Embryoid body (EB) formation is a potent method for differentiating human embryonic stem cells (hESCs). EBs formed on commercially available low attachment plates, however, form asynchronously, tend to vary in size and aggregate, making the differentiation process heterogeneous and uncontrollable. The overall goal of this research project is to establish the conditions required to regularize the formation of EBs for multiple hESC lines, thereby obtaining controlled differentiation to the lineages or cells of interest. Our approach is based on a nano/micro technology platform in which new materials are integrated with microfluidic devices and biomarker sensors. The platform we develop from these components will lead to robust, programmable methods for growing EBs that are far superior to current EB formation protocols. We plan to use this technology platform in combination with a feedback control scheme of the type used to guide complex engineering systems to improve differentiation efficiency. The project consists of two technical aims: 1. Develop a nano/micro technology platform that uses non-adherent surfaces, micromachined three-dimensional architectures and microfluidic chemical delivery actuators to provide a controlled environment for massively parallel growth of uniformly-sized EBs. 2. Apply this technology platform to endoderm differentiation with the objective of improving the differentiation efficiency and yield of endoderm lineage, especially insulin-secreting pancreatic cells. The development of a robust technology platform with programmable microfluidics and feedback control that removes the variability of EB size and shape offers the promise of achieving high throughput, scalable systems for therapeutic applications.
Statement of Benefit to California:
Human Embryonic Stem Cells (hESCs) are promising candidates for tissue replacement therapies for several debilitating pathologies including diabetes, leukemia, and heart diseases, as well as for advancing regenerative medicine research. The formation of spherical aggregates known as embryoid bodies (EBs) is a critical intermediate in the differentiation of hESC into specific cell types needed for these therapies. Unfortunately, current EB processing methods provide low yield, inconsistent sizes, and unpredictable cell types. Achieving uniformly sized EBs with synchronized differentiation kinetics requires new tools to manage and control the environment of hESCs during the EB development stage. In this project, our stem cell researcher team leverages expertise in material engineering and nanotechnology to create robust, repeatable and programmable processing methods that will guide EB formation, growth, and differentiation. These new methods will be superior to current protocols because of the novel engineering techniques capable of monitoring and manipulating at the level of single cell and single embryoid body. This project will yield technology which will remove the variability of EB size and shape and offer the promise of achieving high throughput, scalable systems for therapeutic applications. This work will be of great importance in furthering the state of California’s interest in stem cell biology and regenerative medicine. Specifically, in the near term, the success of our strategy would lead to advances in differentiating hESCs toward insulin producing cells derived from the endoderm. Our work will provide valuable insight into the signaling cues responsible for this differentiation process. Ultimately, the ability to cultivate these cells in a controlled and scalable process would revolutionize the management of patients with diabetes mellitus which is expected to afflict 340 million US citizens by the year 2030. Secondly, the growth of industries interested in the development of hESC based therapies is expected to rise substantially in California given the state’s efforts to promote stem cell research. Translating this work to production scales is necessary and will be enabled through the development of the research tools that we propose.
SYNOPSIS: This proposal seeks to develop a platform for the production of large quantities of embryoid bodies of consistent size suitable for subsequent efficient differentiation toward a desired, in this case endodermal, cell fate. Aim 1 tests nano/micro technology platforms with microcavity arrays of two different designs for their ability to produce massively parallel growth of embryoid bodies from any of a number of hESC lines. These platforms will have non-adherent surfaces, three-dimensional architecture, and microfluidic controlled chemical delivery systems. Aim 2 extends the use of the platform developed in Aim 1 and optimizes the culture conditions that direct the embryoid bodies toward an endodermal cell fate, with the eventual goal of producing insulin-synthesizing cells in a scale useful for human therapy. A built-in feedback control system will minimize the number of experiments required to determine the optimal culture conditions. SIGNIFICANCE AND INNOVATION: Enhanced production of endoderm cells for direct therapeutic use as a source of precursor cells to produce more differentiated insulin-secreting cells would be an important advance. This proposal will develop methods to obtain uniform size embryoid bodies (EBs) by controlling the size of hES cell colonies as they form or by sorting hES colonies already formed according to their size. Once the colonies are allowed to form EBs, they can be sorted again by size if desired. Since reliable and reproducible EB formation is critical for reliable and reproducible hES cell differentiation, and since EBs are microscale in size, it makes a lot of sense to develop microtechnologies for this purpose. Use of microfluidics also adds versatility in how subsequent culture and differentiation of EBs may be performed. Production of insulin producing cells is an important topic relevant to treatment of diabetes. The concept of using microwells to create uniform size hES colonies has been described previously but the idea of using deterministic lateral displacement to sort hES colonies or EBs by size is new. As the investigators acknowledge, reliably and conveniently loading cells into wells, then releasing the colonies once formed may be challenging. Since hES colonies are not always very spherical, sometimes even pancake-like, it is also a bit unclear how well the proposed sorter will work. Sorting EBs by size seems like it should work well. STRENGTHS: Applies state of the art micro/nanotechnology, with feedback control to maintain optimal conditions for culturing embryoid bodies biased toward endodermal development. Shown the technical ability to grow embryoid bodies from human ES cells of uniform size in microwells. The research team comprises distinguished faculty researchers with expertise directly related to the proposed research from materials chemistry for fabricating the nano-platforms, microfluidics, and stem cell biology, both human and mouse. No obvious advantage to non-approved hSC lines. The investigators are all very strong in their respective areas and complement each other well. They have preliminary results that show collaboration. The criteria for success in specific aim 1 is clear (formation of uniform size EBs and synchronized differentiation). EB culture and differentiation using microtechnology seems to be a good match in size, in technological compatibility, and in biological need. WWEAKNESSES: The utility of embryoid bodies as an effective source of differentiated cells of uniform properties and therapeutic potential is likely limited in the long term. This is due, in part, to the complex signaling interactions between different developmental foci that cannot all be monitored, let alone controlled. Limited insights into normal endodermal development and choice of markers may limit the progress of the proposed research. The plans and criteria for success in aim 2 seem less clear than for aim 1. The investigators note that their method will greatly reduce the number of experiments needed to realize optimal culture conditions but the exact methods, strategy, starting point, and readout/criteria for success is vague. The definition of success and explanation of the likelihood of achieving success requires more explanation. Use of microwells for hES colony size formation is not new. On the other hand, methods for efficient seeding of cells into wells and removal of cells from wells when needed is vague. DISCUSSION: Embryoid bodies were generally regarded as not useful as a source of cells for endoderm differentiation to diabetes cells due to the large number of uncontrollable directions that they can take and the difficulties in monitoring in real time. Attempts to follow markers won't necessarily detect the type of changes that the applicant wants to monitor. Reviewer 2 was concerned about the lack of expertise with embryoid bodies, and stated that aim 2 was not clear. Embryoid body sorting was predicted to have many pitfalls. The proposal was regarded as somewhat convincing but marked by flaws.