Pharmacological characterization of human stem cells and their differentiation via the GPCR transcriptome
In envisioning how stem cells could be used to replace lacking or deficient cells in the central nervous system (CNS), we recognize that one of the best way would be to induce stem cells present in the brain to differentiate into the lacking or deficient cells. When considering Parkinson's disease, one of the best way to develop a therapy would be for mature neurons that can replace lacking dopamine-expressing cells. The issue is how to drive stem cells into becoming full-fledged neurons. This project proposes a new way at reaching this goal. Every cell in the organism expresses a battery of G protein-coupled receptors (GPCRs). The GPCRs bind all the small molecules that direct intercellular interactions. Importantly these include all the neurotransmitters and neuropeptides., making the GPCRs the prominent regulators of brain function. Binding of the GPCR ligands initiate a cascade of intracellular reactions that change the transcriptional activity of the recipient cell. Most notably GPCR activation can initiate differentiation, i.e. they may be able to induce a stem cell to become a neuron. Each cell in the organism, expresses a few dozen different GPCRs. Although not determined thus far, stem cells too will harbor several GPCRs, probably at least as many as the adult cells. If we can find the stem cells GPCRs we may be able to use them to transformed these cells into neurons. The first issue is to find all the stem cells GPCRs. This will be done using a well developed technique, RT-PCR. Using it we will catalog all the GPCRs express in different stem cells. We will then activate the GPCRs that we have found on stem cells to determine whether they can induce the stem cells to differentiate into neurons. There are already data that a few GPCRs have this ability. Our aim will be to discover how many can do it and whether there is some commonalities between the ones that have this ability. We will also determine whether the exhibit differences in the neuronal phenotypes and thus define the cascade of events that leads to this phenotype. Knowing which GPCRs can induce neuronal phenotypes and at which step in the differentiation process a particular GPCR acts will have direct impact on therapies. GPCRs are the most sought targets in drug development and many drugs acting on GPCRs already exist. Our research may point at some of these drugs as potential mediators of neuronal regeneration and possibly helpers in therapies aiming at replacing lacking neurons.
Statement of Benefit to California:
The success of this proposal would have a positive outcome for California. It approaches the use of stem cells from a pharmacological stand point. In doing so it leads to therapies that do not require surgical intervention. Instead it may point at existing drugs as potential agents for neuronal regeneration. This would result in immediate applications. It may also point at new targets that should be pursued pharmaceutically.
SYNOPSIS: The authors propose to investigate the contributions of G protein-coupled receptors (GPCRs) in embryonic stem cell differentiation. These studies will examine GPCRs expression in hES cell lines. Most cells express a few dozen different GPCRs on their surface, and most GPCRs are activated by small molecule ligands, although there are important cases where the ligands are larger polypeptides. The question to be addressed here is which ones are expressed by hES cells? Eight (8) cell lines developed by WiCell will be utilized, and these cell lines will be tested for the expression of 260 GPCRs. These GPCRs are chosen to be non-somatosensory (i.e., presumably ligand-dependent) and non-orphan, with the assumption that their specific expression will be directly related to their differentiation potential. Ligands for subsequent activation studies will be available in many cases. The PI anticipates that hES cells will express about 30 to 50 GPCRs. These will be grouped according to shared as well as unique expression patterns among the entire panel or subsets of the hES cell lines. The hypothesis to be tested is that distinct GPCRs and downstream pathways may induce and/or suppress discrete programs of differentiation. The first Aim will catalog the GPCR expression profiles in hES cells by selectively amplifying various GPCRs by PCR. In the second aim the investigators propose to assess the activation of GPCRs using appropriate pharmacophores (e.g., leukotriene receptor B4, LTB4, and vasoactive intestinal protein, VIP) to investigate their effects on neuronal differentiation pathways. Cell growth and markers diagnostic for different neuronal and glial differentiation pathways will be analyzed over time. The PI also proposes to analyze the roles of shared GPCRs by treating cells with pools of ligands, and subsequently identifying which ones promote a given effect by a sib-selection strategy. Subsequent studies will analyze the exact spectrum of neuronal markers that may be induced by individual and combinations of GPCR ligands. SIGNIFICANCE AND INNOVATION: The actual studies in this proposal are not particularly innovative or original from a technical standpoint, but they are highly significant and appropriate to the questions addressed. Specifically, the repertoire of GPCR expression at the RNA level will be defined in 8 distinct hES cell lines. This amounts to a cataloging endeavor; however, few similar studies have been reported. As noted by the investigators GPCRs have been implicated in neuronal differentiation, and it is clear that GPCRs play numerous important biological roles in adult as well as in developing organisms. The potential for the development and application of therapeutics to facilitate neuronal differential of stem cells may have broad