An important aspect of understanding stem cell biology is to have a basic understanding of the processes that balance stem cell self-renewal and differentiation. Stem cell proliferation and differentiation signals are at least partially regulated by direct contact between cells. For example, stem cells normally reside in a specific microenvironment, or “niche”, that integrates specific cell-cell contacts in order to translate information from the environment into proliferation patterns. In this SEED proposal we plan to investigate the role of Eph/ephrin signaling in hESC growth and differentiation. Eph receptor tyrosine kinases and their ligands, ephrins, are large gene families that initiate signal transduction pathways which lead to changes in cellular adhesion, proliferation, and migration. Both Ephs and ephrins are expressed on the surfaces of cells, thus restricting their interactions to sites of direct cell-cell contact. It is known that Ephs and ephrins are expressed in hESCs and are therefore in the right place to be involved in regulating hESC proliferation and differentiation decisions. To better understand how Ephs and ephrins might be involved in hESC growth regulation, we plan to characterize the expression of Ephs and ephrins in hESCs during different stages of growth and neural differentiation to determine if Eph/ephrin signaling is used to regulate proliferation and differentiation of hESCs. The characterization of the role that Ephs and ephrins play in hESCs will provide insights into how stem cell proliferation is regulated in culture and will likely be applicable to how stem cell niches are organized in vivo. This understanding may allow for the development of standard culture conditions that will optimize both self-renewal and homogeneity of cells. This will in turn lead to more efficient large-scale production of stem cell populations and also methods for maintaining a self-renewing state in culture. Conversely, in a therapeutic setting, even a small number of undifferentiated cells could result in tumor formation; therefore, we also need to understand how to prevent self-renewal of stem cells.
This proposed research will benefit the State of California and it citizens by addressing the molecular signals that are involved in triggering human embryonic stems cells to divide, die, or differentiate into neurons. Human embryonic stem cells have great potential to be used in cell replacement therapies once their growth and differentiation programs are understood. Our work studying the role of a major class of signaling molecules, Ephs and ephrins, is likely to shed light on how these signals are generated and responded to in culture. Understanding how this process is regulated at the molecular level may allow for the development of standard culture conditions that will optimize both self-renewal and homogeneity of cells; which in turn can lead to the large-scale production of stem cell populations and methods for maintaining a self-renewing state by reversible blocking of hESCs. It is also important to make sure that upon differentiation, no stem cells are left behind, because they may be able to give rise to tumors when put into the body.
SYNOPSIS: The topic is the role of Eph-Ephrin function in the proliferation and differentiation of hESC to neurons. The PI suggests that specific combinations of Eph receptor and Ephrin ligands may be important at different stages of neural development and that this can be described and then manipulated. The rationale derives from mouse studies clearly showing the signaling pathway regulates different stages of neurogenesis. While EphA1 and EphrinA3 were shown to be expressed in subsets of hESC cultures, there is no direct evidence for function in the hESC system.
Aim 1: Characterize the expression patterns for Ephs and Ephrins during ESC culture and during neural differentiation, using RT-PCR and IHC. There are 14 Ephs and 9 Ephrins, the hope is to find interesting patterns.
Aim 2: Deregulate specific pathways, first by adding Eph and Ephrin Fc-reagents to culture, or by expressing them on MEFs. Forced expression of dominant negative reeptors alleles or siRNA.
Aim 3: Test if EphA1 expression is associated with cell cycle-dependent cell adhesion.
SIGNIFICANCE AND INNOVATION: Eph-Ephrin signaling is likely to be important during neurogenesis and certainly one place to start is by describing expression patterns. In time the project could derive important insight into stem cell proliferation and renewal, specific stem cell niches, and also the ability to ensure full differentiation of ES cells to avoid tumor formation. The pathway is particularly interesting in that both ligand and receptor are membrane bound so the focus is on local cell interactions.
STRENGTHS: The strength is simply the likelihood that this will be an important regulatory pathway and gene family sets that will need to be understood in the context of hESC growth and differentiation. The PI has experience in the field in the context of murine visual development, although there is no experience growing ES cells or performing the neural differentiation program.
WEAKNESSES: One weakness is breadth of taking on both entire sets of gene families. Expression patterns in the EB are likely to be very complex and over-lapping and so the RT-PCR data may not be very relevant. The IHC experiments will rely on commercial antibodies that may or may not provide specificity. Fc reagents will cross-react and it seems unlikely that this or the MEF idea will be able to ask questions beyond affects on hESC growth, rather than regulation during EB-derived neurogenesis. In contrast, the last aim is particularly narrow and it was not well explained why the focus on ephA1
The proposal should be focused on the descriptive studies, which would likely take 2 years (the first 6 months already are devoted to just learning to grow the cells), or provide justification for functional analysis of a specific pathway.
DISCUSSION: The PI is a productive, young investigator with a good publication record, formerly a post-doc in J. Flanagan's laboratory. This lab is a leader in Ephs and ephrins and axonal targeting in general. He is now looking at the dependance on these receptors for differentiation and looking for a novel function(s) of these receptors ourside of the nervous system. This work should provide important descriptive data. The hypothesis that Eph-Ephrin signaling might be important in hESC's seems well supported, and the function of this pathway outside of the neural system is completely unknown. One of the discussants noted that they would love to see the same approach used in looking at new roles for the netrins.
The applicant proposes to employ PCR and immunocytochemistry approaches - there is a question as to whether the reagants to be employed for immunocytochemical studies exist and for those that do, whether they will have high enough resolution to distinguish among individual members of this large family.
The proposal lacks focus, is not well put together and may be unrealistic for a 2-year project but this is the best guy to do it.