Stem cells have unique potential to cure human disease. Their ability to give rise to virtually any type of cell has given hope to the public for curing all types of ailments. For this reason, stem cells continue to capture the headlines of the mass media and esteemed scientific journals. Embryonic stem cells (ESCs) are probably the best known stem cell available to researchers. Most of our knowledge about these important cells comes from studies in animals, particularly in mice. Recent animal studies have revealed amazing success at curing neurological diseases such as Parkinson's Disease (PD). It is no coincidence that progress has been made first in the area of the brain. The nervous system seems to be the default pathway for many stem cell lines that have been studied. The research proposed here endeavours to discover why the cells follow this pathway. The cells we will study are those that have the most benefit to patients, human embryonic stem cells (hESCs). We know that as stem cells become nerve cells (when they mature or differentiate), they develop special abilities as part of their function in the brain. They must carry electrical signals to function. These electrical properties are akin to the signal carried to your TV by a cable. The signal has to propagate to your TV through the signal cable. Neurons do the same thing using special proteins called ion channels. Ion channels are proteins stuck in the cell membrane that allow small ions like sodium and potassium to move back and forth across the cell membrane to change their electrical state. These channels have been implicated in many human diseases. Their proper functioning is critical to human health. The research proposed here seeks to understand how these channels affect the differentiation of hESCs. That is, we want to find out exactly what type of ion channels are made by stem cells as the mature into brain cells. We also want to know when the ion channels are made by the cells. Thus we hope to discover the function of ion channels in the differentiation of these important cells. Once we have this knowledge we can begin to manipulate the channels to affect stem cell differentiation or their cellular fate. That is, we want to be able to make the cells mature into the type of cell we need. There are many known molecules that are known to affect ion channel function. Some of these open the channels and some of them block them. By using these agents, we can manipulate the function of the ion channels to make the stem cells follow a certain fate. This knowledge will be another tool scientists can use to manipulate the stem cell fate. The more we can fine tune the differentiation of stem cells, the better we will be at designing therapies to cure any number of human diseases.
Statement of Benefit to California:
The research project proposed here seeks to understand how ion channels affect the differentiation of human embryonic stem cells (hESCs). The ability to direct the differentiation of these cells has high potential for use in alleviating a variety of diseases. This will potentially have tremendous medical benefit to the people of California. Funding of this work will also employ and allow the training of up to 4 University students, one technician, and one faculty collaborator in human stem cell work. This training will benefit the State of California by creating trained stem cell workers that will continue to keep our work force well trained and competitive in this growing field.
SYNOPSIS: This applicant proposes to study ion channels in hESCs that are differentiating into neurons. The Principal Investigator (PI) will be using Western-blotting, immunocytochemistry and RT-PCR to identify the channels, and will use patch clamping and other methods to characterize the channels. The PI will determine whether the expression and modulation of ion channel activity has an effect on controlling stem cell differentiation. The work aims to characterize ion channel expression profiles in differentiating hESC neurons using both NIH-approved and non-approven cell lines. These will be compared against human umbilical cord mesenchymal stem cells that are differentiating into neurons. SIGNIFICANCE AND INNOVATION: The goal of this application is to evaluate the expression of ion channels during hES cell differentiation into neurons and then to test whether blockade of the channels expressed has any influence on subsequent differentiation. There is a need to characterize the channel distribution changes in developing hESCs and also in human umbilical cord mesenchymal cells. The proposal suggests some important work, including the electrophysiological characterization of channels on developing cells, but this is not a particularly innovative or original proposal. STRENGHTS: The investigator is experienced with the proposed methods and is likely to be able to carry out the proposed experiments rigorously. The PI has published several studies of channel expression in other cells, and the applicants have already begun similar experiments using cells derived from human umbilical cord matrix (HUCM). Importantly, they demonstrate the ability to culture and record from these cells. WEAKNESSES: There are several scientific concerns with this proposal. The investigator does not consider the question of heterogeneity and how to deal with the likely situation of having a mixed population of cells that would be very hard to analyze/interpret. The applicant also does not discuss which specific antibodies will be used to evaluate neural differentiation or channel subunit expression. Moreover, the criteria for concluding that neural differentiation has occurred seem problematic. The cells in Fig. 2 and Fig. 5 are described as neurons based solely on their phase morphology, which is not adequate for drawing this conclusion. Another weakness of these studies is that they are not particularly innovative. In addition, the Investigator has not been very productive since completing his PhD in 1998 and doing a postdoctoral fellowship from 1998-2001. The PI has published only four papers since 1998. DISCUSSION: The success of this application is critically dependent upon the reliable identification of the cell types being evaluated. The proposal needs to lay out specific criteria for assigning identity to each cell that is recorded. Also, it would be helpful if the applicant provided some data to suggest that it is feasible to carry out detailed electrophysiological analyses of the cells and strategies for dealing with mixed populations of cells.