Posted in | Neurology

Direct Reprogramming of Somatic Cells into Induced Neurons (Part 1 of 3) Webinar

The first of a three-part webinar series on direct reprogramming presented by Dr Benedikt Berninger

Review conceptual and technical aspects of direct reprogramming of somatic cells, such as fibroblasts and astrocytes, into induced neurons.

About the Presenter

Dr Benedikt Berninger is currently a Professor of Physiological Chemistry at the University Medical Centre of the University, Mainz. He received his doctoral degree in 1996 at the Ludwig Maximilians University in Munich for work on activity-dependent regulation of neurotrophin gene expression.

Benedikt then joined the lab of Professor Mu-ming Poo, as a postdoctoral fellow, at the University of California, San Diego, to study fast actions of neurotrophins in synapses and growth cones.

After a brief stay at the Karolinska Institute to acquaint himself with the rapid developments of the neural stem cell field, Benedikt returned to Munich and obtained a position as a lecturer and senior lecturer at the Ludwig Maximilians University, Munich.

In 2012 Benedikt received a call to the Johannes Gutenberg University of Mainz. His laboratory focuses on lineage progression of adult neural stem cells and on direct conversion of brain resident cells into induced neurons.

Webinar Topics

  • The concept of direct lineage conversion via transcription factor-driven reprogramming
  • Direct conversion of fibroblasts into different types of induced neurons
  • Direct conversion of brain endogenous cells (glia and pericytes) into induced neurons
  • Mechanistic principles underlying direct reprogramming

Webinar Transcript

Hello, and thank you for joining us for today's webinar: Direct Reprogramming of Somatic Cells into Induced Neurons. It's the first webinar in this series. It's my pleasure to introduce today's presenter, Benedikt Berninger. Benedikt is currently a Professor of Physiological Chemistry at the University Medical Centre of the University, Mainz. Benedikt received his doctoral degree in 1996 at the Ludwig Maximilians University, Munich for work on activity-dependent regulation of neurotrophin gene expression. He then joined the lab of Professor Mu-ming Poo as a postdoctoral fellow at the University of California, San Diego, to study fast actions of neurotrophins in synapses and growth cones. After a brief stay at the Karolinska Institute to acquaint himself with the rapid developments of the neural stem cell field, Benedikt returned to Munich and eventually obtained a position as a lecturer and senior lecturer at the Ludwig Maximilians University, Munich. In 2012 he received a call to the Johannes Gutenberg University of Mainz. The work of his laboratory focuses on lineage progression of adult neural stem cells and on direct conversion of brain resident cells into induced neurons.

Joining Benedikt Berninger today is Aris Krikelis, Research Area Marketing Coordinator here at Abcam, who will highlight an exclusive promotion for the webinar participants. If you have any questions throughout this presentation, we invite you to submit them on the right hand side of your screen in the Q&A panel. Questions will be answered during the troubleshooting portion of this webinar. At this time, I'd like to hand the presentation over to Benedikt Berninger.

BB: Thank you, Sarah, for this kind introduction and thanks to Abcam for organizing this webinar, and thank you, the audience, for attending this webinar about Direct Reprogramming of Somatic Cells into Induced Neurons. Hopefully, during the talk it will become clear why I'd like to start the webinar with a citation from the Erlkönig, a poem by the German poet Goethe: 'If you are not willing, I'll use force'. It's just what we are telling in a metaphoric way to somatic cells when we reprogram them into neurons. This webinar will cover the following topics: first, I will try to explain, in a few slides, the basic concept underlying direct lineage reprogramming; that is the reprogramming of one cell type into another without going through a pluripotent intermediate. Then we will discuss the most common strategy to identify the right reprogramming factors that can trigger lineage reprogramming. This will be followed by an overview over the achievements during the last few years of converting fibroblasts into induced neurons; a line of research that has gained considerable momentum.

