Early last semester a headline in the journal Nature caught my eye: Neanderthal-like “Mini-brains” Created in the Lab with CRISPR. I bookmarked the article and promised myself I would get back to it soon–staying current is important for my research and keeps my teaching up to date and fresh.
Only a month later Nature, again, caught my attention with another intriguing headline: Scientists Grow Tear Glands in a Dish—Then Make Them Cry. Instead of grading or revising lectures, I spent the rest of my day reading about organoids that mimicked brains (human and Neanderthal), tear glands, intestines, kidneys, the female reproductive tract, and more.
Why are organoids (artificially grown cells) important? What can they teach us? What questions might people of faith have about this technology?
Three-dimensional (3D) tissue culture assays were first reported when I was in graduate school in the early 1990s. My dissertation research depended heavily on two-dimensional (2D) tissue culture and I was well aware of its limitations. It was an imperfect representation of the environment of cells in a 3D organism, so we confirmed conclusions from in vitro experiments in a lab animal whenever possible. When I found that altered hamster cells grew in soft agar, an indication of tumorigenicity, we injected some of them into hamsters to confirm that they did, in fact, give rise to tumors. When the peptides I was working on blocked blood vessel cell migration, an indication that the peptides inhibited new blood vessel growth (a process necessary for tumor growth), we confirmed that the peptides blocked blood vessels from sprouting from the limbus of a rat cornea.
3D tissue culture looked to be an assay that better mimicked a cell’s environment in a whole organism, but the methods were difficult and did not work for all cell types. I followed progress in 3D tissue culture but did not use it for my project.
After graduate school, I kept doing some experiments that required 2D tissue culture, but shifted the focus of my research to include viruses and molecular genetics so I lost track of the progress scientists were making in 3D tissue culture…until those two headlines grabbed my attention. As I dug in I found much had changed.
Organoids were declared the method of the year in 2017 by Nature. As they are currently and most commonly defined, organoids are three dimensional cultures derived from stem cells. When given the appropriate environment, they approximate natural organ/tissue development and maintenance. Because organoids mimic in vivo organs, they are used to study aspects of that organ in a laboratory culture dish. The stem cells used are pluripotent (embryonic or induced) or adult stem cells derived from various organs.
Although organoids are not as complex as actual organs, the extent to which they mimic actual organs is illustrated by the tear gland organoids of the second attention-grabbing headline. In this study, scientists grew stem cells from lacrimal (tear) glands in 3D culture. In our bodies, the production of tears is controlled by the autonomic nervous system—an “involuntary” part of our nervous system involved in “fight or flight” and “rest and digest” responses. Emotions are registered by a part of our brains called the amygdala. When the amygdala registers intense emotions, it sends a strong neural signal to another part of the brain called the hypothalamus. The hypothalamus, a key piece of the autonomic nervous system, then causes the release of a neurotransmitter at the tear glands and they respond by secreting tears. In the organoids, scientists stimulated secretion of tears by adding a similar neurotransmitter to the 3D cultures. Tear production caused the organoids to swell because they lack ducts but when scientists transplanted them into mice, they matured and even developed ducts. The scientists working on these organoids hope their work offers the possibility to treat eye disorders.
The data coming from experiments with tear-gland cells illustrate how well organoids can mimic organs, but the data coming from experiments with brain organoids are perhaps the most captivating. Two particular studies evoke the most provocative ethical questions. The first is the set of experiments represented by the Neanderthal brain headline that caught my attention.
Modern humans are more genetically similar to extinct Neanderthals and Denisovans than we are to any living primate. In fact, only 1.5 to 7 percent of the human genome is not found in our early Neanderthal or Denisovan ancestors. Because these populations interbred, Neanderthal and Denisovan DNA can be found in most modern human genomes today.
