The use of human pluripotent stem cells for cell-based therapeutics is predicated on the ability to convert these cells into functional equivalents of those lost in disease or injury. However, there is only scant evidence that either human embryonic stem cells or human induced pluripotent stem cells make differentiated progeny that are functionally equivalent to those found in tissues. Our preliminary results, gathered over several years suggest that in fact human pluripotent stem cells may make authentic tissue derived cells, but they appear to be most similar to cells found only during very early fetal development. As a result, it is unclear if these cells will suitably replace tissue derived cells in postnatal therapies. We have also uncovered several genes whose expression appears to distinguish mature tissue derived cells and those generated from human pluripotent stem cells. We have designed this project to determine whether manipulating expression of those genes in pluripotent derivatives can bring them closer to postnatal tissue derived cells. We also propose to discover small molecule compounds that can have the same effect. Upon successful completion of these aims, we will bring to the community compounds that allow for appropriate maturation of all types of pluripotent derivatives. Therefore, these reagents will facilitate cell based therapeutics enormously and perhaps allow for successful transplantation of pluripotent derivatives for the treatment of a wide variety of diseases and injuries.
For human pluripotent stem cells to reach their full potential in cell based therapeutics it is absolutely essential that their differentiation to particular cell types produces a product with the functional capacity to replace lost tissue. As California is a leader in the adoption of pluripotent stem cell therapies, it is vital that it also be a leader in the generation of derivatives from pluripotent stem cells that accurately mimic cells found in tissue. Our data suggests that instead of generating cells that would normally be found in adult tissue, human pluripotent stem cells instead produce cells found during very early fetal development. Not only does this suggest that cell based therapies with such cells might be hampered, but in fact the proliferative nature of these cells could even make them dangerous. We propose a series of experiments that will test the idea that manipulating the gene expression of pluripotent derivatives during differentiation will make cells that more accurately reflect those found in tissues that would typically require stem cell based treatments. We will develop tools that will be easily applicable for a wide range of cell types and yield mature, functional cells that not only mimic tissue derived cells, but can also functionally replace cells that are lost in disease or injury. This work will be particularly beneficial to ongoing efforts in California to apply pluripotent stem cells in therapeutic settings, and will therefore greatly benefit Californians requiring such treatment.
This proposal is focused on the goal of generating mature cell types from human embryonic and induced pluripotent stem cells (hESCs and hiPSCs). While cells of many different tissues have been derived from hESCs and hiPSCs, in most cases they resemble cells of early fetal development rather than fully mature cells. The applicant presents preliminary data demonstrating that certain genes are differentially expressed in cells differentiated from human pluripotent stem cells compared to their mature, tissue-derived counterparts. The applicant hypothesizes that these genes may act as a barrier to effective differentiation of pluripotent stem cells into mature cell types. There are two Specific Aims: 1) to determine the function of the identified genes during differentiation of hESCs and hiPSCs; and 2) to screen a small molecule library to discover inhibitors of these genes that could improve differentiation protocols.
Significance and Innovation:
- This proposal is highly significant and addresses a fundamental problem in the field, namely how to produce cells from hESCs and hiPSCs that most closely resemble the cells they are designed to either replace, in a therapeutic setting, or mimic, in a disease model.
- Reviewers described this as a high-risk, high-reward proposal. If the genes that the applicant proposes to study turn out to be key determinants of cell differentiation and maturation, this will advance the field tremendously. However, some reviewers were not convinced that this is the likely outcome.
- The proposal is not particularly innovative and does not employ novel approaches or technologies.
Feasibility and Experimental Design:
- Reviewers expressed mixed opinions regarding the feasibility and experimental design of this proposal.
- Some reviewers appreciated the substantial preliminary data demonstrating the feasibility of the approach.
- Other reviewers raised significant questions about the interpretation of the preliminary data. They suggested that the observed differences between cells derived from pluripotent stem cells and their natural counterparts could be due to the very different environments in which they develop, rather than the activity of the proposed genes. Indeed, they found it unlikely that inhibition of just one gene or gene family would overcome the barrier toward acquisition of mature cell phenotypes.
- Reviewers noted that preliminary data demonstrating that gene knockdown leads to the acquisition of a more mature differentiation state would have strengthened the application significantly.
- Aim 2 depends entirely upon the success of Aim 1.
- The applicant makes a convincing case for the screening strategy and assay proposed for Aim 2.
Principal Investigator (PI) and Research Team:
- The PI has limited but high profile publications and has made important contributions to the field.
- The PI has an impressive track record and is embedded in a strong group at the host institution.
- The PI and research team have all the necessary expertise to successfully perform the proposed experiments.
- One reviewer noted that the screening work proposed in Aim 2 could benefit from an experienced collaborator, while another felt that the necessary resources and expertise would be supplied by the small molecule screening core facility.
Responsiveness to the RFA:
- This proposal is responsive to the RFA as it focuses on the basic mechanisms of human stem cell differentiation.
- A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers noted that this is an underrepresented area of study that is important for the utility of hESCs and hiPSCs. They acknowledged that the application is flawed and risky, but appreciated that it is hypothesis-driven and addresses a significant problem in the field. The motion carried.