Then I will present you work mostly from my own group, which has focused since quite some time on the possibility of converting cells present in the brain into neurons. First, we will see that astrocytes are an interesting target for lineage reprogramming into neurons. Towards the end of the webinar, we will then look at an example of cells present in the adult human brain that can also be reprogrammed into neurons; and thus demonstrate, and further open potential therapeutic windows of opportunity. During this talk, whenever I refer to work from colleagues, you will find the citation as indicated here. This figure, for example, is from a review by Zhou and Melton. What is lineage reprogramming? Conrad Hal Waddington coined a classical metaphor for the process of cell differentiation during development: An epigenetic landscape characterized by valleys of different levels, and hills to separate them. A cell is modelled by a marble ball rushing down from the top to the valleys. The pathway that it takes decides in which valley it will finally settle, providing a metaphor for the acquisition of a specific cell fate.

Since valleys are separated by hills, cells cannot change easily their fate. The green marble represents a pluripotent cell, while the blue marble, let's say, a fibroblast. The question now is how to push the cell uphill again? In the case of reprogramming towards a pluripotent state, such as is achieved by induced pluripotent stem cell technology, you need to push the marble all the way up the way it came. In case of direct lineage reprogramming, often referred to as trans-differentiation, the idea is to directly move from one valley to another, and that's acquiring a new cellular identity. Clearly, this is a very nice image, but how does it really work? Independent of the mechanisms involved, both reprogramming strategies promise to revolutionize medicine. Essentially, they represent alternative ways of obtaining desired cell types like heart cells, liver cells and, of course, neurons and glia.

For the first time, it is possible to obtain large quantities of cells of human origin. These in turn can be used for disease modeling and drug discovery, but hopefully one day also for cell-based therapy of the many devastating diseases such as Alzheimer and Parkinson's Disease.

Now, back to the mechanism. Simplified, we can look at it this way. Each cell fate is characterized by a regulatory network of factors, typically transcription factors that maintain a specific state through reciprocal feedback interactions. These can be called program, let's say, green. Now, to change to program red we must activate new notes in the network that were inactive when the cell was enacting the program green. This may be done by overexpressing transcription factors, or microRNAs. During successful reprogramming, this will now lead to the progressive stabilization of a new network of interacting factors, depicted in red, and the deep stabilization of interactions that characterized the state green.

What is the basis for selecting the right factors? As Amomoto and Arlotta from Harvard University discuss in the recent Science Review, a good choice for the right reprogramming factors is inspired by development. Factors that drive neuronal specification in the developing CNS, are good candidates for reprogramming other somatic cells into neurons in vitro and perhaps also in vivo. The other big question concerns the cellular target or which cell type you want to reprogram. This can include cells outside the CNS, such as skin fibroblasts, but if we want to move reprogramming into the in vivo setting, we must select cells that reside in the brain, such as glia, or, as you will also see, non-neural cells such as pericytes. Back to the factors. One way is to identify transcription factors, and one way to identify transcription factors in microRNAs that can be used for reprogramming, is to screen for them through microarrays or RNA sequencing within the embryonic brain tissue.

From these you can now obtain an arsenal of viral factors, lentiviruses or retroviruses, and it's actually a starting cell population of choice. After some time during which reprogramming should take place, you start to evaluate the outcome and conduct functional assays, such as patch clamp recordings in case you try to obtain neurons to prove that cells really change their identity. What are the criteria for a cell to fulfil, to be called a neuron after reprogramming? First, the phenotype should be stable even once the reprogramming factors are switched off. Of course, at the same time, the properties of the cell of origin should have been lost by the cell, otherwise this would indicate incomplete reprogramming. In case of neurons, it is very important to also derive cells of a specific subtype. For instance, if you want to model Parkinson's Disease, you may want to specifically generate mid-brain dopaminergic neurons, such as occur in the substantia nigra.

