Investigation of the Potential of Mouse Embryonic Stem Cells to Develop into Neuroendocrine Cells in Vitro
New Faculty I
Neuroendocrine cells are a set of specialized neurons located in the hypothalamus. They are called neuroendocrine because rather than forming synapses with other neurons, their major function is to release their product, neurohormones, into the blood circulation to act on their endocrine targets. The neuroendocrine system consists of eight neuronal populations, each expressing a specific complement of neurohormones and receptors. They are important because they control most of our vital functions, such as growth, reproduction, nutrition, sleep, stress responses and homeostasis via hormonal balance. Moreover, the neuroendocrine hypothalamus is susceptible to a variety of developmental diseases or syndromes such as Kallmann’s, Prader-Willi, or Rubenstein-Taybi. Although neuroendocrine cells are an important component in the regulation of homeostasis and behavior, very little is understood about the mechanisms that control their differentiation. This is mainly due to a lack of an in vitro model of development. The differentiation of embryonic stem cells in culture recapitulates a number of normal developmental processes that occur in mammalian embryos. This proposal aims at coaxing embryonic stem cells into becoming neuroendocrine in order to provide an in vitro model for further investigating the cellular and molecular mechanisms that control differentiation of hypothalamic neuroendocrine cells in early embryonic development. The biology of mouse stem cells mirrors that of human stem cells and, therefore, this work in mouse stem cells will ultimately provide a methodology for studying human embryonic stem cell differentiation into neuroendrocrine neurons, and thus serve as a means for therapeutic investigations. Finally, this project will also provide the opportunity to generate a core of highly skilled undergraduate students trained to maintain and manipulate embryonic stem cells, who can use their skills in laboratories of both academia and biotechnology immediately upon graduation. This will provide them with critical skills in the expanding scientific field of stem cell biology research, to which the state of California is financially, politically, and socially dedicated.
Statement of Benefit to California:
The State of California and its citizens would benefit at two levels from this proposed work. The first is therapeutic and the second is educational: 1- Therapeutic benefits: Hypothalamic neuroendocrine cells are a set of specialized hormone-secreting neurons that integrate and control several homeostatic processes that are required for our survival. Disruption of the development of neuroendocrine systems thus has the potential of perturbing important physiological processes with lifelong consequences. However, little is know about the mechanisms that control the development of such a critical component of homeostatic and behavioral control. The success of this work with mouse stem cells will ultimately provide a template for research into the development of human neuroendocrine cells and, therefore, serve as a means for therapeutic investigations of a variety of neuroendocrine-related developmental diseases or syndrome, including obesity and high blood pressure. 2- Training of a skilled workforce in the field of stem cell research: This project will also be used as a template for semester-long individual projects in several upper division laboratory classes including cell and developmental biology, and research methodology classes that are part of our undergraduate curriculum. This project will, therefore, provide the opportunity to generate a core of undergraduates trained in techniques of stem cell culture as well as fluorescence microscopy imaging of cells, who can function in laboratories of both academia and biotechnology soon after graduation. In addition, embryonic stem cells are of great interest to science and medicine because of their potential use as a tool to grow and replace damaged tissues in regenerative therapies. Therefore, training my students to successfully grow, maintain and manipulate embryonic stem cells in culture will provide them with critical skills in the expanding scientific field of stem cell biology research, to which the state of California is financially, politically, and socially dedicated.
SYNOPSIS: The overall goal of this proposal is to establish a model system for the study of neuroendocrine and hypothalamic cell fate determination by promoting the in vitro differentiation of mouse embryonic stem (ES) cells into neuroendocrine cells. It is proposed that a better understanding of how neuroendocrine cells develop may serve as a means for generating cells for treating syndromes in which there is a functional deficiency of these cell types. Another stated goal is to train students in stem cell biology research. Based on the preliminary results which indicate that ES cells have the ability to differentiate into cells expressing chromagranin A in the presence of retinoic acid and forskolin, the PI proposes in the first aim to confirm the neuroendocrine phenotype of newly-derived chromagranin A expressing cells by looking at the expression of eight neuroendocrine hormones. In aim 2, he proposes to establish mouse ES cells expressing GFP under control of the chromagranin A promoter, sort GFP-expressing cells by FACS, and then test, in aim 3, the role of other known inductive factors in directing differentiation to specific neuroendocrine lineages. In aim 4, he proposes to investigate the neurosecretory activity of the differentiated cells using fluorescent dyes. The last stated aim of this application is to provide a training platform in the techniques of maintenance and manipulation of stem cells for undergraduate minority students at the Dominican University of California, an undergraduate and masters teaching institution. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The proposal deals with an important cell component (neuroendocrine cells) at an important biological functional boundary in the human body that has received little attention and where knowledge about early development would be extremely valuable. It plans to utilize modified cells to track cell fate and to study the fundamental biology of the neuroendocrine cells. However, specific human diseases that are caused by or associated with neuroendocrine deficiencies are very rare, and therefore the spectrum of human diseases potentially impacted by potential discoveries is low. The research plan is poorly developed and does not involve hypothesis-generating or hypothesis-addressing experiments, it is simply a list of methodologies. One of the stated aims of this application is to provide stem cell science research training to students, mainly minorities. This may explain why the research design is presented more as laboratory exercises than as hypothesis-driven research.The proposal aims to establish an in vitro model which could be a significant challenge for the project and there is no mention of other potentially relevant in vitro models. Preliminary data, although limited, are broadly supportive, and whilst conversion of mouse ES cells to neuronal cells appeared to have been evaluated primarily on morphology, chromogranin A expression did appear to have been demonstrated. While a chromagranin A reporter mouse ES cell line does not exist, and this reagent would be novel, the remainder of the proposal is not innovative. Furthermore, the PI appears to have limited experience with ES cell culture, methodologies, and stem cell biology. The proposal focuses on murine ES cells but does not investigate or propose any work with human ES cells. The funding may provide a suitable training vehicle for undergraduate students, however, it is not likely to generate high impact factor results. In general, it is felt that the proposal is more suitable for an exploratory one or two year funding mechanism. