Jaenisch Lab Research Summary

Our long-range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. An important question is to understand the different epigenetic conformations that distinguish differentiated cell states and to define strategies to transdifferentiate one differentiated cell type into another. Embryonic stem cells are of major significance because they have the potential to generate any cell type in the body and, therefore, are of great interest for regenerative medicine. A major focus of our work is to understand the molecular mechanisms that allow the reprogramming of somatic cells to an embryonic pluripotent state and to use the potential of patient specific pluripotent cells to study complex human diseases.

Embryonic stem cells and the control of self-renewal

The transcription factors Oct4, Sox2, and Nanog have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have, in collaboration with the Young lab, identified Oct4, Sox2, and Nanog target genes using genome-scale location analysis. We found, surprisingly, that Oct4, Sox2, and Nanog co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also show that Oct4, Sox2, and Nanog collaborate to form regulatory circuitry in ES cells consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how Oct4, Sox2, and Nanog contribute to pluripotency and self-renewal.

Nuclear Cloning and the Reprogramming of the Genome

A major issue raised some 50 years ago in seminal frog cloning experiments was the question of whether nuclei of terminally differentiated cells can be reprogrammed to generate animals after nuclear transfer. We have shown that cloned mice can be derived from mature B and T cells or from mature neurons by nuclear transfer, demonstrating that the genome of terminally differentiated cells can be reprogrammed to direct development of a new animal.

Transplantation of functional cells for tissue repair represents an exciting strategy for possible treatment of patients suffering from degenerative ailments such as Diabetes, Parkinson’s or heart disease. A major impediment is the availability of suitable cells that could be used for transplantation and would not be rejected due to immunological incompatibility. The promise of nuclear transplantation (sometimes called “therapeutic cloning”) is to provide “customized” embryonic stem cells that could be used for patient specific therapy. We have performed a "proof of principle" experiment in a mouse disease model to provide evidence that therapeutic cloning combined with gene therapy represents a valid strategy for transplantation therapy. However, the complexity and inefficiency of the NT procedure makes it unlikely that “therapeutic cloning” could be used in a routine clinical setting in the foreseeable future.

In vitro reprogramming of somatic cells to a pluripotent state

The recent success in reprogramming somatic cells to pluripotent Induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. A major focus of our work is (i) to study the molecular mechanisms of somatic reprogramming and to devise efficient approaches for the reprogramming of mouse and human somatic cells; (ii) to derive patient specific iPS cells for the generation of tissue culture models of major human diseases; and (iii) to establish proof of principle experiments for the eventual therapeutic use of iPS cells.

(i) Molecular mechanisms: The study of induced pluripotency is complicated by the need for infection with high titer retroviral vectors resulting in genetically heterogeneous cell populations. We have generated genetically homogeneous “secondary” mouse and human somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. This system facilitates the characterization of reprogramming and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors. For example, the secondary system has allowed us to define the role of stochastic epigenetic events and of cell proliferation during the reprogramming process. Also, we have been able to generate iPS cells and mice from different somatic donor cells such as mature B cells, intestinal cells and neural precursors.

(ii) Patient specific iPS cells: Most current methods for reprogramming human somatic cells preclude consideration for cell replacement therapies since they rely on the delivery of the four reprogramming factors by retroviral transduction, which carries the risk of tumor formation. Also, a concern is that the low level of provirus expression that is consistently detected in the iPS cells may affect other biological characteristics such as differentiation potential. An important issue of the field is to generate vector free iPS cells.

To decrease the possibility of provirus-mediated insertional mutagenesis we have generated human iPS cells with a single-copy proviral insert by using a polycistronic vector to deliver the reprogramming factors. In addition we have shown that fibroblasts from patients with sporadic Parkinson’s Disease (PD) can be efficiently reprogrammed using vectors that could be excised by Cre-mediated deletion thus generating Parkinson patient-derived iPS cells that are free of the reprogramming factors. The cells maintained all of the characteristics of a pluripotent ES cell-like state after removal of the transgenes. Importantly, genome wide gene expression analysis revealed that the factor-free iPS cells clustered more closely with embryo-derived human ES cells than with the parental virus-carrying iPS cells, consistent with the notion that the presence of vectors may affect the properties of iPS cells. A major goal is to establish in vitro differentiation systems that allow us to study the pathogenesis of neurodegenerative diseases such as Parkinson’s, Alzheimer disease or ALS in the Petri dish and to eventually isolate small molecules that could be used for therapy.

