Stem cells are the building blocks during development of organisms as varied as plants and humans. In addition, adult or “tissue” stem cells provide for the maintenance and regeneration of tissues, such as blood and skin throughout the lifetime of an individual. The ability of stem cells to contribute to these processes depends on their unique ability to divide and generate both new stem cells (self-renewal) as well as specialized cell types (differentiation).
In some tissues, cells that have already begun to specialize can revert or “de-differentiate” and assume stem cell properties, including the ability to self-renew. De-differentiation of specialized cells could provide a “reservoir” of cells that could act to replace stem cells lost due to wounding or aging. This proposal seeks to uncover the mechanisms that are utilized to regulate the process of de-differentiation and to compare these to the mechanisms that endow stem cells with the ability to self-renew.
A thorough understanding of the factors that regulate self-renewal programs will be essential for the expansion and long-term maintenance of adult stem cells in culture, a necessary step towards the successful use of stem cells in regenerative medicine and tissue replacement therapies. Furthermore, understanding the mechanisms by which partially differentiated cells can reacquire self-renewal potential and how these programs are utilized during the normal course of tissue maintenance and repair could provide powerful strategies for regenerative medicine by stimulating inherent self-repair programs normally present within tissues and organs.
We plan to identify and characterize genes and proteins that are involved in regulating the ability of specialized cell types to revert back into a more immature cell that can act like a stem cell. Information revealed by these experiments will likely prove useful in understanding both how tissues can be maintained during aging and/or repaired after damage. Subsequently, this knowledge could be developed into powerful strategies for regenerative medicine by stimulating inherent self-repair programs normally present within tissues and organs. In addition, these experiments may provide some insight into how some tumors may be initiated, leading to cancer. Lastly, in the course of these studies, we will be generating ES cell-like cells from spermatogonial stem cells. Although we will initially work with mouse tissues, our ultimate goal would be to adapt these techniques to human spermatogonial stem cells, which would then be used as a source for generating human ES cells. We would make these cells readily available to other investigators and companies in hopes of accelerating the pace of discovery.
SYNOPSIS: This proposal by a young investigator at the Salk Institute targets the dissection of molecular and cellular events underlying the development of the fruit fly adult male germ cells. These cells have stem cell properties as they have the ability to divide and generate both new stem cells (self-renewal) as well as specialized cell types (differentiation). In addition to these two unique properties, once differentiated, these cells can revert or “de-differentiate” and assume stem cell properties, including the ability to self-renew. De-differentiation of specialized cells could provide a “reservoir” of cells that could act to replace stem cells lost due to wounding or aging, a process called reprogramming in other species. The proposal seeks to uncover the mechanisms that regulate the process of de-differentiation, and to compare these to mechanisms of self-renewal.
The PI has established two independent methods for inducing conditional loss of stem cells in order to trigger de-differentiation. She will either utilize temperature sensitive conditional loss of Stat92E function (loss of both germline and soma) or heat shock-dependent over-expression of the bag of marbles gene (loss of germline). Upon release of these blocks, the germline stem cell (GSC) population (or soma) recovers. It is hypothesized that a two-tier regulatory scheme is necessary to provide tight control over a dynamic pool of stem cells. The PI specifically predicts that Polycomb group (PcG) proteins will repress terminal differentiation genes, and stem cell identity and self-renewal will be regulated by post-transcriptional mechanisms such as small RNAs. Three Aims will test candidate factors, screen for new factors, and attempt to translate the system to mammalian spermatogonia stem cells (SSC).
1) Using the male gonad conditional loss of stem cell or soma assays, de-differentiation will be evaluated in mutants for the PcG pathway (such as PRC1 or PRC2) and for the RNAi machinery. A dominant modifer screen will also be carried out by crossing deficiencies onto the Stat mutant background.
2) The goal is to identify factors involved in regulating the transition of mammalian SSC into cells resembling embryonic stem cells (ESC). An in vitro de-differentiation assay will be developed for murine SSC, using Ngn3:GFP mice to purify the cells. Lentiviral transduction will be used for loss of function (shRNA) or gain of function (cDNAs) experiments to characterize candidate regulators or for screening.
3) Similar to experiments in Aim 1, a combination of genetic, and molecular approaches are presented as tools to decipher how somatic stem cells, curiously called Cyst progenitor cells (CPCs) can de-differentiate into spermatogonia. The source of the renewing cells and factors that facilitate it will be identified.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Significance and innovation of this proposal is based on the argument that a thorough understanding of the factors that regulate self-renewal programs will be essential for the expansion and long-term maintenance of adult stem cells in culture. This represents a necessary step towards a possible use of stem cells in regenerative medicine and tissue replacement therapies. One reviewer felt that the main question and incertitude remains about the relevance of these findings to the human case. While it is clear that this work will inevitably enrich our knowledge about insect germ cells’ programming and reprogramming, and from a fundamental point of view this is beautiful, most of the funding so far from Drosophila has not been directly relevant to the human case. In fact, as Drosophila represents a unique developmental program, even when compared to other insects, some of these findings might not even apply to other insects.
However, other reviewers conveyed that this is an exciting application from a young investigator proposing a bold, straight-forward approach to a controversial topic. Her background and innovative use of fruit fly genetics for this work may lead to insights not possible with other systems at this time. Specifically, the ability to de-differentiate has clear relevance to tissue regeneration, and also to the initiation of cancer stem cell fate. The fly system provides an outstanding molecular genetic model to test candidate factors, and perhaps most importantly, screen for novel regulators of this pathway. The potential to translate this to a mammalian stem cell system would provide major significance. This is an excellent proposal using a very well controlled and characterized stem cell and regenerative model with the potential to find novel regulatory pathways.