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. El Majdoubi’s MS and PhD neuroscience education in France was supported by a scholarship from the Moroccan Ministry of Research and Education. He had post-doctoral experience at the University of Pittsburgh, and at UCSF. He is currently an Assistant Professor in the Department of Natural Sciences and Mathematics at Dominican University of California. He has relevant recent training to support the technical work, and a demonstrated track record in neuroendocrine research, and has letters from several supportive mentors in stem cell science. Whilst there is expert support periodically, there is a lack of local mentoring and a lack of postdoctoral workers who could support this work. There is good support in other important technologies. The development plan for the PI will be challenging to implement, as he would be the first scientist at this institution to establish ES cell technology, which means that mentoring is at a distance. The development plan does have formal funding support to enable contacts with expert investigators. Furthermore, there is clear and strong local support for the PI, and the proposed work will be important for the institution to achieve a critical mass for ES cell research. The applicant is clearly enthusiastic, committed to high quality education of the institution’s students and has a good background. His career goals list the importance of his efforts in bringing the Dominican University into the ongoing stem cell research in California, and his personal goal to train upper level undergraduate students, mostly minorities, in stem cell bench science. He plans to remain committed to a dual role of teaching and research. The PI has already stimulated a graduate training program which has yielded high levels of recognition for some students working with stem cells. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. El Majdoubi has the strong support of major figures in the host organisation, and has been given protected time to conduct his research, funded to date through small seed grants. He has been provided with appropriate laboratory space and the institution will continue to protect his time if he can keep his research funded. The institution appears to greatly appreciate Dr. El Majdoubi’s efforts to bring them into the stem cell research arena. DISCUSSION: During the discussion, the panel focused on several points raised by the reviewers, reflecting the unique aspects of this application. First, with respect to scientific merit, the panel agreed that the research proposal lacked depth since it is not hypothesis-driven, and that it just amounts to a list of experiments without clear identification of questions to be answered. The preliminary data are very limited, and the proposal falls short on innovation. Furthermore, the PI proposes to only utilize mouse ES cells, not human ES cells, he does not have much prior experience with stem cell research, and his plan for mentoring is weak. Therefore, the overall assessment of the potential scientific impact of this application was modest. On the other hand, a panelist drew attention to the fact that this proposal is unique in exploring neuroendocrine development, an understudied area. Consequently, results from this proposal may provide useful information for translational research targeting the treatment of hypothalamic neuroendocrine deficiencies. However, another panelist pointed out that such diseases are rare, and that therefore the potential clinical impact of the proposed research would be low. Finally, the panel considered the merit of the PI’s aim to create a training platform for minority undergraduate students with the goal of preparing them for careers as skilled research assistants in ES cell laboratories. Some panelists felt that this was a creative aim, and that because of the PI’s demonstrated dedication to minority education and his institution’s strong support for this endeavor, this application deserved to receive the benefit of the doubt. Others, although in agreement with this notion, regarded this application to be more appropriate for other potential CIRM funding programs. PROGRAMMATIC DISCUSSION: A motion was made to recommend that this application be moved to tier 2 – recommended if funds are available. The discussion centered around two programmatic advantages of this application. First, it was reiterated that the neuroendocrine focus of this proposal is unique and it therefore has the potential to increase the scientific diversity of the CIRM-funded portfolio. However, a discussant doubted that uniqueness alone, without sophistication of the science, is sufficient to justify funding. If that were the case, an applicant would just have to pick an unusual project to receive funding. The second programmatic argument addressed the potential for increasing the diversity of CIRM-funded institutions and for bringing stem cell science to minorities, since 70% of the student body at the applicant’s institution belongs to under-represented populations. However, the point was raised that this application may be more appropriate for other future CIRM-funded programs. One discussant argued, based on the potential for supporting an under-represented institution targeting minority education, attracting a PI new to the stem cell field, and diversifing CIRM’s scientific portfolio, that this application fulfills the mission of this RFA. Another panelist emphasized that when considering the three review criteria for this RFA, this investigator and this institution cannot compete. Finally, a discussant considered that this grant would have an enormous impact on the career of this PI, and that his institution is highly supportive of his efforts. This was countered, though, by the argument that there are other grants this review committee does not recommend for funding despite the potential impact on the PI’s career. The motion to recommend that this application be moved to tier 2 passed.