Tools and Technologies I
Embryonic stem cells have the ability to become any cell in the body and are capable of continuously renewing themselves. Therefore they have the potential to provide an unlimited source of replacement cells for treating many medical conditions where cells have been lost to injury or to degenerative processes associated with disease and aging such as diabetes, heart disease, Parkinson’s, Alzheimer’s, multiple sclerosis, and macular degeneration for example. However, a safe way of storing human embryonic cells and cells that are made from them is needed so that replacement cells are available for physicians to treat their patients and for researchers to have a standardized source of cells for developing therapeutic treatments. Freezing to liquid nitrogen temperatures is an effective way of preserving cells for future use and has traditionally been used by the scientific and medical community. For example, cancer cells, sperm cells, skin cells and even early embryos are routinely frozen to store samples for future use and can be safely used years or decades later provided the appropriate freezing solutions and protocols are followed. Embryonic stem cells can be frozen and stored using traditional methods but they typically do not survive well and there have been few studies to optimize and standardize freezing methods that would allow long term storage without altering the properties of the cells while at the same time preserving their viability. Moreover, scientists recently have developed ways to convert stem cells to the multitude of cells that appear during embryonic development. These embryonic precursor cell lines are intermediate cells having properties between embryonic cells and the fully developed cells that make up our body tissues. They represent a rich source of cells for researchers to study embryogenesis and to devise ways to make any desired body replacement cell starting from embryonic stem cells. However, more effective methods are also needed to store these cells so that a cell bank can be made available for stem cell researchers to use on demand. We propose to develop new and improved methods of freezing stem cells and embryonic precursor cells. We will test novel freezing methods and new formulations containing a medically approved blood substitute and non-traditional preservatives that have already shown promising results for freezing tissues in experimental animals. We will optimize freezing of both human embryonic stem cells and embryonic precursor cells that we will derive from embryonic stem cells in our laboratories. The resulting methods and formulations will help researchers and patients by providing a way to safely and effectively store embryonic cells for future use in the laboratory and in the clinic.
Statement of Benefit to California:
With the passing of proposition 71 in 2004, California is destined to become the nation’s leading center for regenerative medicine. However, before cell replacement and other regenerative treatments can come to the bedside there needs to be a solid foundation of core technologies on which to build regenerative therapies so that they can become routine medical practices. One of these core technologies is known as cryogenics or the ability to safely freeze biological material for future use. We propose to develop new and improved methods of freezing stem cells and embryonic precursor cells. We will test novel freezing methods and new formulations containing a medically approved blood substitute and non-traditional preservatives that have already shown promising results for freezing tissues in experimental animals. We will optimize freezing of both human embryonic stem cells and embryonic precursor cells that we will derive from embryonic stem cells in our laboratories. The resulting methods and formulations will benefit California researchers and patients by providing a way to safely and effectively store embryonic cells for future use in the laboratory and in the clinic.
The central goal of this proposal is to optimize and standardize tools and protocols for cryoprotecting human embryonic stem cell (hESC) lines and their derivatives. The principal focus will be on the use of an FDA approved serum replacement agent that has been successfully used to preserve other cell types and tissues. In the first aim, the applicants will optimize conditions for use of this agent in preserving hESC lines. Various combinations of the agent and assorted other factors will be tested, and the protected cells will be thawed and evaluated by a wide variety of biological assays. The applicants also intend to generate a library of embryonic progenitor cells (hEPs) to optimize and standardize conditions for cryoprotection of the resulting lines. Reviewers recognized the overall importance of cryopreservation but were unconvinced that the technology proposed here would significantly impact the field. The proposal was ambitious but lacked many details, including a discussion of potential difficulties and possible alternative approaches. There were many technical and conceptual errors, and the fundamental controls for validation were not included. Finally, reviewers noted a lack of specific expertise by the research team in the area of this effort. In terms of impact, the proposed tools do not directly address a fundamental scientific issue in stem cell biology, but they do address an important need that could accelerate the pace of discovery, development, and translation to the clinic. If successful, better cryopreservation methods as well as improved thawing techniques could be extremely useful to scientists in a diversity of biological disciplines. In spite of this potential, reviewers noted that the proposed tools lacked novelty and thus were not likely to produce improvements at the level that would be needed to advance the field. In terms of feasibility, reviewers agreed that the investigators proposed a well considered and appropriate list of parameters and readouts for evaluation. However, the applicants provided very little consideration for potential difficulties that could arise, and no alternative methods were proposed in the event of failure. In addition to this, there were multiple deficiencies in the research plan. Of greatest concern to the reviewers was the fact that only one hESC line was chosen for testing. In addition, no functionality assays were proposed, and the rationale for focusing on the agent of interest was never fully discussed. Several technical details were also of concern. For instance, the use of Trypan Blue for assessing cell viability, which is a notoriously unreliable and outdated method. Reviewers also questioned the small number of concentrations of the agent that were assayed for efficacy. Many of the experimental approaches were sparsely detailed, which made it impossible for the reviewers to evaluate their merit. The applicants were considered qualified to perform most of the work in this proposal, but reviewers expressed concern that only one person on the team had any experience with cryoprotection, and this was likely from an engineering perspective rather than a biological one. It was suggested that the team would benefit from some additional hands-on proficiency with hESC culture and cell biology.