This proposal is supported by strong preliminary data. In Aim 1, the ability to evaluate de-differentiation (or regeneration) at the single cell level generates unique opportunities. The screen for miRNA regulatory factors is complicated because the mutants are lethal and so only heterozygotes can be evaluated. It would be nice to have a conditional approach for this class. Also, the focus on miRNA seems rather like a leap of faith.
The concept that mammalian SSCs can convert back to ESC-like cells is very recent and remains somehow controversial as similar attempts in the human context have not shown similar conversion. The analysis of mouse SSCs in Aim 2 seems a bit out of place and was perhaps used to create the impression of relevance of this proposal to the mammalian system. As acknowledged by the author, the lab does not have mouse expertise and relies on the advise of Dr. Niels Geijsen (MGH) for troubleshooting. Human ESC will not be used for this work. There is no connection between this aim and Aim 1 or Aim 3. A combination of candidate gene, as well as genomic approach is suggested to accomplish the aim, however as all the assays remain in vitro, and might not correlate with the in vivo situation this aim is weak. By contrast though, another reviewer felt that the potential to translate fly genetics to a mouse system is of high potential value.
Aim 3, to characterize the de-differentiation of somatic cells within the Drosophila testis, is relatively solid as it takes advantage of powerful genetic approaches so fruitful in the past to understand insect development. This is the crux of the application and the PI has described a logical series of studies that build on her background with this system and have a high likelihood of yielding valuable information The main caveat of the approach is the lack of CPC molecular markers which might make the screen more challenging. It will be of interest to determine if the germline and soma respond by the same or different mechanisms.
Overall, while this represents a good proposal and is scientifically sound, its relevance to regenerative medicine remains unclear. Considering the documented major divergence of molecular mechanisms involved in stem cell regulation even between human and mouse, it is difficult to project that the work in the fly would be relevant to the human situation. From a basic science point of view however, there is no doubt that the successful accomplishment of the goals of this proposal will significantly increase our knowledge of Drosophila cell fate regulation. To enhance that contribution one reviewer recommends that the PI drop Aim 2 which is clearly out of place and focus to the area of her expertise.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Jones is a talented investigator with many important contributions to her field, as shown by her list of publications. She is an Assistant Professor at the Salk Institute (2004) within the Laboratory of Genetics. She was a productive graduate student with K. Munger at Harvard University working on the E7 oncoprotein (4 first author publications), followed by two post-doctoral positions in the UK and at Stanford, where she trained in the fly gonad system with Margaret Fuller, and set up the system to study STAT signaling in the male germ line. She published a co-first author Science paper on that work (2001), and had several other co-authorships. Her CV is not long, but represents strong publications in excellent journals. Although there is a major gap in publication of original research papers from 2004 to 2007, she has published one senior author paper since starting her own laboratory (Cell Stem Cell, in Press). She is relatively well funded for her current projects on the GSC niche and ageing in the fly gonad, including NIH K18 and R01 grants. Dr. Jones is well-suited to carry out the proposed studies in Aims 1 and 3 of this grant.
Dr. Jones has demonstrated a clear commitment to a career in stem cell biology since starting her own laboratory at the Salk. She clearly states her intent to translate the results she can obtain using fly genetics, which provides unique strengths, into mammalian stem cell systems. She is interacting already very well with the stem cell community in San Diego and beyond. Her mid-term evaluation is approaching and it might be useful to develop a more formal senior mentoring plan, although she has excellent support from colleagues including Rusty Gage and Inder Verma.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The Salk Institute provides an outstanding research environment, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. There is an ongoing expansion of current facilities and growth in the area. Dr. Jones was provided with excellent independent space and startup. She has strong support from her Chair, mentor, and collaborator, Dr. Verma. She has no significant administrative or teaching duties and is focused fully on her research program.
DISCUSSION: There was consensus that this represents a scientifically excellent proposal, using an advanced genetic system, the fruit fly Drosophila, to investigate self-renewal of germline stem cells and de-differentiation of specialized cells. Based on the track record of this solid young investigator, it can be trusted that she will be able to accomplish the proposed goals. She is well funded and has strong support from her institution. The PI has a tremendous publication record, disrupted though by a gap from 2004 to 2007. A panelist pointed out that this can be justly attributed to her starting her own lab.
A specific scientific issue was raised regarding the fact that the Drosophila mutants to be anlayzed are homozygous lethal, and that consequently there is a need to analyze heterozygotes, with no evidence that they present with a phenotype. Therefore, there may be a need to develop conditional mutants. A discussant also raised the concern, that the hypothesis of miRNA involvement in the de-differentiation process is a leap of faith.
Although overall this is a beautiful basic science project, questions were raised regarding the importance of doing the work in Drosophila and whether these studies would ever be relevant to human stem cells. Some argued that Aim 2, which involves the study of SSC derived from mouse testes, and as such has more relevance to the human stem cell field than the Drosophila work, appeared to just have been added to comply with the goals of this RFA. Moreover, the PI has no prior experience working with mice. However, the work on mouse SSC was also discussed as a major plus in that it represents the one good system in the stem cell field in which adult cells with ESC-like characteristics have been isolated, and consequently the proposed studies could have an important impact for understanding how to isolate ESC-like cells from testicular biopsies. To date, there has been only one publication describing this phenomenon, and thus this field needs more attention. To expedite the important mouse work, the panel recommended that Aim 1 and Aim 2 be performed in parallel.