Turning Everything Into Brain?

Abstract

The application of stem cell technology to create individualized therapies for patients promises to revolutionize medicine. Recent studies show that mature, differentiated cells can be reverted to a pluripotent state, thereby creating induced pluripotent stem cells (iPSCs). Such iPSCs can be differentiated into many tissue/cell lineages, and are useful both for modeling human diseases and possibly creating personalized cell-based therapies. A recent Nature paper reports another significant advance that bypasses the iPSC step, by demonstrating direct conversion of differentiated fibroblasts directly into induced neuronal (iN cells) (Vierbuchen T, Ostereier A, Pang ZP, Kokubu Y, Sudhif TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010;63(7284):1036-U450). The major differences between these two techniques are highlighted in Table 1. In the case of “pluripotent reprogramming”, differentiated cells are induced through in vitro genetic manipulation to become pluripotent (able to form all cell types except germ cells) via de-differentiation. The regulatory genes required for reprogramming cells to iPSCs have been defined. Major limitations are that pluripotent cells cannot be induced in an in vivo environment, and carry significant risk of teratoma formation due to induction-related destabilization of the cellular epigenome and genome. In contrast, although iN cells are also formed though genetic manipulation of master regulatory genes in differentiated cells, they can be induced in vivo (2), especially if the desired cell type naturally exists in the same tissue environment. iN cells are also hypothesized to be less teratogenic because these cells are created via trans-differentiation, and do not pass through an undifferentiated state like iPS cells. One notable challenge for the direct conversion process is that the genes necessary to direct such drastic cell type change are largely unknown, and will need to be identified for each differentiated cell type in future studies. Table 1 Reprogramming Strategies (Adapted from Zhou et al.) In this study published by Vierbuchen et al, the authors identified a combination of optimal transcription factors, delivered via lentiviral vectors to mouse embryonic or tail-tip fibroblasts, that induced these cells to develop neuron-like morphology and behavior (Figure 1). Within two weeks post-infection with only three selected neuronal fate determination genes (Ascl1, Brn2, and Myt1l), the iN cells developed complex neurite morphology, generated both spontaneous and stimulus-induced action potentials, and exhibited expression of pan-neuronal markers and neurotransmitters such as glutamate and GABA. These and other tested transcription factor genes were previously shown to be important for neural development. Additionally, iN cells were able to form functional synapses with a pre-existing cortical neuron network and with other iN cells when cultured with primary astrocytes. Characterizing changes in action potential height, neurite morphology, resting membrane potential, and other measurements over time demonstrated that iN cell development is highly analogous to normal cortical neuronal development. Overall, this is the first study that shows conversion of differentiated fibroblasts directly into cells with mature neuronal biological properties. Similar experiments have induced muscle specific properties in fibroblasts (3). Some caveats to consider are that this pioneering study was done in vitro and it remains to be seen if similar results can be attained in vivo. One possible method might be to develop optimized viral targeting of cell-specific surface markers to provide induction specificity in generating the desired cell type(s). Further, it will be important to ascertain whether such direct cell fate conversion remains stable over the long term without additional manipulations. The use of lentiviral vectors carries the risk that some cells incorporate multiple copies of the regulatory genes into their genome which may result in late genetic instability. However, newer methods have been developed to bypass genome manipulation to produce iPS cells through direct application of transcription factor proteins (4) or through episomal vectors (5). Perhaps these methods could be adapted to create iN cells. Finally, this study shows results obtained in mice, therefore the viability of this technology with human cells remains to be proven. Figure 1 Characterization of mouse embryonic fibroblast (MEF)-derived induced neuronal (iN) cells The clinical implications for this work span the fields of regenerative medicine and neurosurgery. Foremost is the possibility of creating an individualized, renewable source of lineage-specific cells for studying disease, testing therapies and possibly for cell-based treatments. It is difficult and impractical to obtain and culture differentiated neuronal cell types from patients, especially after trauma, stroke or degenerative disease processes. iN cells may be useful in creating regenerative therapies such as creating iN cells for repair after strokes, or creating motor neurons to treat amyotrophic lateral sclerosis or spinal cord injury. The distinct advantage is that such cells are patient-derived and would avoid immune rejection. This work demonstrates a new paradigm that may be useful in creating novel biological, individualized therapies, especially for repairing and restoring the function of the nervous system.