Philip Ball's 'mini-brains' have cleared their first major hurdle: his skins cells have been successfully transformed into ‘artificial stem cells’. Here, he documents the next stage of his journey to growing a second brain in a dish.
“I’m a step closer to getting my second brain. My prospective ‘mini-brains’ have cleared the first major hurdle: the skin cells or fibroblasts taken from my arm have become successfully transformed into ‘artificial stem cells’, so-called induced pluripotent stem cells or iPSCs.
Formerly elongated tissue-forming cells, they are now more compact, smaller and mostly just nucleus – the compartment, visible under the microscope, where the chromosome reside. If all goes well, UCL neuroscientists Selina Wray and Christopher Lovejoy will be able to coax these stem cells into neurons to begin in earnest to grow my mini-brain.
(In following convention by calling them such, I am defying the advice of developmental biologist Robin Lovell-Badge of the Francis Crick Institute, who deplores the use of the term. Robin insists that what you get in this process is a piece of tissue - what some call an organoid – lacking the detailed structure of a real miniature organ like the one that grows in an embryo. Yet these tissues show many signs of “wanting” to become real brains; they are simply deprived of the right environmental signals to guide them. At any rate, I respect Robin immensely and beg his pardon.)
This project, dubbed ‘Brains in a Dish’, is a part of Created Out of Mind, the resident team at The Hub, Wellcome Collection attempting to inform and change public perceptions about these neurodegenerative conditions through a range of artistic and scientific responses. Selina and Chris grow brain organoids from iPSCs of people who have a genetic disposition to some early-onset neurodegenerative conditions, in order to better understand the role of genes in the development of those conditions. Four Created Out of Mind members, including me, who are having brain organoids grown, will document our thoughts and responses in various ways, to communicate both the science and the subjective experience of this rather remarkable process.
"If all goes well, neuroscientists Selina Wray and Christopher Lovejoy at University College London will be able to coax these stem cells into neurons to begin in earnest to grow my mini-brain."
The biochemical alchemy of reprogramming cell fate is at the heart of that process. It involves persuading a skin cell – a mature, ‘differentiated’ cell type apparently committed to a specific tissue – that it is in fact a stem cell. In this post I’m going to delve a little into how that trick is done.
Until early in the new millennium, no one knew if that act of persuasion was possible, and many researchers figured that it was not. Cell differentiation was regarded as a one-way process. It was only through the extraordinary diligence of Japanese biologist Shinya Yamanaka and his coworkers that we discovered otherwise.
The point is that, even though all our cells contain a full complement of genes – a complete genome, encoded in DNA and partitioned into 23 pairs of chromosomes – they behave according to very different ‘programs’. Skin cells do what it takes to make and maintain skin; muscle cells have a totally different job, contracting and relaxing in response to nerve signals. Pancreatic cells make insulin and other hormones; there are cells specialized for making bone, and others for dissolving it, and so on. These specialized duties are attained as stem cells in the developing embryo differentiate and acquire different fates.
Some researchers once thought that differentiated cell might actually lose the genes they no longer need for the role to which they are committed – or at least, that those redundant genes were permanently deactivated. It’s now known that much of this deactivation or ‘down-regulation’ – where genes are more or less muted – results from chemical modifications of the respective stretches of DNA. Certain chemical groups, called epigenetic markers, get attached to the DNA molecule, where they act as signals for the cell to ignore (or conversely, to switch on) the respective genes. There are other ways to regulate genes without actually changing or removing the DNA itself, for example according to how the long DNA molecule is packaged up with proteins in chromosomes. These changes are induced at particular stages during the growth of an embryo, often in response to chemical or other types of signal received from surrounding cells or from the environment. In short, the development of an embryo from a ball of identical cells into a mosaic of specific tissues is a highly complicated, dynamic and responsive process that is far from fully understood.
Much of the regulation of genes is done by other genes – called, naturally enough, regulatory factors. Most are so-called transcription factors, since they affect the process of transcription, the first stage in the conversion of a gene to the protein it encodes. In this way, genes “talk to” each other in complex networks, and this communication is what orchestrates the biochemistry and development of a cell.
