Embryonic stem (ES) cells have great promise for treating many human diseases and it is believed that this cell therapy someday will revolutionize medicine. However, the rejection of the introduced ES cells by the patient’s immune system is a big challenge lying ahead when applying the currently available human ES cell lines to clinical trials. The recent breakthrough of creating human embryonic stem cells (ESCs) from human embryos by therapeutic cloning has offered a resolution and highlights the possibility of making so called “autologous” cell lines specific to individual patients. However, this technique is not without problems because the successful rate of cloning is extremely low. Furthermore, the technical difficulties make it hard to be broadly employed in common research and clinical facilities. We thus propose to develop a simple and efficient method to replace therapeutic cloning in creating personalized stem cells. We will achieve this goal by using a two-step “cell reprogramming” procedure. Specifically, cells are collected from the skin of the patient and are converted into embryonic stem-like (ESL) cells using defined ES factors, which will induce a complete cell reprogramming in skin cells by activating a panel of ES-specific genes and silence genes specifically expressed in skin cells. These ESL cells have the same features as ES cells and are capable of self-renewing and differentiating into all the adult cell types for clinical cell therapy. Most importantly, they are stem cells derived from the patient and will not cause any side-effects related to immune rejection by the patient’s defense system. As a result, these patient-derived stem cells may function better when implanted in diseased organs in cell therapy than currently available ES cell lines that are cloned from early embryos. Thus, there is every reason to hope that this revolutionary new approach will result in radically improved ways to create human stem cells for treatment of disease.
Statement of Benefit to California:
This project proposes to develop an efficient cell rejuvenating method to create patient-specific pluripotent cells used in cell regeneration therapy. We believe this project will make the following contributions to the stem cell research for the state of California. 1. The project will provide a simple and useful complement, rather than human therapeutic cloning, to reprogram somatic nuclei in creating customized stem cells. This approach is cost-effective and time-saving, and it may eventually lead to an alternative approach for creating genetically tailored human embryonic stem (ES) cell lines for use in stem cell research and treatment of human diseases. 2. These pluripotent cell lines are patient-specific and are unlikely to be rejected by the patient’s immune system when transplanted into the body. Thus, they may be much safer than ES cells that are derived the embryo when applied to clinical cell therapy. 3. Identification and characterization of reprogramming factors in ES cell extracts will benefit biomedical and genetic studies aimed at understanding how to reprogram differentiated cells to an embryonic state and thereby increase their developmental potential. 4. With this powerful in vitro cell reprogramming technique, it is highly possible to create a stem cell bank in California that holds thousands of ES cell Lines with varied HLA types. These stem cell lines will be immediately available for basic research and clinical studies.
SYNOPSIS: The PI proposes to use in vitro reprogramming to generate ES-like (ESL) cells from fibroblasts. The idea is to eventually generate patient-specific ESL from somatic cells to avoid immune rejection and generate differentiated cell types. The project appears to derive from preliminary experiments (not shown) indicating that the forced expression of Sox2, Oct4, and Nanog (or the S/O, myc, Kif4 combo) fails to reprogram human fibroblasts, unlike in mouse. Therefore the PI proposes a “two-step” process, by which the somatic cells will first be transduced to express SON, followed by cell permeabilization and exposure to “factors” in ES nuclear extracts that will facilitate reprogramming. The first aim is to show that this works. Dr. Hu will generate an expression construct to express SON and GFP using CMV and TK promoters and IRES elements. Following subsequent exposure to ES cell nuclear extracts, cells will be cultured in hanging drops and aggregates placed back into normal ES cell culture conditions. Experiments in the second aim will test if these are equivalent to bona fide human ES cells by looking at markers, microarrays, teratoma formation, cytogenetic analysis (karyotypes) and investgation of methylation patterns and imprinting. Experiments in the 3rd aim will seek to define the “factors” in nuclear extracts that work. The primary approach is an expression cloning protocol to screen cDNAs. SIGNIFICANCE AND INNOVATION: The project is based on approaches that have been shown to work in the mouse ES cell system, and it is innovative to apply both together to the human system. The significance of achieving patient-specific ESL from somatic cells is very high, since this could provide a tremendous source of pluripotent cells. Establishing ES-like cells from skin biopsies is innovative. Whether it can be done and whether those “pluripotent” cells thus derived will function as ES-like cells is at present unknown. STRENGTHS: The major strength of the proposal is the concept of generating these cells based on what is known in the mouse system. In principle it is an important problem and with high potential payoff. There is some evidence from mouse fibroblast lines that such reprogramming can take place. It is not clear how similar the resulting cells are to mouse ES cells, however. WEAKNESSES: It is not clear why the PI proposes to use a more complicated two-step process rather than just optimizing a one-step process. Combining two very inefficient protocols seems unlikely to generate a very efficient process. If the expression of SON or SOMK is not sufficient to work in the human system, it might be more productive to determine why this is the case, and fix it directly. For example, are there other candidates than SON that make sense to test in the human system? Perhaps expression levels just need to be enhanced. It was very unclear what is meant by pre-programming compared to re-programming. What’s the difference? Presumably one needs to interfere with the network of stemness regulators and it would be interesting to find out why the human system differs. However, the identification of the novel factor in “step 2” (aim 3) will only work if it is one factor, and it is not clear how at any rate the PI will have optimized for “step 1” before applying this next process. What is the benchmark to know that the cells are pre-programmed successfully, and how does this differ from how the cells will be assayed for phenotype following step 2? It is unlikely that expressing 3 transcription factors in skin fibroblasts followed by incubating cells with extracts from the H7 human ES cell line will turn the fibroblasts into ES cells. Perhaps it would be more appropriate to screen an H7 cDNA expression library for additional molecules that would confer ES properties on the fibroblasts. However, rather than fishing for a novel factor (for which there is only negative data to provide evidence that it exists), it might be more productive to define how the human ES cell differs from the mouse ES in terms of this stemness network. Ideally, one would want to examine cells grown from (new) skin biopsies rather than human cell lines, since these would be the cells used to personalize the technique, and also these would have the advantage of not having been in culture for long periods, during which many changes in gene expression could take place. DISCUSSION: There was no further discussion following the reviewers' comments.