12    Prospects

Chapter 11 ended on an optimistic note regarding the future of neural cell therapies. In this final chapter, I want to briefly consider an alternative outcome: that cell therapies might be superseded.

A recurring theme in this book has been that predicting the future in biomedical science is a particularly pointless task given how often breakthroughs appear apparently from nowhere. How many of us predicted the appearance of iPS cells, yet see how dramatically that development has changed the landscape. There are, however, a number of endeavors on the horizon that are likely to come of age in the next few years, and they might have the effect of pushing to one side the transplantation therapies we have considered in this book. Two seem to me jointly to point in a genuinely fresh direction. The first of these is direct reprogramming.

Direct Reprogramming

The work of Gurdon, Thomson, and Yamanaka revealed something quite remarkable: if a cell can be induced to express the appropriate factors, then its fate can be fundamentally transformed. In the case of iPS cells, terminally differentiated cells—from blood, skin, or endothelium—were reprogrammed into pluripotent cells: that is, from cells with the most restricted of fates to cells with the most expansive. This was a shock to conventional embryologists, who had come to consider certain developmental steps irreversible. It was believed by many that once cells had been channeled during early development into one of the three primary germ layers (ectoderm, mesoderm, endoderm) then that step could not be reversed. Reprogramming destroyed that argument, but it raised an even more provocative question: if the correct genetic formula could be found was there any cell transplantation that could not be engineered?

The technique of iPS cell reprogramming takes a differentiated cell backward in development. From there, the cell can move forward again from the pluripotent state to become any of the various differentiated progeny to which such a cell would normally give rise (figure 12.1). The new question was: could reprogramming move a differentiated cell sideways; to another differentiated cell, for example, or a progenitor cell with a different fate? Could a fibroblast be turned directly into a neuron or a muscle cell? Or could it be turned into a neural progenitor cell or a bone marrow stem cell?

Figure 12.1

Pluripotency and reprogramming. During normal embryogenesis, the fertilized egg gives rise to a ball of cells called the “blastula,” within which is a cluster of pluripotent stem cells called the “inner cell mass.” These pluripotent cells generate all the cell types that make up the body. Reprogramming is the process whereby differentiated cells, such as skin fibroblasts, can be turned back into pluripotent cells. Direct reprogramming turns cells of one differentiated type (such as fibroblasts) directly into another (for example, neurons).

Remarkably, the answer to all of these questions turns out to be yes. As ever in science, there were straws in the wind long before biologists realized this was truly the case. Long before Yamanaka, a team in Seattle had shown that fibroblasts could be turned into muscle cells with a single gene.1 The gene in question, MyoD, we now know to be a member of a group of transcription factors (bHLH genes) intimately involved in cell fate decisions in diverse tissues—heart, muscle, and brain. At the time, however, the molecular control of cell fate was largely unknown, and the existence of families of transcription factors was only starting to emerge as a consequence of the early genome sequencing efforts. Colleagues, I recall, found this fate switch a troubling finding, but consoled themselves with the thought that these two cell types—fibroblasts and muscle cells—were actually pretty close embryologically, and anyway, strange things sometimes happened in tissue culture.

We have already met this phenomenon, “transdifferentiation”—the switching of cell fates—and noted that it has had a colorful history. While there were clear examples in vivo of cells apparently jumping from one fate to another, these were largely limited to “lower vertebrates” and involved closely related lineages. So, for example, if the limb of an amphibian is severed, cells within the stump dedifferentiate into progenitor cells (the “blastema”), which then regenerates multiple different cell types—muscle, dermis, bone—and thereby reconstitutes the lost tissue. In some species, heart cells (cardiomyocytes) can also dedifferentiate in response to damage, then redifferentiate following expansion to replace the heart tissue, and similar jumps have been observed in various tissues.2 But these naturally occurring reprogramming episodes did not necessarily suggest that unrestricted reprogramming might be achievable experimentally.

