Human embryonic stem cells (hESCs) are pluripotent stem cells that have the dual ability to self renew and to differentiate into multiple cell types in the body. Growth and expansion of pluripotent hESCs require a balance between survival, cell death, self-renewal and differentiation signals. Despite the identification of some of the growth factors believed to be involved in hESCs self-renewal and proliferation, hESCs are extremely difficult to propagate and their survival in continuous culture in vitro remains a major challenge. Without improvement in this critical research area, the growth and expansion of undifferentiated human stem cells, which have the highest potency in generating the differentiated cell types, will remain a major obstacle to attaining the goal of human stem cell transplantation. This is because for any human cell therapy to succeed, it is necessary to prepare sufficient amount of undifferentiated human cell stem cells to test out the experimental conditions as well as subsequent transplantation into patients. This critical step is currently the bottleneck for advancing stem cell biology and therapy. This proposal is aimed at discovering the cellular basis on why undifferentiated hESCs, in contrast to mouse embryonic stem cells and human embryonic carcinoma cells, grow so slowly and are so difficult to sustain. We will study and compare a major cellular defense system of human stem cells. Our study will utilize several undifferentiated hESCs, as well as hESCs that have been induced to differentiate into neural stem cells, the latter are critical for future transplantation studies for the cure of neurological disorders such as Alzheimer’s and Parkinson’s diseases. In this application, we also propose a simple procedure whereby upon modification of the growth medium the hESCs may be able to grow more rapidly with a higher survival rate. If successful, our discovery can be applied to the cure of many types of diseases amendable by stem cell therapy. The CIRM seed grant is most appropriate for this study as it is testing a novel concept, has direct translational potential into therapy, and will include human stem cell lines currently not approved by the NIH.
Statement of Benefit to California:
This project has a broad benefit to Californians since we propose to evaluate basic biological functions of the human embryonic stem cells in culture. We hope to gain a better understanding of how the cells are responding to culture conditions and use this knowledge to improve culture conditions. Understanding the mechanisms governing human embryonic stem cell growth and stable propagation is vital to the success of this field. Our goal is to provide better and more reliable culture and scale-up preparations of human embryonic stem cells and their differentiated progeny. Unless we overcome the current limitations with current culture and scale-up protocols, the therapeutic potential of human embryonic stem cells will not be realized. This is an absolute prerequisite for achieving the goal of utilizing human stem cells for therapy of human diseases. Cost savings to California’s tax payers will be realized when human stem therapy can be achieved through stable growth and expansion of human embryonic stem cells.
SYNOPSIS: The proposal rests on the hypothesis that the stress, unfolded protein response (UPR)is impaired in hES cells. Aim1 will determine whether H9 hES cells activate the UPR response and aim 2 will characterize and compare the basal ER chaperones and UPR in a panel of ES lines (both NIH and non-NIH). Finally, aim 3 will investigate whether chemical chaperones that suppress ER stress promote survival and growth of hES cells. INNOVATION AND SIGNIFICANCE: Improved growth conditions for maintenance and expansion of hES cells would be a benefit to the field. The application is novel in that the hypothesis that ER stress may impair hES cell growth has not previously been explored. This may open up a new area for further study and facilitate use of hES cells, thus the studies have both scientific and potential biomedical importance. STRENGTHS: The investigator is very experienced with direct relevant expertise working in a strong scientific environment with clear evidence of collaboration with an hESC expert already in place. The specific aims are feasible and compelling. The applicant has preliminary data showing that Grp 78 is essential for the proliferation and survival of the ICM in the mouse and preliminary data on human UPR components. There is the potentially novel discovery of the importance of the ER stress proteins in selective aspects of human ES cell and related stem/progenitor cell functions. WEAKNESSES: The evidence to date in support of the hypothesis is meager. In part, it relates to apparent differences in levels of proteins in murine vs hESC. How valid this comparison is an issue. Focus may be too narrow, what about other members of the URL family? Attempts to find new members of the ER stress protein family or identify subset that are differentially expressed and to directly explore their function would enhance the proposal. Gain and loss of function via RNAi and BAC rescue would enhance proposal. DISCUSSION: Although the general idea that members of pathway may be important, the proposal focuses on only one member Grp 78, for which evidence of involvement in development of stem cells is "less than compelling".