Finally, these cells must exhibit the electrophysiological characteristics of your desired type of neuron. A real breakthrough study came from the lab of Marius Wernig in Stanford. In this study, published in Nature 2010, Vierbuchen and colleagues showed that fibroblasts can be converted into neurons by false expression of defined transcription factors. The remarkable thing about the study was that fibroblasts are of mesodermal origin, while neurons, of course, derive from the neuroectoderm. So the champ of the marble ball, using the metaphor of Waddington, must have occurred over several valleys to allow reprogramming from one germ layer to another.

To screen for the right factors, Wernig and his lab used a mouse line in which neurons are labeled by GFP driven from the Tau promoter. They then isolate the fibroblasts from skin, none of which were green fluorescent at the outset. They then transduced these fibroblasts with various lentiviruses encoding distinct transcription factors, and found that some combinations resulted in the appearance of green fluorescent cells. Indication that some cells had been converted to neurons.

This complex slide shows that the best combination Vierbuchen and colleagues identified, was one of three factors abbreviated BAM. BAM stands for Brn2, a POU domain transcription factor, Ascl1, a basic helix-loop-helix transcription factor and Myt1l, a zinc finger transcription factor. Interestingly, Ascl1 alone already induces neuronal properties, but co-expression of the other factors strongly promotes a degree of differentiation, as you can see here in the acquisition of more mature electrophysiological properties. Now, in this study, the Wernig lab found that the BAM combination induces degeneration of neurons that use the transmitter glutamate and, to a lesser extent, GABA. But for studying and treating Parkinson's Disease, it would be valuable to obtain dopaminergic neurons. So the lab of Vania Broccoli, from the San Raffaele Institute in Milan, set out to reprogram fibroblasts into dopaminergic neurons.

They utilized the mouse line in which GFP is expressed in cells, but have an active TH promoter. TH or tyrosine hydroxylase is an enzyme in the dopamine synthesis. They found that another triad of transcription factors succeeded in converting fibroblasts into green fluorescent cells. Conspicuously, Ascl1, also called Mash1 ranked again among them. The other factors were Nurr1 and Lmx1a, well-known to play a role during the development of dopaminergic neurons in the embryo.

This slide summarizes the attempts of various labs all over the world to generate different subtypes of neurons from fibroblasts, both of mouse, but, importantly, also human origin. These include now also motor neurons, which could be shown to innovate skeletal muscle upon transplantation into chick embryos: a study from the Kevin Eggan lab in Harvard. Just to give an idea about potential applications, we see here a strategy to use lineage reprogram fibroblasts to model Alzheimer's Disease.

The lab of Arthur Abelowitz from Columbia University, transduced fibroblasts from healthy controls and from Alzheimer patients with the familial form of the disease, and obtained human-induced neurons, as you can see here. They found that the induced neurons from patient-derived fibroblasts secrete much more of the Aβ40 fragment than the induced neurons from controls. Thus indicating the pathological, a better processing can be studied in the system. Importantly, these differences only emerged upon reprogramming interneurons, and were not discernible when looking at the original fibroblasts. This makes a strong case for the utility of lineage through program cells for diseased modeling. From all these studies, Ascl1 emerged as a key player of reprogramming fibroblasts into induced neurons. More recently, work from the Wernig lab now provides some insight why this is so. It turns out, as depicted by the model, that Ascl1 acts as a pioneer transcription factor that is capable of opening closed chromatin, and recruit the other factors to induce neuronal gene transcription.

However, they also found that Ascl1 cannot do this feat in all cell types, as shown here. Apparently, keratinocytes exhibit a non-permissive chromatin and, hence, cannot be reprogrammed by the BAM combination of factors. The permissiveness, in turn, seems to be defined by a triad of chromatin modifications, which are depicted here, so those sides marked by a trivalent state, Ascl1 combined. While if the chromatin is found in another state, no binding occurs and, thus, also no chromatin opening, and ultimately no reprogramming.

In the following, I will now discuss our own line of research. This slide shows the basic idea of our working dream, as I like to call it. The idea is to reprogram cells endogenous to the brain into neurons of all kinds of flavor. Ultimately, the restored deceased neural circuits from within, rather than through transplantation, and the starting point for this endeavor has been for us the astrocyte.

