Ligand-Targeted Phage Particles as Tools for Progenitor Cell Tracking, Gene Delivery, and Biomarker Discovery
Tools and Technologies I
Human pluripotent stem cells offer the potential of a continually self-renewing source of human cells that can become any tissue or cell type needed to treat human degenerative diseases such as heart failure, diabetes, macular degeneration, MS, ALS, Alzheimer's, and Parkinson's as well as spinal chord injury and other wound healing. Until recently, human embryonic derived stem cells were the principal source of pluripotent cells. New techniques like reprogramming adult human cells point the way to more plentiful and cost-effective cells for research and development. However, our understanding of how to efficiently make specific cell types such as heart or skin cells on demand starting from pluripotent stem cells and how to produce sufficient clinical grade quantities of cells needed for therapy is currently quite limited and presents a significant challenge for researchers in the field of regenerative medicine. Our proposed research offers to directly address this issue by developing research tools and techniques for understanding and monitoring differentiation of human pluripotent stem cells. As pluripotent cells grow and change in a dish in the laboratory they progress to cells that are programmed to become various mature cells based on environment signals. We propose to create targeted bio-probes that will recognize and label only very specific populations of early precursor stem cells. The bio-probes will leave an indelible color coded mark on their target cells so that we can track the progression of these cells over time and determine what type of cells they become in response to interaction with other cells and other environmental cues. We will use time lapse photography to make movies showing color coded precursor cells as they grow, migrate, divide and change into functional mature body cells such as skin or beating heart cells. The ability to mark specific populations of progenitor cells will allow researchers to also isolate them to study their growth and development under various conditions aimed at producing desirable mature cell types from the progenitors. The new tools we propose to develop will help researchers distinguish one stem cell derivative from another so that well characterized cells can be prepared for disease therapies that are free of unwanted cell types.
Statement of Benefit to California:
About half of California's families have a child or adult who has suffered or will suffer from a serious medical conditions that could potentially be treated with stem cell therapies whether it be cell transplantation, the use of drugs tested on stem cell derived cells or drugs that affect endogenous stem cells to promote healing. The proposed research will result in new tools and technologies for identifying, isolating and tracking progenitor cells that are derived from pluripotent stem cells. Recent advances allow more ready access to pluripotent stem cells through reprogramming however there remains a critical need to understand how efficiently derive cells for therapy or drug testing. The innovative and versatile tools we propose to develop would allow California researchers to to tag lineage specific cells derived from any pluripotent stem cell line at any point in development. Multiple probes can be used to study two or more lineages at the same time. The technology can be used to target progenitor cells with a known biomarker interaction or to identify new surface markers or combinations that distinguish one type of precursor cell from another. These tools will make it easier for researchers to understand how to derive useful therapeutic cells from embryonic and other types of stem cells in the laboratory, to determine how to make and scale up pure cell populations that are free of unwanted cells and thus facilitate the translation of research to the development of cells and regenerative medicines for treating the patient population of California. An added benefit to California will be that the movies we create of differentiating ES cells will have important educational value for the general public's understanding of stem cells and their potential for curing disease. There are many other benefits to California researchers that will result from the development of the proposed progenitor targeting probe technology among them are improved assay development for differentiation agents, characterization of cells for transplant, and monitoring the fate of transplanted cells. Our proposed tools would therefore benefit the California economy by advancing the biomedical industry.
This is an application to develop reagents for targeting and tracking pluripotent stem cell-derived progenitor cells based on their surface marker profile. The first aim of the proposal is to develop a model tracking system using phage particles targeted to a specific growth hormone receptor. Phage particles will be tracked in three different ways, including by conjugation to quantum dots. The second aim is to apply the technology for biomarker identification by finding a pool of ligands that target human embryonic stem cell (hESC)-derived progenitors. The applicant will then use ligand-targeted phage tracking methods to screen the selected ligands, in order to identify new ligands that target surface biomarkers on keratinocyte progenitors. Time-lapse microscopy will be used to track and map targeted cell fate, migration and patterning. The research in this proposal addresses a fundamental bottleneck in the stem cell field, characterizing hESC derivatives and mapping their fate. However, reviewers commented that the tools proposed in this application are unlikely to be useful to the stem cell community. There are more robust strategies for tracking cells using genetic engineering that would be more advantageous for following cell fate. In addition, the preliminary data was not strong enough to suggest that the specific experiments in this proposal would yield interpretable results. The principal investigator and collaborators on this project were considered excellent, but there were concerns regarding the number of on-site personnel that would be available to conduct the work. The characterization of hESC derivatives and map of their fate is a fundamental issue in stem cell biology. Although this group will begin with a proof-of-concept in a limited system, the follow-up aims would help to refine the techniques for a wider use. If this method was effective for optimizing directed differentiation of hESCs, it could have an important impact. However, there is little biological rationale presented in the application to justify the use of phage technology compared to other approaches to track hESC-derived somatic cell populations. The ligand-directed phage delivery to cultured mammalian cells is a technique developed by the PI and one of the collaborators on the project. This delivery approach has been improved for in vivo application by other groups (which the applicants do not acknowledge), but reviewers commented that it still has not found wide acceptance and utility in biomedical research. Reviewers were uneasy about the outcome measures for this proposal, and therefore concerned about its feasibility. Three different methods of labeling targeted phage would be tested: fluorescent particles (quantum dots), quantitative measurement of phage DNA, and phage gene delivery of a fluorescent tag. The applicant presents some preliminary data suggesting that they can introduce these particles into hESCs, but the level of detection appears to be low. Reviewers commented that if only one of the phage-tracking methods works the time-lapse microscopy analysis might be compromised. In addition, reviewers commented that the application lacked preliminary data on the time-lapse analysis and the quantitative measurements of tracking cells from the progenitor stage through differentiation into a more mature cell type, nor does it contain any convincing explanation of how cell-cell interactions will be analyzed. Finally, it is not clear why the applicants chose to go after neural progenitors in Aim1 and keratinocyte progenitors in Aims 2 and 3. The principal investigator (PI) has an excellent record and experience in this area, and has been part of an initiative to apply phage display to biomedical research. The collaborators listed on the project are excellent, although more experienced with adult progenitors than hESCs. Reviewers expressed concern that the PI is relying on an assembly of team members from outside of the laboratory; the applicant institution will contribute one research associate to perform all the experiments with the PI. The budget seemed appropriate or a little high for a study largely based on phage display, a relatively inexpensive methodology. Overall, reviewers were not enthusiastic about this proposal. They felt that the technology was not particularly useful, and were concerned that the outcome measures had not been adequately thought out.