All three genomes contain a gene called NOVA1, which encodes a protein that binds RNA and regulates a process called alternative splicing. Splicing pre-mRNA molecules allow cells to produce different, but related, proteins. NOVA1 (Editor’s Note: The term “NOVA1” can refer to a gene or a protein. It is italicized as a gene and not italicized as a protein.) functions as a master regulator for alternative splicing. This gene and its protein product are important: when mice lack any functional NOVA1, they die shortly after birth due to programmed cell death of motor neurons in their spinal cord and brainstem. In humans, altered NOVA1 activity is associated with neurological disorders. In short, NOVA1 is a protein that is critical for proper nervous system development and function.
Of great interest to scientists is the fact that there is a difference between the human NOVA1 sequence and those of Neanderthals and Denisovans. Here was a protein, important in nervous system development and function, that showed a human-specific sequence!
The next question was obvious. Does the human-specific NOVA1 variant contribute to making humans distinct? To begin to address this question, scientists used CRISPR/Cas9 to alter stem cells so that the modern human version of NOVA1 was replaced with that of Neanderthals. Then they gave the cells carrying the Neanderthal NOVA1 gene the environment they needed to develop into brain organoids and compared them to fully human brain organoids. Scientists immediately saw a difference. Human brain organoids are smooth and spherical in shape. The brain organoids carrying the ancient version of NOVA1 were smaller in size and had rough, complex surface morphology. When scientists dug deeper they discovered additional differences. The organoids containing the Neanderthal gene proliferated more slowly and showed more cells undergoing programmed cell death.
Because NOVA1 controls alternative splicing, scientists looked for genes that were differentially expressed when comparing human organoids and those carrying the Neanderthal NOVA1 gene. They found more than 250 genes with differential expression and more than 100 genes that were differentially spliced. Many of these genes are involved in the development of the nervous system, synapse formation, and generating neural networks. It was perhaps this observation that is the most interesting. Scientists assessed the impact of synapse differences on the activity of the neural networks they observed and found more heterogeneity in the electrical signals sent by cells in the brain organoids containing the Neanderthal NOVA1 gene. In short, this remarkable set of experiments demonstrated that substituting the ancient form of the NOVA1 gene in human brain organoids resulted in changes in genes that are involved in nervous system development—specifically genes that influence cell proliferation and synapse formation between developing neurons. Scientists concluded that this underlies the traits that make modern humans distinct from their most closely related, extinct relatives, and that this single amino acid change may have significantly contributed to human evolution.
If you are still with me you know that organoids raise a number of important ethical, moral, and theological questions. Organoids are grown from stem cells. Christians disagree about the use of stem cells. Some Christians allow only for the use of adult stem cells, some for limited use of embryonic stem cells. Others believe any use of stem cells is inconsistent with their understanding of human personhood and still others support any use of stem cells that can be connected, even indirectly, to helping humankind. There are countless books and essays written about the use of stem cells, many from Christian perspectives. Read them! They are important. However, the use of stem cells is not what I would like to focus on in this essay.
Also, the conclusions drawn from experiments that use organoids have important implications for evolutionary theory, perhaps most importantly for human evolution. Christians disagree about how the scientific evidence for evolutionary theory fits with their biblical hermeneutics and this is especially true when it comes to human evolution. These questions, too, are interesting and important and I encourage you to read the countless books and essays that address them. But this is also not the conversation I want to focus on in this essay. Instead I want to focus on another important and interesting question raised by organoids, especially brain organoids: the question of consciousness.
When stem cells are placed in an environment that mimics the environment of brain development, they differentiate into various types of brain cells that self-organize into functional neural networks. These neural networks produce detectable brain waves. Neural networks form when neurons mature and become interconnected; they form synapses. This process takes about ten months. Scientists detected the brain waves produced by the brain organoids after only two months. They describe the electrical signals as sparse and exhibiting a similar frequency and pattern seen in very immature human brains. As they continued to monitor the developing organoids, they detected an increase in brain wave activity and range of frequencies. The brain waves alternated between a resting state and network-synchronized firing that resembled what is observed when EEGs are performed on preterm infants. These observations suggest that the brain organoids continue to develop their neural networks and form additional synapses. In fact, over time neurons that responded to more than one neurotransmitter formed and the frequency of brain waves was measured at two to three per second. Brain wave frequencies of greater than one per second are a hallmark of human brains.
