The aim of California Stem Cells Initiative is to develop new therapeutical approaches by utilizing human embryonic stem cells (hESCs) to renew themselves and to differentiate into a variety of cell types, thus enabling the engineering of specific tissues to treat diseases that cannot be currently cured. To realize the potential of hESCs in regenerative medicine will require (1) the establishment of conditions for the expansion of these cells into a sufficiently large quantity and (2) the development of protocols to differentiate them into specific cell types and generate the desired tissues. Experimental manipulation of the environmental cues, such as chemical signals and physical stresses, to which stem cells are exposed, will lead to the discovery of conditions that specifically direct hESC growth and differentiation.
Studies on factors affecting stem cell growth and differentiation tend to focus on one or a few elements in the microenvironment, e.g., some proteins in the matrix underlying the cell or growth factors brought to the cell from the circulation or neighboring cells. The proposed research will develop a platform that will allow the concomitant screening of thousands of conditions consisting of combinations of various molecules for the systematic selection of the optimum conditions for hESC growth and differentiation. This platform is based on a microarray technology using robots to place 1200 spots of individual and combinations of proteins in a precise pattern on a glass slide surface. The hESCs attached to these spots will interact with different molecules in the patterned spots to elicit specific cellular responses. In addition, we will subject the cells on the arrays to well-controlled mechanical forces imposed by fluid shearing. Thus, we will combine mechanical and molecular stimuli in a controlled manner to study the responses of hESCs to physicochemical modulations in their microenvironment in terms of their signaling behavior and cellular fate, i.e., growth and differentiation.
The application of hESCs for regenerative medicine requires the establishment of the optimal physicochemical microenvironment that allows us to control and direct the growth and differentiation of these cells. Our proposed research focuses specifically at this critical need. We will develop a ‘systems’ approach to understanding the response of hESCs to multiple factors in the microenvironment. The results will lead to the definition of the optimal microenvironment parameters for the control of differentiation of hESCs into specific cell types such as cardiovascular cells, neuron cells, cartilage/bone cells, etc., for the treatment of many important human diseases. Hence, this project has fundamental importance and broad applications. This is a most cost-effective way to pursue hESC research for the improvement of human health and quality of life, and the resulting technology advances may provide financial gain for the people in California.
Millions of people suffer from diseases and injuries that cannot be cured by current treatments in clinical medicine, e.g., cancer, heart diseases, Alzheimer’s diseases, Parkinson’s diseases, and spinal cord injury. Recent knowledge of the potential of the pluripotent stem cells has opened the door of regenerative medicine, which will enable scientists/physicians to develop new strategies for treating patients with cell-based therapies to overcome the inadequacy of the conventional chemistry-based treatments. Most current stem cell research studies focused on the pathogenesis of a single disease, aiming at improving the treatment of the disease. Success of such research activities requires a platform that can control and direct the fate of the stem cells in terms of proliferation and differentiation. The aim of our proposed research is to establish a platform that allows the systematic investigation of the role of the physicochemical microenvironment in modulating the fate of various types of stem cells. The result will provide a common foundation needed for stem cell research directed at a broad range of diseases.
Our proposed platform will allow the concomitant screening of thousands of parameters (separate or combinatorial) to select the optimal conditions for stem cell proliferation and differentiation. Thus, we will develop a ‘systems’ approach to understanding the response of human embryonic stem cells to the microenvironment. The results will lead to the definition of the optimal microenvironment parameters for the control of embryonic cell differentiation lineages, such as cardiovascular cells, neuron cells, or cartilage/bone cells, etc. With the modulation of the microenvirnment parameters at sublocations on a scaffold material, multiple cell types differentiation can be triggered from a single source of human embryonic stem cells, thus leading to the creation of the desired functional tissues to repair the degenerated organs.
About half of California’s family suffered or will suffer from degenerated diseases and injuries. The results from our proposed studies, by providing the fundamental knowledge on how to manipulate human embryonic stem cell fate, will significantly facilitate stem cell research for virtually all diseases. The success of stem cell-based therapies will markedly reduce the future health care cost and ease the financial burden of California residences. In addition, the outcome of this project will lead to the development of a biotechnology platform to provide benefits to the advancement of California biotechnology. The patents, royalties and licensing fees that result from the advances in the proposed research will provide California tax revenues. Thus, the current proposed research provides not only the essential foundation for the scientific advances in regenerative medicine to improve human health and quality of life, but also potential technology advancement and financial profit for the people in California.
SYNOPSIS: This application from a premiere bioengineering lab has some ambitious aims centered on understanding the role of mechanical forces, extracellular matrix, growth factors and glycans in directing stem cell fate. Just this last sentence would be pie-in-the-sky except that the group has developed high throughput techniques for handling large, similarly combinatoric problems, but not in hESC. The specific aims are (i) to test a huge array of microenvironments for endpoints of adhesion, proliferation, maintenance of pluripotency, apoptosis (ii) to develop an array platform for presentation of growth factors and glycans and look at all the endpoints discussed in the first aim and (iii) to analyze shear flow effects on maintenance of pluripotency and endothelial cell differentiation.