One reason why we have focused to a large extent on astrocytes, is their close lineage relationship with radial glial. The cell type that generates neural cells during the developmental period of neurogenesis is often referred as to neural stem cell. However, at the end of neurogenesis, radial glial loses its neurogenic ability and transforms into astrocytes, which continually divide to produce parenchymal astrocytes. Now, a central question pioneered by Magdalena Götz and which we have followed for many years, is whether these parenchymal astrocytes can still be enticed to generate neurons. We have tested this hypothesis by the following in vitro paradigm. Astrocyte cultures are prepared from the cerebral cortex of postnatal mice of five to seven days of age. The cells are plated on flasks, then expounded for a week. Then the cells are re-plated and transduced with retroviruses encoding neurogenic transcription factors. In particular, we examined the reprogramming capacity of the proneural genes neurogenin-2, Ascl1 and the homeobox transcription factor Dlx2, for reasons which become clear in the following.

The protocol is described in detail in the indicated publication. The main rationale of using these factors is to direct astrocytes towards generating distinct types of neurons. When we overexpressed neurogenin-2, we found that astrocytes were very efficiently converted into neurons. Notably, neurogenin-2 transduced astrocyte cultures were abundant in immunoreactivity for vesicular glutamate transporter, indicating that neurons derived from neurogenin-2 transduced astrocytes acquire a glutamatergic identity. Consistent with this, patch clamp recordings of these cells reveal glutamatergic synaptic transmission. Finally, these astrocyte-derived neurons typically exhibited morphology reminiscent of pyramidal neurons, displaying synaptic spines that were targeted by pre-synaptic terminals.

Why do neurogenin-2 transduced astrocytes convert into glutamatergic neurons? Actually, neurogenin-2 is expressed in the developing dorsal telencephalon where it contributes to the specification of glutamatergic neurons. There it is induced by Pax6 within the ventricular zone. Hevner and colleagues describe the cascade of transcription factors that are expressed during cortical development, and are hallmarks of glutamatergic neurogenesis. Subsequent to expression of Pax6 the transient induction of Tbr2 here in green. In the subventricular zone, and then Tbr1 in orange in the cortical plate. Notably, in neurogenin-2 transduced astrocytes we can see a similar sequence of transcription factor expression. In fact, first we see the transit upregulation of Tbr2 - as shown here - in cells that do not yet express the neuronal marker beta-III tubulin. Later on, expression of this transcription factor is lost, and Tbr1 is expressed instead. Thus during the process of conversion, neurogenin-2 transduced astrocytes recapitulate key steps of glutamatergic neurogenesis.

This slide shows still images of a time lapse movie following the conversion process of neurogenin-2 transduced astrocytes. The green fluorescence is due to genetic fate mapping indicative of a previously active GLAST promoter in an original astroglial cell. At the beginning, there is no or little report of fluorescence driven from the retrovirus encoding neurogenin-2 and DsRed. While the transgene expression commences, the astrocyte is dividing one last time and then kicked out of the cell cycle, and both daughter cells differentiate into neurons still marked by a genetic fate mapping. An important criterion for true reprogramming is the stable acquisition of the new identity. To test this, we utilized a construct encoding a fusion construct of neurogenin-2 with the ERT2 domain, rendering neurogenin-2 activity strictly dependent on treatment with Tamoxifen. Therefore, efficiently converted into neurons when Tamoxifen was supplied for four consecutive days. Strikingly, when the treatment was then stopped and the cells were then analyzed after 14 days, they had not lost their neural identity, as you can see here, but still exhibited a neuromorphology in MAP2 and Tbr1 expression. Thus demonstrating that the new phenotype was stable.

