Despite predictions of transdifferentiation being a technology of the future, Dr. Marius Wernig’s lab at Stanford has recently discovered a method of reengineering neurons directly from fibroblasts by the the forced expression of transgenes. This is the same method by which induced pluriptent stem cells (iPSCs) are produced, and transdifferentiation, the engineering of cells so they change their type without preceding through an intermediate stage, has been suggested as the logical progression from iPSCs. Its success suggests that, along with iPSC technology, cellular differentiation and cell fate are far more flexible than previously thought. The new discovery that somatic cells can be turned into completely different cell types by a cocktail of a few genes revolutionizes previous thought about the unchangeable nature of fully differentiated cells, and adds new theories as to how cell fate is determined. The research also importantly shows that reengineering cells with transcription factors can create the complex structures and functions of somatic cells, not simply undifferentiated stem cells. A combination of three transcription factors that are specifically found in the brain were shown to be able to convert both embryonic and postnatal fibroblasts directly into neurons, in much the same way iPSCs are created, but without preceding through any pluripotent stem cell intermediate. The lab tested nineteen potential genes to see what combination could efficiently convert mouse fibroblasts into neurons in vitro. The gene Asc11 alone could generate immature neurons with undeveloped properties. Another eighteen genes were then tested in combination with Ascl1, and five genes (Brn2, Brn4, Myt1l, Zic1, and Olig2) substantially improved neuron development. While none of these five genes generated induced neuronal (iN) cells when tested individually, it was found that the three factor combination of Ascl1, Brn2, and Myt1l produced the highest quantity of neurons with the most mature action potentials. As a result this combination was concluded to be the ideal grouping of factors for inducing neuron phenotypes.
Induced Neurons: Are they just like normal neurons?^
A difficulty in evaluating whether transdifferentation is successful is that unlike the relatively simple phenotypes of undifferentiated cells, somatic cells have far too many characteristics that must be tested to ensure that complete reengineering occurred. Induced neuronal cells (iNs) express neuron-specific proteins, generate action potentials and form functional synapses but it is still not known how, if at all, the iNs differ from normal neurons. The cells expressed three neuron marker proteins, MAP2, NeuN, and synapsin, and produced spontaneous action potentials, which are the distribution of charged ions across a neuron membrane that causes a signal to be propelled down the neuron’s axon and to the next neuron. Action potentials promote neural communication as they allow a message to cross a cell before neurotransmitters become involved in its movement between two cells. These action potentials were blocked when a sodium ion inhibitor, tetradotoxin, was introduced, as would occur in normal brain tissue. In normal brains, the concentration of sodium across the membrane of the neuron is essential for the cells to generate action potential and relay electrochemical messages through the neuron and onto the connecting cells. The cells’ expression of functional membrane channel proteins that allow sodium ions to flow in and out of the neuron supports the claim that iN cells and normal neurons exhibit identical membrane properties.
Problems in iN Generation^
The iN cells had various phenotypes, but did not exhibit all types of nervous tissue. Most iNs were excitatory neurons, while almost none contained periperin, a protein characteristic of neurons in the peripheral nervous system. The majority of iN cells were excitatory cells that expressed markers indicative of cortical identity, i.e. specific to cells typically found in the cortex. Further research may focus on the generation of iN cells of other specific neuronal subtypes, not to mention the generation of iNs from human cells (currently only mouse iNs have been successfully engineered). Additional neural transcription factors may aid in creating neurons of more specific phenotypes.
Like induced pluripotent stem cells, iNs go through a gradual process of reengineering. While immature neuron-like cells can been seen as early as three days after infection, it takes five days for branching neuronal cells to form. Physical maturation continues over several weeks. The efficiency of converting cells into iNs ranged from 1.8–7.7%, which is substantially better than efficiency of iPSC production. However, iNs cannot proliferate like iPSCs, so creating a larger quantity of cells is crucial.
An important question concerning iNs involves their ability to function as neurons by forming functional communication with other cells. This is a crucial requirement if they are to be used as tissue replacement in the future. The researchers tested whether iN cells have the capacity to form functional synapses with other iN cells and whether iN cells were capable of integrating into preexisting neural networks. When iN cells were grown with actual neurons, spontaneous and rhythmic neural activity was observed. The cells could receive synaptic inputs from the normal neurons, demonstrating their ability to integrate into preexisting tissue. It was also shown that iN cells are capable of forming functional synapses with each other.
Transgenes that Engineer Neurons^
Like iPSCs, the gene combination for creating iN cells allows some leniency and variation, but a certain “recipe” seems the most effective. It remains to be seen if different genes or different ratios of the genes that Dr. Wernig’s lab identified will even further improve efficiency of iN production. While Ascl1 alone is sufficient to induce some neuronal traits, such as expression of proteins that generate action potentials, the addition of Brn2 and Myt1l creates more mature cells with increased efficiency up to 19.5%. The highly efficient production of iNs makes it unlikely that they are merely formed from rare stem or precursor cells in the starting cell population, as great care was taken to exclude neural tissue in the isolation of the initial cell population, and no neurons or neural progenitor cells were detected in the culture. Future studies are nonetheless needed to unequivocally demonstrate that cells that have their own unique morphologies can be directly converted into neurons, and that the iNs are not mere derivatives of stem cells.
Why certain neuronal subpopulations (such as the cortical neurons) are more favored than others is another aspect of iN technology that remains to be researched. It may be that high expression of neural cell-fate determining factors directs certain cell types to form, so they are reengineered more often. Different cell types are produced during development by sets of transcription factors that cause cell type specific proteins to be produced. Each cell can be thought of as a person who walks by a lot of doors, but only has one key (specific transcription factors) that allows him to open one door, i.e. the cell can only become the type specified by its transcription factors.
iN cells are a possible alternative to iPSCs for generating patient-specific neurons. The generation of iN cells is fast, efficient, and has the major advantage over iPSCs that it does not go through a stage of pluripotent stem cells that are susceptible to tumor production. The iN cells could also provide new methods for studying cellular identity and neural development. They have potential uses in neurological disease-modeling, drug discovery, and regenerative medicine. Formerly, transdifferentiation was never thought of as anything besides a futuristic version of cell engineering that would take many years to accomplish. The Wernig lab has shown that cells with more complex morphologies can indeed be generated directly from other cell types using much the same method as iPSCs, and although much remains to be tested, this new technology may revolutionize cell therapies as more cell types are derived.
For Further Reading^
Vierbuchen, et al. “Direct conversion of fibroblasts to functional neurons by defined factors.” Nature. 25 Feb. 2010, 463 (7284):1035-1041.
Well written, fairly accessible article. Some parts a bit technical but very nice section on the next steps for iN.
A. Lanctot 2011.