Principal Investigator Rudolf Jaenisch
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.
(iv) Generation of human ES cells with properties of mouse ES cells: A major impediment for realizing the potential of human ES cells for the study of diseases is the difficulty to grow and to genetically modify the cells. Thus, in contrast to mouse ES cells, human ES cells have a low single cell cloning efficiency, depend on TGFβ and activin instead of LIF/STAT3 for self-renewal, are very inefficient in homologous recombination impeding the generation of gene targeting and have to be passaged mechanically instead of by trypsin to avoid chromosomal aberrations. In addition, female mouse ES cells are pre-inactivation with both X chromosomes being active (XaXa) whereas conventional human ES cells have already undergone X inactivation (XiXa). We have succeeded in converting conventional human ES cells into a pluripotent state that resembles mouse ES cells by all the criteria mentioned above. It is hoped that these new human ES cells will overcome the many obstacles that presently impede the use of human ES cells for disease research.