As alluded to before, employing other transcription factors causes astroglia to convert into other types of neurons. When we overexpress the homeobox transcription factor Dlx2, or the basic helix-loop-helix transcription factor Ascl1, we obtained neurons that exhibited action potential discharges typical for interneurons generated in the medial ganglionic eminence, where these two transcription factors are also expressed during development. As you can see here, some cells were found to respond to step depolarization with a high frequency action potential firing, similar to fast-spiking interneurons. Others, by contrast, exhibited a phenotype reminiscent of so-called burst-spiking interneurons. Consistent with a GABAergic neuron identity, we also observed GABAergic synaptic transmission.

So far our work indicates that brain-resident cells such as astrocytes can be converted into neurons, but how about the adult human brain? Here, so beautifully depicted by Michelangelo. Does it also harbor cells that are permissive to neuronal reprogramming? To address this question, we collaborated with Christian Schichor from the Neurosurgical Department of the Ludwig Maximilian University, Munich. We obtained cortical tissue that was removed during the course of brain surgery to treat patients for vascular malformations of pharmacoresistant epilepsy.

We then prepared cultures from this tissue, but we observed only very few GFAP or S100β positive astrocytes, while the majority of cells were negative for all glial and neuron markers tested, suggesting that these cells may be non-neural. In fact, it turned out that these cells expressed markers characteristic of brain pericytes, such as PDGF receptor beta, smooth muscle actin, NG2 and also CD146 not depicted here.

What are pericytes? These are cells typically tightly associated with brain microvessels, where they regulate induction and maintenance of the blood/brain barrier, and also control the diameter of the microvessels, thereby regulating local cerebral blood flow. Indeed, in our tissues we found these type of cells are indeed present, and associated with the vasculature as can be seen in the immunohistochemistry for NG2 and PDGF receptor beta.

The question is now whether these cells, these pericytes are also permissive for direct lineage reprogramming? To address this question, we plated these cells on microwells, transduced the cells one day after and then switched to a neuronal culture medium as low oxygen. In fact, we found that low oxygen conditions greatly facilitate reprogramming. Interestingly, when we tested the abovementioned factors Ascl1 and neurogenin-2, we found that very little happened. We observed a modest decrease in cell cycling and some induction of the neuronal marker beta-III tubulin, as quantified here. But without any neuromorphology, thus we tested co-expression of SOX2, another transcription factor, which figures prominently amongst the Yamanaka reprogramming cocktail, but it's also well-known for its role in neuro stem cells. So we hypothesized that SOX2 may render permissive to neuronal reprogramming.

Consistent with this, SOX2 alone, as you can see here in these green cells, did not cause any obvious change compared to controls. But when it was co-expressed with Ascl1 we found that roughly a third of the cells, as quantified here, acquired a neuromorphology besides neuronal map expression. Here I want to show you a time lapse movie of a human brain pericyte-derived cell undergoing neuronal reprogramming. First, we sorted the culture for self-expressing PDGF receptor beta. We then infected these cells with two retrovirus encoding SOX2 and Ascl1, and you will see sometimes the images depicting the fluorescence, the reporter of fluorescence indicating the expression of the two transcription factors. The movie shows that advanced used cells undergo morphological changes, at some stage looking like a multiple cell or projecting processes, but often retracting them again. We are currently investigating whether this reflects the progressive resolving of conflict of cell fates, which, in many cases, results in cell death instead of successful lineage reprogramming.

In this case, however, the cell finally acquires a neuronal identity and morphology. What is also remarkable is the rather protracted time course of the conversion process, which takes nearly two weeks to be complete. So now you still see the cell migrating a lot, and rapidly altering its processes and morphology, but in a few days, as indicated here, the cell will finally acquire a stable neuronal process and beta-III tubulin expression.

What neuronal phenotype do SOX2 and Ascl1 reprogrammed pericytes acquire? We have evidence that the majority of the successful reprogrammed cells become GABAergic, as can be revealed by immunostaining for the transmitter GABA. Very importantly, parasite-derived neurons exhibit also function properties of true neurons, they can fire action potentials in response to step depolarization. Moreover, when co-cultured with neurons from the embryonic mouse cortex, pericyte-derived neurons are recognized as synoptic targets and receive functions in optic input.