implications for the treatment of neuronal degenerative diseases. Therefore, a comprehensive catalog of GPCR expression in hES cell would be of considerable value to the field. The studies in the second Aim, which are more functionally oriented, are also of value. STRENGTHS: A major strength of this proposal lies with the PI, Olivier Civelli. He is highly qualified and has a strong record in screening orphan GPCR libraries. The laboratory is extremely well equipped and has accumulated a good proportion of the reagents required to carry out the proposed work. Another major strength of this proposal is its straightforward nature, which is highly likely to yield valuable yet descriptive information. As such, these studies are an important first step in analyzing the functions of GPCRs in hES cell biology. In particular, the PIs focus is to identify GPCRs that may be important for neuronal differentiation. There is clear precedent that GPCRs are critically involved in almost all tissues and numerous aspects of tissue physiology and homeostasis. Since the PI will analyze 8 different hES cell lines, it may be possible to establish hES cell GPCR expression signatures. In the second Aim functional studies are proposed to evaluate the ligands corresponding to the identified panel of GPCRs. Primary emphasis will be given to identify those that induce neuronal or glial differentiation. The pharmacological properties (i.e. appropriate doses) for many of these ligands are known, and the experiments will be conducted in triplicate in multiwell formats. Overall, the proposed studies are generally strong, and will likely provide valuable information. WEAKNESSES: The proposal is non-hypothesis driven and at times highly underdeveloped, which often makes the positive aspects also the negative aspects. For example, a broad analysis of GPCR expression in hES cells is clearly warranted, but the danger is that too much data will be obtained and that quantitative aspects of GPCR expression levels in individual hES cell lines may complicate data interpretation. The investigators selected to use a more arbitrary, binary decision to select which GPCR reverse transcribed in the reaction is categorized as “expressed”. How will the PI define a significant level of expression? Will the RT-PCR reactions be quantitative? Perhaps a quantitative, realtime PCR procedure (qPCR) would be more objective. While it is appreciated that steady-state mRNA levels do not always correlate with the functional contributions of a given gene, qPCR may help objectively ascertain background noise from legitimate expression. In fact, it may be that a much broader range of GPCRs will be expressed by undifferentiated hES cells, albeit at very low levels. How will the PI define biologically relevant levels of expression? In addition, it may be that mRNA levels do not reflect the protein levels. This is not considered adequately. For example, there are proteomic data sets that already exist or are being established for hES cells. It would have been useful if the PI proposed to interrogate these for actual protein expression. Another example of the positive aspects also being the negative aspects is that the investigator has chosen not to screen orphan receptors, even though they may have an equal potential to regulate neuronal differentiation. Indeed, it is understood that the identification of orphan receptors would make subsequent functional experimentation difficult owing to the lack of activating ligands. However, the identification of orphan GPCRs that are critical for neuronal differentiation might spawn studies to search for endogenous ligands, an endeavor that the PI is well positioned to pursue. It also should be noted that some aspects of the studies in Aim 2 are of an exploratory nature. Will feeder layers be used, and if so, how may potential paracrine effects of the feeder layer in response to the ligands be ruled out? More importantly, the source and the nature of the ligands for this Aim were not stated. If the nature of the ligands arises from results of Aim 1, then the dependency of aims has the potential for a fatal risk. If the ligands are derived from a library of compounds and are to be utilized in a high-throughput screening sense, then it should be stated. However, it is not clear whether the investigator can carry and propagate the pluripotent lines in a high-throughput sense, and, although not required for SEED grants, no preliminary data are given to support whether the investigators can demonstrate that the differentiation markers are robust enough to be used in a screen. DISCUSSION: This proposal aims to study (presumably ligand-dependent, non-somatosensory) GPCRs in the differentiation of hESCs under the assumption that expression of a GPCR is related to differentiation potential. Reviewers differed in their overall enthusiasm for the proposal. Overall, there was acknowledgement of the possible significance, but the proposal was not thought to be innovative in its use of techniques to approach the questions asked. While the development of therapeutic agents may be facilitated as a consequence of the proposed studies, the decision to ignore orphan receptors was a significant weakness. Orphan receptors may be critical for neuronal differentiation and focusing on them would play to the unique strengths of the applicant. In addition, the applicant did not address the possible complications of results that may be reasonably expected as a consequence of paracrine effects caused by the feeder layer. This type of study might benefit from electrophysiology data, but no such studies are presented. The qualifications of the principal investigator are a key strength of this proposal, but the poorly developed scientific content significantly dampened enthusiasm. This project may be well suited for NIH funding.