Transdifferentiation of fibroblasts into neurons by cell reprogramming

LOPEZ LEÓN M.; HEREÑÚ CB; REGGIANI PC; GOYA RG

Bethesda, MD

Otro; Brain Disorders in the Developing World Meeting.; 2014

National Institutes of Health (NIH)

In 2006, Takahashi and Yamanaka reported that the transfer of the four pluripotency genes Oct4, Sox2, cMyc and Klf4 (known as the Yamanaka genes), to somatic cells can reprogram them, taking the cells to a stage in which they behave as embryonic stem cells. The possibility of generating this type of cells, known as induced pluripotent stem cells (iPSC), has opened a horizon of hitherto unimagined possibilities for the development of personalized therapeutic strategies. Cell reprogramming is also a powerful methodology to transdifferentiate a somatic cell type into a different somatic cell lineage. In particular, there is a keen interest in transdifferentiating fibroblasts and other somatic cell types to either mature neurons or neural precursors (NP) which can later be used for implementing cell therapy for neurodegenerative pathologies like Alzheimer?s (AD) and Parkinson?s disease (PD). It is known that neurons and NP can be produced by direct transdifferentiation of mature fibroblasts. In one study transdifferentiation was achieved by transferring the Yamanaka genes to tail-tip mouse fibroblasts. This study revealed that a brief (4 days) expression of these four genes takes the fibroblasts to a stage of ?epigenetic instability? which under appropriate culture conditions revert towards NP and neurons. The first step for cell reprogramming involves the transfer of an appropriate set of pluripotency genes to the somatic cells to be reprogrammed. Our research group has established an advanced technological platform for the construction of adenoviral vectors and regulatable gene expression systems as well as for the implementation of the magnetic field-assisted gene transfer technology called magnetofection. Since the main interest of our group is the development of interventive strategies aimed at the treatment of age-related neuropathologies, we have decided to incorporate cell reprogramming to our battery of technologies in order to perform experimental regenerative medicine in suitable animal models. Capitalizing on our expertise in the construction of regulatable adenoviral systems we have undertaken a collaborative effort with Dr. G. Mostoslavsky of the Boston University School of Medicine, with the goal of constructing a regulatable helper-dependent (HD)-adenoviral vector harboring an expression cassette, termed STEMCCA, constructed in 2009 by Mostoslavky?s team. This polycistronic cassette expresses the four Yamanaka genes and therefore allows transdifferentiating somatic fibroblasts into neuronal cells. We report here the construction of a regulatable helper-dependent-recombinant adenoviral vector (HD-RAd) for the simultaneous expression of the pluripotency genes Sox2, c-Myc, Klf4 y Oct4 and the reporter gene for humanized green fluorescent protein (hGFP). These genes are being cloned in a regulatable bidirectional system recently constructed in our laboratory. The system consists of a regulatable bidirectional cassette which expresses hGFP and has an empty multiple cloning site (MCS) available for cloning genes of interest. In this case, the MCS is being used for cloning a tandem harboring the four pluripotency genes mentioned above. Both, the hGFP gene and the pluripotency gene tandem are under the control of a single regulatable (Tet-Off) bidirectional promoter which can be reversibly inhibited by the antibiotic doxycycline (DOX).  We plan to use this new gene delivery tool  for implementing regenerative medicine in the brain of aging rats.