This is also reflected by the decoration of the dendroids with presynaptic terminals, originating from co-cultured neurons. For the pericyte-derived neurons can also produce a functional synaptic output, is still under investigation. This slide summarizes this last part of the webinar. Pericytes from the adult human cortex can be expounded in vitro, and reprogrammed into functional-induced neurons. Of course, the ultimate goal for such studies is to perform a similar lineage conversion in vivo. Of course, we are still far away from being able to do this within the context of a clinical trial in humans.

To sum up, we have seen that somatic cells of various origins, including human, can be lineage reprogrammed into induced neurons. These neurons exhibit functional properties, can fire action potentials and form synapsis. Now these cells can be used to start the fundamental mechanisms underlying neurogenesis. Furthermore, we have seen that they can be employed for modelling human neurological diseases, such as Alzheimer's Disease. Of course, the final goal would be to recruit these cells for brain repair.

While the future looks bright for lineage reprogramming, there are several issues to be addressed to fully employ the potential of this approach. First, we need to examine the possibility of conducting lineage reprogramming within the brain in vivo. Several recent reports have actually provided evidence that brain resident cells can be indeed reprogrammed within the in vivo context. We will follow this theme in a future webinar.

Another crucial aspect concerns the authenticity of the reprogrammed cells, neurons. How close are they to the neurons that occur in the brain? It is very likely that we need to improve reprogramming protocols to obtain cells of authentic neuronal fabric. Finally, a profound understanding of the molecular mechanisms underlying reprogramming, and knowledge of the epigenetic barriers that interfere or block lineage conversion, will allow for developing even better reprogramming strategies; and, thus, also feed the aim of obtaining authentic neurons.

Thus, I come to an end, and I want to thank Abcam again for giving me the opportunity to discuss our work here in a broader context. I'd also like to thank my own team, especially Marisa Karow and Rodrigo Sánchez, who did the work on pericytes, Christophe Heinrich who spearheaded the astrocyte work, and Felipe Ortega for the time lapse movies. Special thanks also to Christian Schichor from the Neurosurgical Department of the LMU, Munich and Magdalena Götz and her team for a longstanding collaboration on astrocyte reprogramming. Likewise, I'd like to thank Francois Guillemot, Timm Schroeder, Chichung Lie and Marcus Costa for fruitful collaborations over the past years. Last, but not least, the funding agencies. So this is my current team in the University of Meinz, and we are looking for talented young people to join us for a PhD or postdoc. Thank you.

AK: Thank you, Benedikt. Please don't hesitate to submit your queries as we guide you through a brief presentation of related products and resources from Abcam. Next slide, please.

On our website, you will find a wide range of posters, pathway cards and protocols available free for download. Among them are a popular IHC application guide, as well as a new avenues for brain repair poster produced in collaboration with Paola Arlotta, Benedikt Berninger and Alejandro Schinder. This poster summarizes some of the methodology used in the reprogramming of somatic cells, so glial into neurons with or without a pluripotent intermediate.

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Finally, if you've enjoyed this webinar, we would like to invite you to register for Part II of the series. This is presented by Alejandro Schinder on March 11th 2014, and the topic is: Adult Neurogenesis and the Plasticity of Hippocampal Networks. Thank you for your attention. I will now pass you back to Benedikt who will answer some of the questions you posed to him during this break.