SIGNIFICANCE AND INNOVATION: Microarray-based, discovery-driven approaches to the study of cell differentiation based on the type of matrix polymers and proteins that cells are adhered to have been shown to be fruitful for a variety of cell types. In this proposal, the investigators will expand this type of microarray technology to also include more soluble factor environments such as growth factors and glycans. This type of study may enable rapid accumulation of knowledge concerning what combinations of factors regulate hESC adhesion, proliferation, maintenance of pluripotency, and differentiation. Furthermore, the investigators will combine the effect of fluid mechanical environments to further regulate cell proliferation, maintenance, and differentiation. The combined effect of chemical and mechanical forces on embryonic stem cell differentiation is understudied and interesting, and there is a potential to identify better conditions for differentiation and maintenance of hESC.
STRENGTHS: The PI is an elder statesman of bioengineering and brings his experience with high throughput engineered microenvironments and microfluidics to bear. Significant established infrastructure is available for the work. Other engineering groups are doing similar studies of ECM and other arrayed proteins on hESC fate, but this approach is original. The investigators have significant preliminary results and an excellent track record of research in microarrays, shear stress, growth factors/inhibitors, and glycans. Use of a specific hES cell line that tolerates dissociation strengthens the proposal to perform microarray experiments to screen differentiation conditions. The effect of shear stress on hES cell differentiation is an understudied area of research that is interesting.
WEAKNESSES: The work is ambitious for the time-frame, especially given that no single person is devoted full time to pushing the project forward. Since the combinations of conditions examined are so large, and there is some fluctuation in patterning, it would help to have an estimate of the numbers of replicate experiments the PI anticipates. In this respect, the reviewers are not given sufficient information to judge the feasibility of completing the studies in the 2 year time frame. In fact, how the combinatorics of Aims 1, 2, and 3 are handled is not made clear at all, and this makes the proposal difficult to review. Will the glycans in various combinations be combined with the other ECMs and trophic factors in spots on the arrays? Will the shear stress be applied to cells on only a subset of matrices? Just as clinical studies require a power analysis to help reviewers, a similar presentation would have improved the application. In addition, these studies really are screens - whether the research will fundamentally change the way cells are maintained or differentiated will require a hand-off to stem cell biologists. For example, expression of certain markers of differentiation does not mean that the cells generated are fully functional. Nonetheless, even if Aim 1 gets accomplished successfully, and little progress is made on the other aims, the research will yield important information.
One concern that was not acknowledged is that different concentrations of the many proteins to be arrayed could have fundamentally different effects. There is no plan for looking at different concentrations, although the applicants have the luxury of using newly derived lines (for example, approved lines generally don’t adhere to 5 ug/ml laminin but do adhere to 500 ug/ml laminin.) The arrays may be presenting different doses of ECM or other proteins, but this is not clearly stated. In addition, the biology collaborator suggests that isolation of certain proteins from Matrigel may be possible for the purposes of the grant, whereas in the body of the grant this possibility is presented as a relative certainty. This may slow the effort more than anticipated.
Although the applicants have some preliminary data suggesting that hESC will grow for a week on the glass arrays (though how well is not clear), and they anticipate no technical difficulties, that assumption may be a bit naïve. For example, experienced hESC hands would probably anticipate difficulty getting cells off of a variety of matrices in the same conditions for flow studies - it is likely that the conditions for getting cells to single cell suspension from the arrays may be different depending on what the cells are sitting on. The release of cells is needed for the flow cytometry studies proposed. There are alternative ways to approach this problem, but the problem has not been acknowledged. Similarly, there will inevitably be a learning curve for interfacing these cells to the arrays and choosing the proper incubation conditions, which are not discussed at all even though there have been previous studies of hES cell differentiation on microarrays of different biomaterials (e.g., the Langer lab). Also, the proposed well gasket format of the microarray for regulating the extracellular matrix-growth factor-glycan environment (the soluble factors) does not seem compatible with fluid shearing.
DISCUSSION: This is a highly innovative, ambitious proposal from one of best bioengineering labs in the country, which is well known for studying shear stress on cells. They propose to study a huge array of microenvironments, but they already have preliminary data on hESCs. It is highly innovative to bring this work to hESCs. However, the applicant falls into trap of equating the expression of biological markers with having the desired cell type. Other weaknesses include the lack of a plan for handing off the cells after development to a well-established stem cell laboratory, and previous studies in the field on hESC differentiation on microarrays of different biomaterials were not referenced. In addition, while the well gasket format of the microarray was well described, one reviewer did not see how fluid shearing was simultaneously compatible with the arrays of glycan microenvironments. There was a question of whether this work is NIH-fundable, but panel members noted that those older lines can't get into the arrays for shear stress testing, thus the Melton lines may be more suitable for array placement than presidential lines.