What Yamanaka showed is that, if just a small number of the right transcription factors is added to mature differentiated cells, the genes that were previously silenced – for example, by epigenetic modifications – can be reawakened. This means that the cells can be transformed back into a stem-cell-like state, from which they can then be induced – perhaps by adding other transcription factors – to grow into a different tissue type. In other words, gene function is not irreversibly lost in differentiation; it is just made dormant. Cells reprogrammed this way are called iPSCs; ‘pluripotent’ here means that the cells have the potential to become any tissue type in the body. Whether such cells are truly like normal stem cells in the early embryo remains unclear, as it’s not known if all epigenetic marks get erased in the process.
Yamanaka’s initial experiments for making iPSCs, in the mid-2000s, required 24 genes to be introduced to switch the cells back. But subsequent work by Yamanaka’s group and others whittled away at the number of genes required until iPSCs could be created using just four of them.
"It’s not implausible that even neurons might be grown to replace brain tissue damaged by injury or neurodegenerative diseases, although that’s still a distant prospect at best."
The discovery has revolutionized – the word is not too strong – medical cell biology. It means that tissues and perhaps complete organs – or at least smaller but functioning organoids – can be grown afresh for transplantation, using a patient’s own cells. It might become possible to replace malfunctioning kidneys or heart tissue with tissue grown this way from, say, easily harvested skin cells. It’s not implausible that even neurons might be grown to replace brain tissue damaged by injury or neurodegenerative diseases, although that’s still a distant prospect at best. Such iPSCs can even be grown into gametes (eggs and sperm) for assisted reproduction, potentially addressing infertility associated with a failure to produce sperm or poor-quality eggs. But this too isn’t going to happen any time soon, not least because little is known yet about the long-term safety of such procedures. If iPSCs retain some imprint of their ‘past life’, for example, that could create complications further down the line.
How is the reprogramming done? Transcription factors don’t need to be somehow inserted into the chromosomes themselves. They can be added to cells as separate pieces of DNA, typically formed into rings called plasmids. To get the plasmids into the cells, they have to be transferred across the cell membrane without destroying it. Yamanaka originally did this using specially engineered viruses that ‘infected’ the cells with the genetic material. The standard way of doing it now is to apply pulsed electric fields to a suspension of cells, which opens up temporary gaps in their membranes: a method called electroporation. Alternatively the plasmids can be enclosed in artificial membranes made of molecules closely related to those of real cell membranes. These can merge with the cell membranes like soap bubbles, disgorging their contents into the cell.
This, of course, makes a very complicated and delicate process sound like simple cooking. Selina and Chris have done it often enough to have a good chance of success, but that’s never guaranteed, and so I was relieved to hear that the conversion of my fibroblasts to iPSCs has gone well. You can see if it has worked partly just from the microscope images, because the different cell types have different shapes. Fibroblasts are elongated, while iPSCs are more blobby and relatively small, dominated by the darker regions of the nucleus.
Because we’re dealing with living things here, the cells aren’t fully predictable and are liable to go their own way if not carefully looked after. It’s within a cell’s nature, you could say, to differentiate: it’s as though the cell “wants” to specialize. So iPSCs are apt to do that if the conditions aren’t just right. In particular, they retain their stem-cell-like state only if grown in clusters of the right size. If they become isolated in the culture medium, they will pretty soon “choose” a fate – and curiously, that fate is typically a neuron, as though this is the default fate. I noticed one such cell in one of my own samples, growing the branching appendages that typify neurons. On the other hand, iPSCs don’t like to be too gregarious: if the clusters are too large then they’ll start to differentiate too. To someone like me trained as a physical scientist, this is disconcerting. We get used to dumb matter that only changes if prodded hard enough. It’s a reminder that biology isn’t so much about the deterministic unfolding of a genetic program as about the sensitive interplay between innate tendencies and feedback from the environment.
Which is a way of saying that I’m very glad my cells are in safe hands. Selina and Chris froze the pieces of me during the Christmas break. Now we’ll see if they will play ball in turning into mini-brains".
This is the second post from Philip Ball in a series of articles, reflecting on his experiences as part of Created Out of Mind's 'Brains in a dish' project, which aims to stimulate curiosity about the healthy and ageing brain, what might happen when a neurodegenerative condition like a dementia occurs and the cutting-edge technologies providing new insights into dementia.
Read the 1st post here: Building a new brain for dementia research