BB: Yes, I got a few questions and one question was asked by Dean, and he asked about whether we have any experience with direct reprogramming of keratinocytes to neurons? So we ourselves don't have any particular experience with keratinocytes, but I showed you one slide from the laboratory, or a model of the work from the laboratory of Marius Wernig, where they tried to reprogram keratinocytes. They found that keratinocytes have a different chromatin state that doesn't permit Ascl1 to bind, so what they thought or what they believe is that a keratinocyte may not be permissive to neuron reprogramming. Now, of course, that is perhaps valid for if you only test these facts, and Dean asks, actually, whether there will be any other factor combinations that would allow reprogramming of keratinocytes? So I would speculate, but we haven't tested this, that perhaps co-expression of SOX2 may be a way of overcoming the apparent non-permissiveness of keratinocytes. What we think is that perhaps SOX2 is, even on a greater scale than Ascl1, capable of opening closed chromatin and thereby facilitating reprogramming. So I think this combination of SOX2/Ascl1 has not yet been tested on keratinocytes, I cannot give you a positive answer, but I think that might be a way to try this.

So this actually connects me to the next question by Ohud, and the question was: What are exactly the epigenetic barriers that might be responsible for interfering with reprogramming? So, actually, a very interesting study from within invertebrates has shown that, indeed, the epigenetic state plays a major, important role in regulating the permissiveness of reprogramming. So what I'm referring to is work from the laboratory of Oliver Hobert from Columbia University in New York, and they studied C. elegans. What they found was that when they overexpress transcription factors that drive the specification of specific subtypes of neurons in other cells in C. elegans, these do not by themselves cause the cell to convert into neurons. However, they found that when they, at the same time, knocked down proteins that are chromatin remodelers or part of chromatin complexes, they found that then reprogramming became feasible and they showed in particular that you can reprogram a cell in the germ line of C. elegans.

If you, at the same time of expressing a transcription factor, knockdown a protein that is part of the inert complex, which is a chromatin remodeling complex. So what we think is that probably in many cells in this section, all cells, the chromatin is encountered in a specific state, and in order to render the cell is permissive to reprogramming, you have to overcome these certain epigenetic modifications that block, for example, the binding of Ascl1 to its target chains, or the binding of other transcription factors to its target chains, then this way interfere with the possibility of reprogramming. Of course, that may be very dependent on the history of the cell, so the more mature a cell is the more it probably accumulated epigenetic modifications that sort of fix it in the valleys. In the certain way you can imagine, or look at it like if you recall the epigenetic landscape of wanting that the valley gets deeper and deeper, and it gets more and more complicated to overcome the hill that then is building up and prevents to change the cell identity.

So another question refers to the possibility of reprogramming in vivo: namely how far are we there? Perhaps you have seen recent applications in by several groups, which show that, indeed it's possible to reprogram cells within the brain, even the adult brain in vivo. The same types that have been demonstrated or suggested to be reprogrammed there, are both of the glial lineage, namely the astrocytes and many of the things that I've shown you here in vitro, apparently seem also to work in vivo and also the MG2 glial population. At this stage, it's I think still premature to be very sure about the cell identity reprogram, but these studies used different genetic fate mapping strategies to demonstrate, or to indicate which cell types are reprogrammed. So the big question is, of course, to which extent then these cells really mature into functional neurons, and to which these cells really can contribute to function. This is, of course, another of the questions raised. This is something that is very difficult to predict, because, of course, if you insert a cell into the circuit, it should acquire very specific neuronal identity in order to exert then also specific functions. I think only the future will be able to show whether we can use these strategies really to improve clinical deficits first in animal models, and then later on, of course, also in clinical trials.

So clinical trials are certainly something that are still far away at the current stage, but it is certainly worthwhile to follow our experimental approach both in vitro and in vivo, in order to test whether at some stage we can really move forward and test, whether by reprogramming endogenous cells, we can reconstitute neural circuits that are damaged or deceased; and, thereby also improve the lives of many people that suffer neurological diseases. So I think these are the questions that I got to address, and with this I would terminate my part of the webinar. Thank you very much, again, for listening.

Thank you, Benedikt and thank you ladies and gentlemen. That does conclude today's presentation. Please note that as you leave the event today you'll be presented with a brief feedback survey. If you could take just a moment to fill that out we would appreciate it. So, once again, thank you for your time today and you may now disconnect.

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