SYNOPSIS: Insufficiency of liver donors is an important problem for patients with acute or chronic end-stage liver diseases. The goal of the proposed work is to use high throughput, arrayed culture substrates to identify optimal ways to differentiate human embryonic stem cells into hepatocytes for transplantation. The focus will be on developing the conditions for improved hepatocyte differentiation from hESCs, using additional co- or tri-culture with other cell types including adult primary hepatocytes. Aim 1 will use robotic printing of extracellular matrix (ECM) proteins and growth factors on glass slides to screen for the optimal ECM for differentiation of hepatocytes. Aim 2 involves microfabrication of multi-cellular co-cultures to identify the optimal co-culture conditions that promote human embryonic stem cell (hESC) differentiation in hepatocytes. Aim 3 will assess the transplantation of pre-differentiated hESC, generated by combining the information gathered in Aims 1 and 2, into NOD-SCID mice to determine their fate after transplantation, including engraftment, survival, and proliferation.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal builds on the applicant's expertise is micropatterning of substrates/proteins for differential adhesion of cells so that multiple matrices/growth factors can be spotted on the same slide for more rapid testing of combinatorial screening. In principle, the approach and development of the techniques to do this solid phase display of growth factors and tri-culture of cells would also be an incredible platform for differentiation to other cell types; thus, the development of the techniques to do this is of great importance, but is not necessarily easy and straightforward. The expertise of the mentors (Zern and Reddi) will be key to the biological issues of this system. The strength of the proposal is that the Principal Investigator (PI) is willing to tackle a thorny problem - the efficient differentiation of functional hepatocytes from hESCs. As such, the proposal addresses a significant medical problem for which there is in theory a significant place for stem cell based therapies. However, as described below, the weaknesses are quite fundamental.
It is difficult to evaluate the feasibility of Aim 1 because no strategy for multiplex analysis is presented. There is only a table with the ECM proteins (six of them including Matrigel), and the PI proposes only one concentration of each ECM protein that will be used. There is no indication of dose-response curves or strategies for combining these proteins. Similarly, a list of growth factors that will be arrayed is presented with no concentration information, no information about gradient patterning, and no information about the combinatoric approaches that will be used for high-throughput screening of optimal combinations of ECM and growth factors. It would have beneficial to have a table with the numbers of conditions outlined in order to evaluate the quality and feasibility of the design. The choice of the size of arrayed spots changes throughout the application, and though this may seem trivial, the size of the spot also determines the critical mass of cells present and may be an important issue. How these sizes were chosen, especially since there is no mention of the sinusoidal architecture of the liver, is something for the PI to consider in future applications.
Several aspects of the experimental design raise further question about feasibility. First, hESC-derived embryoid bodies (EBs) will be dissociated on these printed surfaces and ‘hepatic function’ assessed. The decision about how to dissociate (or not) EBs has not been addressed yet, but given the dimensions of the arrayed spots, it seems essential to dissociate the cells for plating. Second, in the co-culture experiments the PI talks about using a stellate cell line and primary ‘non-parenchymal cells’. What are these? There are many kinds of non-parenchymal cells in liver. Third, though the preliminary data describe the advances made in the PI’s lab, it is clear that some basic considerations have not been covered yet, for example “cell seeding conditions including cell density and incubation times for hEBs”. Fourth, Aim 3 involves a scale up of the results from spotted arrays on glass slides to other culture conditions, and a real strategy for the considerations of scale-up is not presented. Furthermore, two mice per time point is an odd choice of number for a complex end-point of analysis—the survival and fate of transplanted cells of any type. The phenotypic analysis of the differentiated hepatocytes (which are not liver function studies) also is not well-thought out. The protein level expression studies are fairly extensive (though CK 18 is a marker for cholangiocytes, not hepatocytes) and so it is unclear what the laser capture (catapulting?) PCR studies really add. Along these lines a bipotential stem cell would be a good thing to look for in these studies.
A final issue is with the lentivirus carrying alpha1-antitrypsin promoter:GFP construct, which is proposed as a read-out system for identification and purification of differentiating hepatocytes. It is stated that this construct resulted in stable transfection of hESC and thus will be used throughout as the reporter system. However, there are concerns since the recent on-line publication from Dr. Zern transduced differentiated hESCs 10-14 days after EB plating and were only followed for 7 days whereas the implication was that transduced cells were stably transduced. If each protein array has to be infected, how will there be the denominator of transduction efficiency for the different conditions on each plate?
Obviously, hepatocyte differentiation is in need of new approaches but there is no guarantee that this approach will work to increase the efficiency of hepatocyte differentiation. There are many ways to introduce new tools into this area and the PI does not make a compelling case for why his approach is the best one. What if 3-D culture is really the answer, or small molecules - or many other novel approaches including going back to developmental biology and examining developing livers for matrix expression, or even hematopoietic co-culture given what happens in the liver during development?
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI's goal is to occupy a unique niche interfacing stem cell biology and engineering with a fully interdisciplinary lab. He is well versed in microfabrication techniques and has become involved in hepatocyte maintenance and differentiation from stem cells, where he has benefited from close collaborations with Dr. Zern (hepatocytes) and Dr. Reddi (cell-matrix interactions). His training is excellent. Dr. Revzin received his PhD in Chemical Engineering from Texas A&M in 2002 and his post doctoral training with Martin Yarmush and Michael Toner at Mass General in a multidisciplinary, clinically-oriented lab. The PI was appointed Assistant Professor at UC Davis in 2004, and has 4 grad students and 3 post-docs in his lab of 1250 sq. ft.. The engineering facilities and culture facilities all are well-suited to the project with available fabrication and robotics, for example. His assessment of his career development is reasonable and well thought out with the emphasis of being a stem cell engineer.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Overall the institution, as well as the Department of Biomedical Engineering, are strongly committed to stem cell biology. UC Davis has put together several investigators who should serve as a suitable intellectual colleagues for the PI. There is also strong commitment with startup, protected time, and supportive mentoring, particularly from Professors Zern and Reddi who are collaborators on the proposed grant and who will serve as career mentors for the applicant. The PI would have benefited from having these mentors review the grant for clarity of the design plan in order to make it sufficient for reviewers to judge the merits of the research design.
DISCUSSION: This proposal comes from a bioengineer who has started a collaboration with two senior stem cell biologists in an attempt to generate hepatocytes with greater efficiency. Reviewers like the idea and project aspirations, and while the career development plan would strengthen the PI's biology background, there were several problems with the experimental design and clinical portions of the proposal. Aim 1 lists factors but not how they are going to be used, and the multi-cell co-culture techniques proposed in Aim 2 have been tried before. In Aim 3 where pre-differentiated cells will be transplanted into NOD-SCID mice, the readouts for these experiments are lacking, and the markers proposed identify cholangiocytes, not hepatocytes.