Following Yamanaka, however, a simple formulation emerged. If the combination of factors that prescribed a particular fate could be identified, then quite plausibly, expressing those factors robustly might make a cell adopt that fate. While the extreme form of this theory probably doesn’t hold up—that anything can be transformed into anything—nonetheless several quite remarkable steps have been demonstrated experimentally. Among them is the generation of neurons directly from fibroblasts.

The first demonstration of this came from Marius Wernig’s laboratory at Stanford.3 Their experiment reflected directly the approach that Yamanaka had pioneered. They sought the combination of transcription factors that would convert mouse skin fibroblasts directly into neurons, They found it required just three genes (Ascl1, Brn2, and Myt1l), and from this conversion emerged cells with all the significant properties of neurons: they grew a neuronal morphology, expressed the proteins that neurons express, formed synapses, and were electrically active. This was not, however, the first time that neurons had been directly reprogrammed from nonneuronal cells. Magdalena Götz and her collaborators had shown that transcription factors such as Pax6 and Olig2 modulated the capacity of glial cells to generate neurons.4 But generating neurons directly from skin fibroblasts was an enormous leap in embryological terms: from a mesodermal end state (the fibroblast) directly into an ectodermal end state (the neuron), with no stem cell, or progenitor phase in between.

The neurons generated from this initial Wernig study, impressive though they were, were only characterized as generic neurons: no particular neuronal fate had been specified. The question therefore arose of whether specific populations of neurons could be generated. As we’ve seen, if the history of brain cell replacement has taught us anything, it is that we need the precisely correct neuron for each job. Several labs have now derived reprogramming formulas to generate specific neuronal populations, a number of which we’ve discussed in this book. For example, Ernest Arenas and colleagues at the Karolinska Institute in Stockholm have developed a protocol to generate dopaminergic neurons,5 while Andrew Woo and colleagues at Washington University in St. Louis have made striatal neurons directly from fibroblasts.6

As well as indicating that clinically relevant neuronal populations are possible with this technology, these studies add a further wrinkle. It transpires that to achieve an optimal outcome, more than transcription factors need to go into the mix. At several points in this narrative, we’ve implied that cell fate can be determined by the correct combination of transcription factors. But as our understanding of cellular control mechanisms improves, we have discovered further cell components that participate in these processes. One such is noncoding RNAs.

For many years following the discovery of the genetic code in 1961, molecular biologists thought that the only essential role of DNA was to encode genes, which in turn encode proteins. Slightly alarming therefore was the discovery that only 1 percent or so of chromosomal DNA actually encoded conventional genes. The question became then: what is the other 99 percent doing? No less a person than Francis Crick is credited with concluding that it was probably “little more than junk.”7 So the term “junk DNA” entered the molecular biologists’ vocabulary. But, of course, this had to be wrong. Were we seriously suggesting that a cell carried megabase upon megabase of DNA for which it had no use? Rather than deceiving ourselves by calling that 99 percent “junk,” we needed to discover what it was actually doing.

We now know that much of the genome (though still not all of it) encodes RNAs that do not encode proteins. These RNAs have a direct function, rather than just being vehicles for the transport of protein-coding information from the nucleus to the cytoplasm. That function, in many cases, is to regulate the cell’s translational machinery. They change the efficiency with which proteins are produced: proteins, which they themselves do not encode. Unsurprisingly therefore, they influence cell fate decisions, and can thereby influence reprogramming. In both of the direct reprogramming steps just cited, noncoding RNAs added to the mix improve the efficiency of the reprogramming steps.

This direct reprogramming has proven of interest to potential cell therapists for fairly obvious reasons. Instead of the laborious process of generating iPS cells, then taking them through a relatively long, complex process of differentiation, fibroblasts can be turned into the desired neuronal type in a single leap. There are, however, two issues with this approach, one practical and the other theoretical.

