Development of a 3D ESC Culturing Process: Considerations on Stem Cell Fate Decisions Induced by Shear Stress Stimulation (SISSi)
Tools and Technologies I
In recent years the wide-spanning applications of stem cells in biomedical research have been commonly acknowledged. The primary goal of this proposal is to boost stem cell research one level up towards clinical application. Although excellent progress has been made with regard to understanding stem cell specification into tissue-specific cells and also the mechanisms of their self-renewal, major hurdles on the way to the clinic still exist. One of them – the quality controlled manufacturing of expanding cultures of pluripotent stem cells in a FDA conform manner, will be addressed in this proposal. Normally, stem cells are cultured in an adherent manner in culture flasks. Developing manufacturing protocols requires taking the cells out of this 2D environment into suspension culture vessels, as only those allow controlling culture parameters, such as pH or oxygen levels. As stem cell fate is not only influenced by chemicals or growth factors, but also mechanical stimuli, we first have to understand how the novel 3D microenvironment affects the stem cell population. It is possible that such extracellular cues may be translated intracellularly to activate genes that control stem cell maintenance or differentiation, which may preclude suspension culture per se. This proposal therefore aims to identify the effects of physical forces caused by shear stress in a suspension microenvironment on stem cell features. Ultimately, we will be able to devise novel media formulations that contain soluble modulators of such intracellular pathways to counteract shear effects should they negatively influence stem cell behavior. The success of our approach should create a platform on which stem cell treatment of degenerative diseases can build. The use of such manufactured cells would help to reduce the economic burden on the Californian healthcare system associated with conventional medical treatment options as it will allow stem cell therapies to progress one step closer to the clinic. Furthermore, SISSi will potentially create patents, which remain within the state of California and may launch a new product pipeline for stem cell research and therapy.
Statement of Benefit to California:
During the last few years, the profound implications of pluripotent stem cells for biomedicine have been widely recognized. Given the recent advances in many research fields, we are now in a position to move the frontier of stem cell research to the next level towards clinical application – this is the primary goal of SISSi. Understanding cell fate decisions will in turn inform the design of strategies to identify and validate scale-up culture processes and GMP-grade manufacturing protocols. The research proposed in this application will provide tangible benefits to Californians. The success of the approach of SISSi should create a platform on which stem cell treatment of all kinds of degenerative diseases can build. Spinal cord injury, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease and osteoporosis are among those diseases for which the concept of replacing destroyed or dysfunctional cells in the brain, spinal cord or bone is a practical goal. In considering the steps towars realizing these impacts, the stem cells will need to be differentiated or otherwise modified before they can be used clinically to prevent tumor formation or immune rejection. Overall however, the use of such manufactured cells would help to reduce the economic burden on the Californian healthcare system associated with conventional reparative options. SISSi covers aspects of high quality applied research and timely applications to real-world biomedical problems. Therefore, the impact of SISSi is four-fold: it will make important contributions or SCIENTIFIC IMPACT, regarding the definition of clinical-quality stem cell expansion procedures. SISSi achieves this by creating STRUCTURAL and MULTIDISCIPLINARY SYNERGIES inside the team and the outside world. It will create novel Intellectual Property, which will BOOST THE INDUSTRY SECTOR and facilitate transltion into the clinic by setting Californian STANDARDS for the clinical application of stem cells. The intellectual property associated with these advances will be protected with the assistance from the [REDACTED] office of technology transfer. Pursuant to federal statute (Bayh-Dole Act), the organization will grant non-exclusive use rights to the Government. If the results are of significant impact, industrial partnerships will be developed with established companies in tissue engineering and biomedical product development. The funding of this proposal may therefore launch a new product pipeline for sem cell research and therapy in California. Crucially, however, the proposed research program will significantly benefit California by training highly qualified personnel for future careers in academic research or the biomedical industry (i.e. Genentech). These trained professionals will have the skills to develop new therapeutic strategies in a broad range of applications, improving patient quality of life and positively impacting the Californian healthcare system.
This proposal focuses on the translational bottleneck of scale-up of human embryonic stem cell (hESC) cultures, focusing on the effects of shear forces on ESC behavior in a good manufacturing practices-compliant suspension bioreactor. Currently, achievable levels of scale-up of hESC-derived cells for clinical use are limited by the necessity to grow cells at early stages of differentiation on a matrix (adherent cultures). The applicant proposes to optimize conditions in a bioreactor, first using murine ESC and then using a non-NIH registry line that has the property of being easily passaged by trypsinization. A variety of end-points of ESC biology will be evaluated as a function of an engineered microenvironment. The applicant will focus on a few signaling pathways that are known to control stem cell fate, and examine their regulation as a function of shear stress in a suspension culture system. The studies described have the potential to elucidate mechanism of aggregate ESC behavior in response to mechanical cues. As such, the work may have some real impact on overcoming how the cytoskeleton and nuclear matrix are affected by agitation, and reviewers thought that there was some potential for more general impact on the bottleneck of cell manufacturing scale-up. In general reviewers were enthusiastic about the experimental plan, recognizing that shear forces have not been studied much in ESC and are important, but thought that the proposal would have been strengthened by attention to alternative approaches of scale-up. The principal investigator (PI) proposes to do a large (“prodigious”) amount of work but the experiments were felt to be relatively straightforward. One concern is that the experiments, as designed, will lead to a limited understanding of the conditions necessary for scale-up, since this proposal is limited to very defined conditions of the stirred tank system in which the cells will be cultured. The more general underlying premise that shear is an important variable in control of cultured stem cells is not really addressed in a systematic or direct, quantitative way. Reviewers suggested that the comparison of several different stirrer tank type systems might yield much more generalizable information. Further, the system that will be used does not necessarily represent a major technological advance in the field, and comparisons with other systems would make it more likely to lead to development of novel, alternative cell expansion technologies. Although the PI will likely generate information on an important signaling pathway’s regulation in response to mechanical cues, the experiments that would really close the loop, i.e., inhibitor assays of the pathways uncovered, are not well described. In addition reviewers voiced concerns that the preliminary data did not yet strongly support the focus on one particular signaling pathway, as proposed. The PI holds an academic appointment in California and an institute appointment outside the country, and has made a recognized contribution to describing differentiation of ESCs into osteoblasts. Reviewers found the biology in the proposal strong, but felt that incorporation of more engineering into the design would help the applicant’s work.