The traditional tools used today to culture human embryonic stem cells (hESCs) have led to significant hope, but limit their full potential. Newer technologies that could make cultures more consistent, easier to optimize, and healthier would be tremendous boons not only to basic research, but also for drug testing, diagnostic tool development, and clinical therapies across the field. An emerging technology with this potential is microfluidics. Microfluidics employs the concepts and engineering used to make the electronic chips in our televisions and computers, but instead applies them to make miniaturized devices for controlling fluids. Together with innovations developed by our research team, microfluidics provides opportunities for improving hESC cultures that would be impossible otherwise. Our team has been able to use microfluidics to culture neural stem cells, a project that now has ongoing federal and state funding. In the course of this project, we have developed new and highly versatile microfluidic devices that are simply added onto traditional cultures, and a new method for identifying dying cells in live cultures. These microfluidic and imaging tools give us the opportunity to make hESC cultures more consistent, easier to optimize, and healthier. These are the goals of our proposal.
Statement of Benefit to California:
The goal of this project is to use a new technology, known as microfluidics, to improve human embryonic stem cell (hESC) cultures in multiple ways. The general improvements we hope to achieve (making hESC cultures more consistent, easier to optimize, and healthier) should have relevance and applications across the entire hESC field. Thus, if successful, this project should have many benefits to the State of California and its citizens, including to potential consumers, pharmaceutical companies, basic scientists, and others working on diagnostic tools and therapies.
SYNOPSIS: The long-term objective of this proposal is to use microfluidics to optimize human embryonic stem cells (hESCs)cultures. Microfluidics devices can generate single and combinatorial concentration gradients at will, and create a stable microenvironment for cell growth. The PI proposes to apply microfluidic and optical imaging tools to optimize single hESC survival. This project aims to identify concentrations of different neurotrophins to optimize hESC survival, alone and in combination with other agents such as BMPs. INNOVATION & SIGNIFICANCE: The innovation in this proposal is the application of the investigators' microfluidic gradient generation devices to hESC culture optimization. STRENGTHS: There are several strengths to this proposal. The collaboration with Dr. Donovan, who recently identified the role of neurotrophins in hESC self-renewal is one. The team of Monuki and Jeon has a proven track record and know how to work together. The microfluidic gradient generation system is mature and fairly simple to use, and the porting of it to polystyrene substrates is a significant advance. Finally, the microfluidic device uses perfusion, which may result in better control over the microenvironment. WEAKNESSES: There are several weaknesses that moderate enthusiasm for this proposal. First, the proposed work plan is not very exciting and does not seem to require microfluidics. The microfluidic gradient generators are fantastic for studying motility or development in gradients, where the gradient itself is biologically useful. Here the gradient is being used to generate a range of concentrations. There is no reason why one couldn't generate those concentrations in a standard multiwell plate, and it would be easier to multiplex multiple factors in such a plate than in the microfluidic device, since the microfluidic device can vary one factor (or two, if one is kept constant). As noted above, the microfluidic device does add perfusion, which may yield useful information. Also, the gradients created with such devices do not typically span large concentration ranges. For instance, it is easy to make a linear gradient from 0 to 500 ng/ml, but it more useful to make a gradient maps 100x or 1000x changes in concentration linearly across the array. Additionally, the investigators want to modestly extend the work of Dr. Donovan, by adding BMP4 or Noggin to the NTs. The proposal would be more exciting if in two years the investigators could demonstrate a dramatically new result that can only be obtained using their technology. Finally, there is no fundamental reason why these experiments could not be funded by the NIH, rather than CIRM. While the investigators will eventually use non-presidential lines, nothing in their current plan requires the use of those lines, and thus there is nothing to preclude federal funding at this time. DISCUSSION: This proposal aims to study hESCs using microfluidics. While not highly innovative, this proposal describes neat and sophisticated microfluidic culture tools. A focussed approach presented that should give a good, directed payout, but where's the biology here and does it really require a microfluidics format? It seems that the PI would spend most of the time reworking Donovan's studies; and there is no letter of support from Donovan. The progress thus far is purely technical and not very exciting. One technical weakness is that the system doesn't exactly involve a 2D array of growth factors because it only gives the ability to change the first while the second is fixed.