corrected tissue cells). Our central hypothesis is that gene transfer vectors capable of refined patterns of gene expression will provide enhanced capacity to manipulate hESC. We will develop novel gene delivery systems that can insert genes into hESC that will be expressed (”on”) specifically in different types of cells that develop from the hESC, such as cells along the stages of development from the embryo to pancreatic beta cells. The types of genes that will be studied include genes that can make the hESC grow and develop into specific cell types, or genes that produce a cell label (e.g. green fluorescent protein) to mark cells of specific types so that they can be isolated for studies or genes that can eliminate remaining hESC that have not fully developed into mature beta cells. By defining methods to effectively manipulate hESC, we will contribute both critical knowledge and experimental tools to the stem cell field. Moreover, the proposed studies will have direct clinical therapeutic applications for diabetes mellitus and other disorders that may be treated by stem cell therapies.
Statement of Benefit to California:
Development of methods for regenerative medicine using hESC will have wide-spread applications to improve the health and to provide novel, effective therapies for millions of Californians and tens of millions of people world-wide. Regenerative medicine may provide new treatments for diseases including diabetes mellitus, Parkinson’s disease, organ failure and injuries, inherited diseases and cancer and leukemia. The major challenge facing the field of regenerative medicine is to increase knowledge of the processes by which the mature cells of tissues (pancreas, brain, bone marrow, etc.) develop from stem cells, so that clinical approaches can be developed to produce cells suitable for transplantation. This Project will produce and apply novel tools for experimental studies of human embryonic stem cells (hESC) and for the development of clinical therapies using hESC. The central focus is on the development of gene delivery vectors that can transfer and express new genes in hESC, to influence their properties for research or for clinical purposes. These studies will help to advance the capacities for regenerative medicine. All scientific findings and biomedical materials produced from our studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the State of California.
SYNOPSIS: This proposal deals with the use of gene transfer methods to facilitate aspects of human embryonic stem cells (hESCs) to becoming beta islet cells for eventual use in treatment of diabetes by cellular administration. First, cells with be marked with lentiviral vectors. Second, cells will be expanded (via conditional c-myc expression) or eliminated (by selection against pluripotency gene). Third, genes thought to be important for islet cell differentiation will be introduced to promote differentiation. Technical features of the work include use of both lentiviral vectors and non-integrating lentiviral vectors and use of conditional systems with tet-regulation and ligand-induction (c-met).
IMPACT AND SIGNIFICANCE: The major goal of this proposal, i.e. to refine current stem cell therapy approaches by incorporating gene manipulation strategies, is attractive and logical. Successful completion of the described experiments could have very significant impact on the rate of development of the stem cell field towards generation of cells that are highly differentiated and capable of replacing damaged tissue. The principal innovation in this application rests with the gene transfer systems, particularly the non-integrating lentiviral vector and the ligand-inducing (cmet) system for conditional expression. Other concepts are not particularly novel, perhaps save for the use of c-myc expression to conditionally expand the population of cells.
QUALITY OF THE RESEARCH PLAN: This is a very well planned and prepared proposal. The quality of the research plan is generally strong and logical as it is centered primarily on the strength of the PI's laboratory, ie gene transfer technology. The march of experiments from marking and tracing hESCs to their expansion and ablation and finally to their differentiation is first rate. Each of the experimental aims seeks to address a specific translational goal or barrier via a hypothesis that can be experimentally tested, and the PI has already generated important and appropriate preliminary data. A question is whether the approaches outlined here will be competitive with those of other groups already heavily invested in the islet cell field (who are using similar strategies by in large).
STRENGTHS: A strength of the proposal is in the experience and expertise of the PI in lentiviral gene therapy. Additionally, the experimental plan is well designed and complete as it moves through its three specific aims designed ultimately to generate functional pancreatic islet beta cells through use of gene modification procedures involving the Oct-4, Sox17, Foxa2, PDX-1 and insulin genes. The PI addresses both selection of differentiated cells and elimination of primitive hESCs. The PI has good preliminary data on the use of promoters in the vector systems and has already successfully validated lentiviral gene therapy, and fluorescence tracking of lineage specification in hESCs. Furthermore, inducible c-myc experiments--to attempt to expand hESC-derived lineage-specific progenitors--is a very neat idea, and is a model for studying other genes in a similar fashion. Also, the technology could be useful to others and readily transferable.
WEAKNESSES: The strength of the group is in bone marrow transplantation and not in beta islet cell differentiation and it was not made clear how this approach is superior to that of other groups who are modifying conditions for in vitro differentiation of islet cells from hESCs. In parts of the proposal, the amount of experimental detail is very sketchy, so much so that no serious commentary can be made about feasibility of the experimental plan. An example of this appears at the end of Specific Aim #1 where a very brief treatment is given of the work to be performed by Drs. Chow and Asgharzadeh. Presentation of some preliminary work by these two collaborators would have enhanced the application.
For non-hematopoietic tissues, the PI does not spell out precisely how he will prove that a hESC-derived cell is, or is not, actually of a specific lineage or at a specific stage. Additionally, the proposal never really explicitly comes to grips with the possibility that the beta cell product that will be eventually derived may be unusable, i.e. that it might be close to an insulinoma with constitutive insulin release rather than a beta cell with glucose-responsive insulin release. This issue is raised again by mention in Specific Aim #2 that “cells modified to express a transferred proto-oncogene may not be clinically applicable”.
The Preliminary Data section illustrates in Figure 3 that murine islets do not express the InsPr(LA)-eGFP vector nearly as will as seen in MIN 6 cells. The PI states that he has performed work with human islets, but gives no preliminary data in this section. The transient nature of PDX-1 gene expression and lack of insulin gene expression discussed in the final paragraph of the Preliminary Results is concerning.
The proposal would benefit from involvement (collaboration) of additional experts in islet cell biology and with specific lineage biochemical and functional assay skills.
DISCUSSION: The PI has developed a gene transfer technology that the PI wants to use to make beta islet cells. The PI is the right person for the job given the significant experience with lentivirus transformation of stem cells. The vector approach is important in validating the in vivo potential and the notion of an on/off myc is a clever idea. This is a sound proposal based on a particular technology with good investigators, and while there are a couple of interesting aspects to the proposal, the work is largely derivative. There was a question as to the uniqueness of the approaches described and the preliminary data provided is “sketchy”. Scientifically speaking, the cells may not work because the constitutive release of insulin versus glucose-mediated release is not addressed. The applicant would benefit from having more expertise on the team with islet cell expertise since their own strength is in hematopoietic disease. The beta cell expert recruited doesn’t seem to have much data in this grant and his degree of involvment is unclear.