If the therapeutic potential of human embryonic stem (ES) cells is to be realized, the ability to produce pluripotent stem cells with defined genetic backgrounds is essential. Pluripotent cells, through differentiation, have the ability to become any cell type. For basic and applied research, access to human ES cells derived from patients with specific diseases would be very valuable. In a more therapeutic setting, the ability to isolate differentiated cells from an individual patient and reprogram these cells to a pluripotent, stem-like state may ultimately lead to truly personalized medicine. Thus, an understanding of the genes that establish and maintain the pluripotent state of human ES cells is critical to future medical applications. The overall goal of this research program is to establish an experimental protocol to efficiently reprogram differentiated human cells into a pluripotent state. It has recently been shown that the expression of only four genes in mouse fibroblasts reprograms these cells to a pluripotent state. We will pursue a similar strategy using differentiated human cells. Importantly, we have recently developed a new technology for regulating protein expression in human cells, and this technology will allow us to regulate the expression levels of these reprogramming proteins with unprecedented control. The first half of this proposal focuses on the regulated expression of several genes that are known to be involved in the establishment and maintenance of pluripotency in human ES cells. By using our technology to regulate the levels of these proteins in differentiated cells, we will define the expression levels that lead to efficient nuclear reprogramming. The second half of this proposal will focus on epigenetic reprogramming. Epigenetic marks are modifications to DNA and the supporting histones that do not change the actual DNA sequence but that regulate gene expression. We will use our new technology to regulate the expression of proteins that are involved in maintaining the epigenetic state that is characteristic of embryonic stem cells. We believe that expression of these epigenetic modifiers, coupled with the regulated expression of pluripotency-inducing genes, will dramatically improve the efficiency for reprogramming differentiated cells to a pluripotent state.
Statement of Benefit to California:
Pluripotent cells such as embryonic stem cells, through the processes of differentiation, have the ability to become any cell type. If the therapeutic potential of human embryonic stem cells is ever to be realized, the ability to produce pluripotent stem cells with defined genetic backgrounds is essential. Access to human ES cells derived from patients with specific diseases would be very valuable for both basic and applied research. In a more therapeutic setting, the ability to isolate differentiated cells from an individual patient and reprogram these cells to a pluripotent state may ultimately lead to novel treatments for human diseases. Thus, an understanding of the genes that establish and maintain the pluripotent state of human ES cells is critical to future medical applications.
In this proposal the investigators propose to test whether human adult cells can be reprogrammed to an ES like fate. Studies will be done using in an initial phase Oct4, Sox2 and Nanog, with, if needed, Klf4 and Myc. A second series of genes that will be tested are demethylating genes expressed in ESCs. The investigators also plan to introduce the genes into cells such that the protein products can subsequently be eliminated by simple addition of a drug (FKBP12 and TMP), as they hypothesize that this will be needed to test whether presumed reprogrammed cells can differentiate. As far as the the goal of the RFA is concerned, hESCs are used as comparator cell population, even though no direct studies on those cells are being proposed. SIGNIFICANCE AND INNOVATION: This proposal is highly innovative, utilizing a unique regulatable system to modulate the expression of pluripotency-associated genes in human somatic cells with the goal of reprogramming them back to an embryonic state. If this proposal were successful it would obviate the need for human embryos and oocytes to generate human pluripotent stem cells and might also shed light on the molecular mechanisms involved in nuclear reprogramming. Thus, the proposal is highly significant. STRENGTHS: This is a strong proposal wherein investgators will evaluate whether expression of genes known to be important for pluripotency of ES cells, and/or demethylating enzymes known to be expressed in ESCs can reprogram adult human cells to an ES fate. The investigators hypothesize that expression will need to be "tunable", and propose to use technology developed in their lab that allows the protein to be eliminated in a tunable fashion. This will likely be very important in determining the degree to which reprogrammed cells are capable of differentiation. Although it remains to be seen whether simple addition of a few genes to adult cells can indeed reprogram the cells to an ES-like fate, the choice of genes to be added is based on insights in ES pluripotency; recent data from mouse cells that a combination of 4 genes can reprogram mouse fibroblasts to an ES-like state; and the fact that demethylating enzymes will be included is interesting. The investigators are aware of this possibility and propose to introduce a number of other genes that play a role in pluripotency (Ivanobva et al, 2006), as well as genes that are involved in histone modification, a second mechanism underlying epigenetic regulation of pluripotency. The use of a novel regulatable expression system developed by the investigators is one of the major strengths of this proposal. In addition, the research is highly collaborative. Overall, the proposal is well written, well planned, and well supported by preliminary results. WEAKNESSES: There are scientific weaknesses in this proposal. For instance, close inspection of the genes upregulated in hES cells shows that certain homologues (e.g. Klf4) are not upregulated in human that are in mES cells. Most likely, this proposal will need to look at a number of genes in an attempt to identify those with a similar capacity to reprogram somatic cells a la Yamanaka. Moreover, the success of Yamanaka's technique is based on the ability to select for reprogrammed cells using an Fbox15-neo knock-in cell line. Thus only cells that have been reprogrammed back to a status where they would express Fbox15 are drug resistant. This proposal does not appear to suggest or describe a similar strategy. It is highly unlikely that a strategy without selection would work. The proposal is very ambitious, and high risk. However, even if only part of the proposal can be accomplished, significant new insights in methods to reprogram human cells to an ES fate will be obtained. As far as the the goal of the RFA is concerned, hESCs are used as comparator cell population, even though no direct studies on those cells are being proposed. DISCUSSION: The question was raised as to whether this application is responsive to RFA as the goal of the project is to reprogram adult cells to become hESC-like, using hESC as the control. After discussion, the consensus was that the proposal is responsive to the intent of the RFA. The project expands on the reprogramming work of Yamanaka by using a regulatable expression system. Some concern that it would not be easy to identify reprogrammed ES-like cells without a selection procedure. Noted that the success of the Yamamoto reprogramming technique was based on selection - some concern that this project won't work without selection. In spite of concerns, this was considered a 'beautiful grant' with outstanding science.