The practical problem is that, without the stem cell intermediate step, the possibility of expanding the cell population is lost. Neurons, as we know, are postmitotic: they don’t divide. With the iPS cell approach, each reprogrammed fibroblast gives rise to a line of iPS cells that can be infinitely expanded, ultimately giving rise to billions of neurons. But with direct reprogramming, each reprogrammed fibroblast gives rise to a single postmitotic neuron. This does not amount to many cells. A halfway house might be to reprogram from fibroblasts to neural progenitor cells, bypassing the iPS cell, but still giving rise to a dividing cell, which can itself then be expanded to give rise to many neurons. Strategies are now in place to pursue this route.8

The theoretical issue relates to the mechanism underlying the direct reprogramming. Reprogramming iPS-style makes some sort of embryological sense. You make a pluripotent cell, then allow it to differentiate following the various embryological steps it would have taken in vivo. Direct reprogramming, however, makes no embryological sense. Nothing in nature, as far as we know, ever turns directly from a fibroblast into a medium spiny striatal projection neuron. This raises a number of questions regarding the veracity of directly reprogrammed change. Certainly, the reprogrammed cells have properties appropriate to the fate they’ve adopted, but have they abandoned all the indigenous programming that led them to their original fibroblast fate? This largely comes down to the epigenetic question we discussed earlier, and is the subject of current research.9

In Vivo Reprogramming

Direct reprogramming has the potential to enhance considerably the production of appropriate cells for stem cell transplantation. In combination with the final development I want to consider, there is the potential to make stem cell transplantation totally redundant. It would be ironic if reprogramming technology, which has done so much to liberate cell therapy from the constraints of cell availability and scalability, were to make the whole cell transplantation field obsolete, but this final development has the potential to achieve exactly that.

All the reprogramming we’ve considered so far takes place in a tissue culture system. What if cells could be reprogrammed in the patient? While reprogramming was limited to the production of iPS cells, this was not a plausible prospect. Turning a skin fibroblast into an iPS cell while it remained in a patient’s skin would have done more harm than good. Since iPS cells have the potential to form teratomas, an iPS cell in a patient’s skin (or anywhere else in the patient’s body) would quickly give rise to a horrible tumor. But direct reprogramming avoids that risk, and performing the reprogramming directly in the patient also potentially overcomes the expansion problem.

How might this work? Take a population of nonneuronal cells in the brain that had the potential to be reprogrammed into neurons. Why couldn’t the reprogramming vectors be injected directly into the brain, so they could so they could reprogram the nonneuronal cells near the damage site directly into the cell type that were lost? There are now a number of preclinical studies that have started to explore this scenario. For example, in 2014, Magdalena Götz and colleagues in Munich looked at direct reprogramming in the mouse brain following a stab wound.10 They used a sharp blade to induce trauma in the mouse cerebral cortex, which previous work had indicated would activate a number of nonneuronal cells to proliferate in response to the injury. They followed the wound with the injection of a gene therapy vector encoding two transcription factors (Sox2 and Ascl1). Their earlier work had suggested that these two factors would be sufficient to reprogram particular glial precursor cells, known to be activated by the injury, and turn them into neurons. Sure enough, the activated glial precursor cells in the wounded tissue incorporated the vectors, expressed the encoded genes, and new neurons began to appear in the damaged mouse brain as a consequence of this direct reprogramming.

Once again, these first experiments did not seek to generate any specific neuronal type, and that would certainly need to be achieved before a serious attempt could be made at therapy. But as we’ve seen, this specificity problem is being pursued in culture experiments, and is likely to translate in vivo. The big leap, of course, will be from mouse to human. We’ve already seen how easily that can come unstuck. Nonetheless, direct reprogramming of glial cells into neurons actually in the patient’s brain is now a distinct possibility.

In this book, I’ve tried to relate the story of how neural stem cell therapies have grown from a flimsy, tentative idea into a robust and ambitious clinical program. While no cell therapy has yet to be licensed for any brain disorder, several therapies have entered proper clinical trials, which means that quite soon we will discover which work and which do not: whether the scientific and technical innovations we’ve encountered in this book can bring real benefit to patients suffering from intractable neurodegenerative disorders.

