Funding opportunities

Modeling Human Embryonic Development with Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00259
Principle Investigator: 
Funds requested: 
$571 575
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Stem cells have entered the public consciousness as “cells that can do anything” and have been hailed as a panacea in the fight against disease, aging and cancer. Unfortunately, we have only scratched the surface in understanding these cells. Some of the things we think we know are that: embryonic stem cells hold great promise because they do seem to be “cells that can do anything”, but still cannot be isolated from consenting adults, and that adult stem cells, while isolatable, are much more limited in their ability to replenish tissue beyond their organ of origin. In addition, we know very little about human embryonic development for the simple fact that experiments on human embryos has proven to be nearly impossible due to ethical and technical obstacles. Clearly, if we gained a deep understanding about human embryos and human embryonic stem cells, we could not only develop useful clinical opportunities, but also potentially detect and treat errors made during human development. This proposal suggests that in fact we could learn a great deal about not only the therapeutic potential of hESCs, but also human development by exploiting cell culture. We propose to model human embryonic development in order to understand how a particular portion of the embryo undergoes a transformation to become either the brain or the skin. The fact that seemingly one cell type early on in the embryo can form either the complete nervous system or the skin has intrigued scientists for decades, we now hope to understand how this process works and in the process we hope to challenge existing theories of the potential of adult stem cells as well. With a deeper understanding of what makes a neuron a neuron as opposed to a skin cell, we will in fact be able to impart a neural code on a skin cell, and perhaps turn a skin cell into a neuron. If this becomes possible, we could: take a skin biopsy from a patient with parkinson’s disease (a degenerative disorder where dopaminergic neurons are lost), use already established mechanisms for expanding those skin cells in culture, turn on the “neural code” to turn them into dopaminergic neurons, and then transplant them back into the same donor patient. This kind of self-transplant obviates the need for either immuno-suppression therapy which is toxic and sometimes deadly, or for patient-specific stem cells which are, for now, impossible to derive.
Statement of Benefit to California: 
The people and the state of California stands to gain both scientifically and economically from the work proposed here. We propose a plan to augment our working knowledge of a process that, without hESC, we have no other method to address. We are developing a model to describe a fundamental process that occurs during human embryonic development. Obviously, we cannot perform experiments on human embryos, so we are taking advantage of the primitive nature of hESCs in order to model this process in vitro. Clearly, a working knowledge of human embryonic development would be of enormous significance to not only the scientific community, but the world at large. Mistakes during development lead to tragic consequences such as mental retardation, spina bifida, and perinatal mortality. Another benefit of the development of this model system is that currently the process by which hESC differentiate down different cells lines is somewhat of a black box. We know that hESCs can make many different cell types, some of which might be useful clinically for degenerative disorders, but we really do not know anything about how these different cell types come about. If we could gain some insight into how cell fate decisions are made, then we might be able to coerce not only hESCs, but other cell types as well, down a particular lineage. For instance, if we can understand the genetic program makes a primitive cell become a dopaminergic neuron, we could apply this program to another cell type such as a skin cell. Then, we could isolate skin cells from a patient with Parkinson’s disease, a debilitating disease where dopaminergic neurons are destroyed, and turn these cells into dopaminergic neurons and put these neurons back into the patient with the disease. This kind of protocol would obviate the need for immunosuppresive therapy or patient specific stem cells.
Review Summary: 
SYNOPSIS: This proposal aims to test the hypothesis that hES cells can be used to study human embryonic development in vitro – in particular, fate decisions of embryonic ectoderm as it chooses to become neural or epidermal. Specific Aim 1 will generate new GFP, RFP, and YFP reporter lines under control of known neural or epidermal lineage promoters, and following differentiation, FACS and immunophenotyping will be used to determine if these new reporter lines accurately reflect the differentiation state. Aim 2 will apply gene expression profiling on the lines generated in Aim 1 to identify the succession of genes required for neural versus epidermal fate choice, paying particular attention to genes shared amongst the two. Finally, Aim 3 will examine known murine equivalent genes and novel genes identified in Aims 1 and 2 and overexpress them in human embryoid bodies (hEB) to affect growth and differentiation of the hEBs. These cells will then be transplanted in immunocompromised mice to follow their fate in vivo. INNOVATION AND SIGNIFICANCE: The proposed work is highly innovative. The generation of the described reporter cell lines will be extremely useful in developing models of human embryonic development, in particular in comparison of a neural versus epidermal fate. The data generated could have significance toward an understanding of mechanisms involved in transdifferentiation. STRENGTHS: This is an innovative, cutting-edge proposal. hESC and hEB studies offer numerous advantages to studying human embryonic development that cannot easily be studied in human embryos and fetuses, and the choice point to be studied here (ectoderm to neural vs epidermal) is excellent for establishing this model. Recent studies showing transdifferentiation potential of skin cells to brain cells adds credence to the proposed studies attempting to determine such fate choices in hESCs. The choice of trying to generate dopamine neurons vs keratinocytes also seems like a good one to study, based on the extensive body of literature already available for fate choice determining factors of these two populations. This is a gifted young investigator, fresh out of a very productive postdoctoral and graduate training experience. He is working in an excellent academic setting with good collaborations. He has most of the tools in place and experience with working with cells in developmental biology paradigms like this to assure successful completion of these interesting studies. WEAKNESSES: While the initial parts of the grant proposal are well-written and thoughtful, the Research Design section has no structure and would be an easier read if it were separated out into Specific Aims. There is also a lack of detail in some places in the method descriptions. For example, in the neural transplantation section, to simply cite references on the techniques to be used is insufficient – despite the applicant's acknowledgement of relying on Dr. Kornblum here. Another noted weakness is that the studies of Aim 3 aimed at expression of the murine equivalent in mouse ectoderm to see if the candidate genes discovered in Aim 2 will be expressed during the transition of ectoderm to neural or epidermal tissue is interesting, but this potentially time-consuming and maybe-not-simple-to-interpret study does not completely fit with the other proposed studies. DISCUSSION: This PI is exactly the type of young investigator that the RFA intended to attract, although it was noted that the applicant needs to develop better grant writing skills. Reviewers agreed that the development of hESC reporter lines is simply not fundable by NIH, and once created these cells will be in high demand. The reviewers predict success from these studies given the extensive literature available for studying fate choice in dopamine neurons, and the tools and experience already in place. This is a fresh perspective on the development of dopaminergic cells.
Conflicts: 

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