Stem cells are mater cells from which specialized cells and tissues in our body are derived. Stem cells have the ability to regenerate them (self-renewal) and at the same time produce specialized cells (differentiation). Diseases such as cancer can arise when the control of stem cell self-renewal and differentiation goes awry. How the stem cells in our body balance these tasks is not understood. Studies of stem cell behavior in other organisms have identified a number of molecules that regulate the self-renewal and differentiation of stem cells. Many of these molecules are conserved in humans. Whether there is a functional conservation is not known. The goal of this proposal is to test whether conserved mechanisms are used to control stem cell behavior in our body. Information to be obtained from this study has the potential to promote new ways to maintain human stem cells in its multipotent state in culture for long term and to direct their differentiation into specialized cells such as neurons, bone marrow cells, and heart cells, which can be used in transplantation therapies. More excitingly, this knowledge could be used to manipulate the capacity of the resident stem cells in our body to repair or re-grow damaged cell lost to disease or injury. This could revolutionize the ways doctors treat a wide range of devastating diseases such as cancer, heart disease, diabetes, autoimmune, and neurodegenerative diseases.
Statement of Benefit to California:
Stem cells are unspecialized cells that are capable of reproducing themselves over an extended period of time. Under certain conditions, they can be transformed into cells with specialized functions. Stem cells offer the possibility of a renewable source of replacement cells and tissues to treat diseases or conditions such as Parkinson’s, Alzheimer’s, heart disease, diabetes, or spinal cord injuries. To realize the vast potential of human stem cells, we need to have a better understanding of the mechanisms that control their ability to regenerate themselves and at the same time produce specialized cells. The goal of this proposal is to identify molecules that control these unique properties of human stem cells. Information to be obtained from this study could be used to devise ways to faithfully maintain human stem cells in culture for long term and to direct their transformation into specialized cells such as neurons, bone marrow cells, and heart cells for transplantation therapies. This knowledge could also be used to harness the innate capacity of the stem cells in our body to repair or re-grow damaged cell lost to disease or injury. This could lead to new treatment strategies for a wide range of devastating diseases. Thus, the proposed research has the potential to benefit the State of California and its citizens in several ways: 1. It could lead to new cures for cancer, heart disease, diabetes, and neurodegenerative diseases. 2. It could save people's lives and cut health care costs. 3. It could stimulate growth of the biotech industry in California, thus generating new tax revenue for California.
SYNOPSIS: The general goal of this proposal is to understand mechanisms that regulate the pattern of cell division in human ES cells and human neural stem cells, and the role that this plays in stem cell self-renewal and differentiation. The proposal focuses primarily on Numb, which is a protein initially characterized in Drosophila stem cells that is asymmetrically distributed during cell division and is a determinant of cell fate. It is thought to act by antagonizing Notch signaling. The specific aims are to study the localization of Numb in human ES and neural stem cells by immunostaining, and to study the functional consequences of Numb under- and over- expression in these cells. A third aim will be to do similar experiments with one or two Numb interacting proteins. INNOVATION AND SIGNIFICANCE: The study of pathways that mediate the choice of cell fate is going to be very important in developing optimal strategies for the therapeutic use of human ES cells. The Notch pathway and its regulators, including Numb, are among those that will be crucial for understanding differentiation by ES cells or their lineage committed descendants. The applicant has a strong background in studying the molecular biology of proteins that distribute asymmetrically during cell division of neural progenitor cells. The proposal is innovative in its application of findings in Drosophila to the human stem cell system and significant in the potential to elucidate basic mechanisms of stem cell biology. STRENGTHS: The applicant is a talented molecular biologist who, as a post-doctoral fellow, published important papers on assymetric protein distribution during cell division. The application focuses on a pathway that is likely to be important in cell fate decisions in the neuronal lineage. The team assembled by the PI for this project includes Drs. Julie Baker and Theo Palmer, respected human stem cell researchers. The PI and collaborators have access to excellent facilities and institutional support. WEAKNESSES: The reviewers cited several weaknesses in this proposal. Overall, the proposal is not clearly written, it is confusing, and there are contradictions in the plan. It is not clear, for example, if the team will be working on hESCs and primary hNSCs, or if the NSCs will be derived from hESCs. The proposal does a remarkably poor job of explaining why the Numb protein will be interesting to study. Its biology in Drosophila is not described, and thus the applicant has left it to the reader to figure out why it might be important to study in human ES cells. The applicant also implies that Numb has been exclusively studied in Drosophila, but this is not true. Considerable work with genetically engineered mice has already provided substantial insight into the roles it plays in mammalian neurogenesis. Knockout of both Numb and the related Numb-like genes in the mouse leads to severe depletion of neural progenitor cells in the mouse. However, early brain development is normal indicating that these proteins are not required for the initial production of neural progenitors, but rather for their maintenance or self-renewal. Hence, the rationale for studying Numb in pluripotent ES cells is not clear. In Aim 2, the applicant proposes to study the effect of Numb knockdown in human ES cells, but does not discuss the fact that prior mouse work has shown that Numb is redundant with Numb-like, and therefore it is likely that both will need to be targeted. Furthermore, Numb is a Notch antagonist and the applicant fails to cite prior work showing that the Notch pathway is inactive in human ES cells, and is not required until these cells differentiate into lineage specific progenitors. The final part of Aim 2 proposes to look at undefined Numb regulators or interactors in human ES cells. These experiments are very poorly described and inadequately justified. The PI has had a grant for 5 years from the NIH to study the control of asymmetric cell division in neural stem cells in Drosophila. Although the application describes data from studies that appear to be in progress, the applicant has not published any papers on that topic during this 5 year grant period. While the researchers are experienced in the Drosophila system, they have no experience with hESCs or hNSCs. Drs. Palmer and Baker have written letters of support but none of the permanent team are trained in mammalian cell biology or have experience with human stem cells. The proposed experiments entail creating hESC lines with stable expression of an introduced transgene. This is not trivial even for experienced researchers. More worrisome is the lack of careful planning and a basic understanding of the system. For example, the PI proposes to use Oct4 expression as an indicator of cell differentiation. Oct4 is not a good marker for such studies because of the relatively long amount of time its expression continues following initiation of hESC differentiation. DISCUSSION: There was no further discussion following the reviewers' comments.