If we wanted to identify a single measure of how far we’ve come, it would probably be this. Attending conferences on advanced therapies in years past, one used to be overwhelmed by the technical hurdles yet to be overcome. In the last few years, that has subtly changed. Now the question most often asked as we huddle around the conference coffee outlet is: how are we going to make these therapies affordable? Our big worry is that, even if the therapies are proven to work, we won’t be able to produce them cheaply enough to make them available to all who need them. As a scientist, one senses the manufacturers and regulators now looking over your shoulder, but one also has the gratifying sensation of having handed the problem on.

Notes

  1. 1.  Davis, R. L., Weintraub, H., and Lassar, A. B., “Expression of a Single Transfected cDNA Converts Fibroblasts to Myoblasts,” Cell 51, no. 6 (1987) 987–1000. https://doi.org/10.1016/0092-8674(87)90585-X.

  2. 2.  Jopling, C., Boué, S., and Belmonte, J. C. I., “Dedifferentiation, Transdifferentiation and Reprogramming: Three Routes to Regeneration, Nature Reviews Molecular Biology 12, no. 2 (2011): 79–89. http://doi.org/10.1038/nrm3043.

  3. 3.  Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Sudhof, T. C., and Wernig, M., “Direct Conversion of Fibroblasts to Functional Neurons by Defined Factors,” Nature 463, no. 7284 (2010): 1035–1041. http://doi.org/10.1038/nature08797.

  4. 4.  Buffo, A., Vosko, M. R., Erturk, D., Hamann, G. F., Jucker, M., Rowitch, D. H., and Gotz, M., “Expression Pattern of the Transcription Factor Olig2 in Response to Brain Injuries: Implications for Neuronal Repair,” Proceedings of the National Academy of Sciences 102, no. 50 (2005): 18183–18188. https://www.pnas.org/content/102/50/18183; Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K. L., Hack, M. A., et al., “Glial Cells Generate Neurons: The Role of the Transcription Factor Pax6,” Nature Neuroscience 5, no. 4 (2002): 308–315. http://doi.org/10.1038/nn828.

  5. 5.  di Val Cervo, P. R., Romanov, R. A., Spigolon, G., Masini, D., Martín-Montañez, E., Toledo, E. M., et al., “Induction of Functional Dopamine Neurons from Human Astrocytes in Vitro and Mouse Astrocytes in a Parkinson’s Disease Model,” Nature Biotechnology 35, no. 5 (2017): 444–452. http://doi.org/10.1038/nbt.3835.

  6. 6.  Victor, M. B., Richner, M., Hermanstyne, T. O., Ransdell, J. L., Sobieski, C., Deng, P.-Y., et al., “Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts,” Neuron 84, no. 2 (2014): 311–323. http://doi.org/10.1016/j.neuron.2014.10.016.

  7. 7.  Francis Crick, as quoted in Hall, S. S., “Hidden Treasures in Junk DNA: What Was Once Known as Junk DNA Turns Out to Hold Hidden Treasures, Says Computational Biologist Ewan Birney,” Scientific American, October 1, 2012. https://www.scientificamerican.com/article/hidden-treasures-in-junk-dna/.

  8. 8.  See, for example, Lee, J.-H., Mitchell, R. R., McNicol, J. D., Shapovalova, Z., Laronde, S., Tanasijevic, B., et al., “Single Transcription Factor Conversion of Human Blood Fate to NPCs with CNS and PNS Developmental Capacity,” Cell Reports 11, no. 9 (2015): 1367–1376. http://doi.org/10.1016/j.celrep.2015.04.056.

  9. 9.  See, for example, Gascón, S., Masserdotti, G., Russo, G. L., and Götz, M., “Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success,” Stem Cell 21, no. 1 (2017): 18–34. http://doi.org/10.1016/j.stem.2017.06.011.

  10. 10.  Heinrich, C., Bergami, M., Gascón, S., Lepier, A., Viganò, F., Dimou, L., et al., “Sox2-Mediated Conversion of NG2 Glia into Induced Neurons in the Injured Adult Cerebral Cortex,” Stem Cell Reports 3, no. 6 (2014): 1000–1014. https://www.sciencedirect.com/science/article/pii/S2213671114003294.