(iii) Therapeutic potential of iPS cells: One of the most exciting applications of the iPS cell technology is the use of patient specific cells for the treatment of diseases such as Diabetes, Parkinson’s or blood disorders. We have, as a proof of principle therapy study, demonstrated that iPS cells derived from autologous skin cells of a mouse with Sickle Cell Anemia can induce complete recovery when transplanted into the mutant mice. In a second model we demonstrated the integration of iPS derived neurons into fetal brain and the subsequent reduction of symptoms in rats with Parkinson’s disease. Both of these models are encouraging and argue that iPS cells can be used for the therapy of major diseases.

Cancer

Genomic hypomethylation is a widely observed and early step in human tumorigenesis. Using different mutant alleles of the Dnmt1 gene we have shown that hypomethylation results in a substantial increase in the genomic mutation rates, the mutations being caused by enhanced mitotic recombination. These results are significant as they may explain the selective advantage of hypomethylation in early stages of transformation: hypomethylation leading to genomic instability may provide the incipient tumor cell with a mechanism to efficiently delete tumor suppressor genes by LOH. The MTases Dnmt3a and b are the best candidates to cause the silencing of tumor suppressor genes by de novo methylation. Indeed, our results show that Dnmt3b is crucially important for cancer as it promotes the silencing of tumor suppressor genes.

The importance of epigenetic alterations leading to cancer was demonstrated by the reversal of the malignant state. Nuclear transplantation was used to reprogram cancer cells to pluripotent ES cells that were able to generate chimeric mice consistent with the notion that the malignant phenotype of the donor cancer cells was largely determined by epigenetic, i.e. reversible alterations. Current efforts are directed towards using in vitro reprogramming to revert the phenotype of tumor cells including cancer stem cells.

Last updated May 2009.

SELECTED PUBLICATIONS

Boyer, L.A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L., Lee, T.I., Levine, S.S., Tajonar,A., Ray, M.K., Wernig, M., Otte, A., Vidal, M., Young, R.A. & Jaenisch, R. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349-353 (2006).

Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., Lengner, C.J., Dausman, J.A. & Jaenisch, R. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250-264 (2008).

Hanna, J., Wernig, M., Markoulaki, S., Sun, C-W., Meissner, A., Cassady, J., Beard, C., Wu, L-C., Brambrink, T., Townes, T. & Jaenisch, R. Treatment of Sickle Cell Anemia mouse model with IPS cells generated from autologous skin. Science 318, 1920-1923 (2007).

Hochedlinger, K. & Jaenisch, R. Generation of monoclonal mice by nuclear transfer from mature B and T donor cells. Nature 415, 1035-1038 (2002).

Hochedlinger, K., Blelloch, R., Brennan, C., Yamada, Y., Kim, M., Chin, L. & Jaenisch, R. Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875-1885 (2004).

Hockemeyer, D., Soldner, F., Cook, G.G., Gao, Q., Mitalipova, M. & Jaenisch, R. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell 3, 346-353 (2008).

Rideout, W.M., Hochedlinger, K., Kyba, M., Daley, G., & Jaenisch, R. Correction of a genetic defect by nuclear transfer and combined cell and gene therapy. Cell 109, 17-27 (2002).

Soldner F., Hockemeyer D., Beard C., Gao Q., Bell G.W., Cook E.G., Hargus G., Blak A., Cooper O., Mitalipova M., Isacson O., Jaenisch R. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-77 (2009)

Wernig, M., Meisner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E. & Jaenisch, R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 (2007).

Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., Jaenisch, R. (2008b). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA Apr 7 105, 5856-61 (2008).

Mature B cells reprogrammed to stem-cell-like state

Reprogrammed cells reduce Parkinson's symptoms in rats

Reprogrammed adult cells treat sickle-cell anemia in mice

Adult stem cells lack key pluripotency regulator

Scientists show cloning leads to severe dysregulation of many genes