Because the nature of the brain waves they detected resembled immature human brains, scientists used a computer algorithm to compare the brain waves from the organoids to brain waves recorded from premature babies. The algorithm predicted the number of weeks the organoids had been developing in culture, suggesting that the organoids and human brains have a similar developmental course. Nine-month-old brain organoids showed brain wave activity patterns similar to those of the premature newborns.
Why are scientists pursuing these experiments? Since brain organoids model developing human brains, they are an important tool in learning about and finding treatments for a number of brain pathologies including depression, autism, epilepsy, bipolar disorder, Alzheimer’s disease, schizophrenia, and more. Scientists hope to learn why and how neural networks form in brains of people with neurological disorders and to use them to test the efficacy of various treatments. They also hope to use them to inform those working on artificial intelligence systems as well as gaining a better understanding of brain development in general. Additionally, organoids are used to better model the microenvironment of tumor cells, thus providing a tool to predict drug responses. Brain organoids have even traveled to the International Space Station to investigate the effect of microgravity on brain development.
Scientists working in this field are quick to emphasize that they are far from making human brains in the laboratory. The organoids are very small, lack many of the cell types found in human brains, and lack supporting tissues such as blood and blood vessels that are critical for brain function. Scientists argue that the organoids lack mental activities such as consciousness because they are rudimentary—lacking many other brain parts and structures. They concede that the brain waves they observe may not have anything to do with the activities of real human brains.
Of course, as the tools to make brain organoids are refined, scientists will likely find culture conditions that would allow the brain organoids to grow larger and the neural networks more complex. Only five years ago, the ability to create organoids with functional neural networks seemed impossible–yet here we are. It is not out of the question to assume that scientists will improve their techniques, producing brain organoids that are increasingly humanlike. This is a goal since the best model for human brains is one that resembles human brains as much as possible. The best model will provide the best insight into understanding and treating brain disorders.
Not surprisingly, bioethicists are paying close attention to this research. There is consensus among those familiar with the field of brain organoids that the mini organs grown in the lab today are not conscious and are not likely to be in the near future. They lack the size, structure, and complex interconnectivity of human brains. They lack some of the important cell types found in brains and have no sensory input. Even so, it is important to start talking about potential ethical issues sooner rather than later.
It is reassuring to note that the research is conducted under strict regulatory guidelines which provide ethical protections including the extensive rules governing the use of stem cells. There are committees which include scientists, ethicists, experts in the law and society that oversee stem cell use and animal welfare rules—both of which apply to organoid research. The National Institutes of Health hosts regular meetings and workshops that include important and extensive discussions on the ethics of organoid research.
Two of the most difficult topics of discussion among scientists and ethicists alike are consciousness and moral status. Science does not have good tools to assess whether the neural networks produced in organoids possess consciousness and there is considerable work being done to analyze and characterize the brain waves produced by organoids in an attempt to better understand the relationship between brain waves, their type and frequency, and consciousness. The issue is compounded by the ambiguity of the definition of consciousness, which can include anything from wakefulness to sentience to self-awareness. Moral status is not a scientific question and the discussion of moral status should include voices from people of faith.
As I consider both the Neanderthal “mini brains” and the human organoids that make brain waves, I can’t help but wonder what they say about our fundamental humanness. What makes us different from other creatures—those alive now and those that are long extinct? Will that difference, someday, consist solely of a list of genes? What might that mean for our understanding of humanity’s relationship with God? In what ways are we similar to other creatures and, thus, connected to all of God’s creation and should this connection inform the ways we are called to care for creation? Do these experiments help us understand what it means to be made in God’s image or do they muddy the waters—at least for now?
I don’t have answers to these complex questions. I’m certain that Christians who struggle with mental illness or have a loved one with Alzheimer’s disease might find great hope in this area of research. Other Christians may feel that caution is the best posture to take. Wherever you might fall it is important to be aware and have a basic understanding of organoid research so that Christians can speak into the conversations about consciousness, moral status, being human, and the image of God with knowledge and integrity.
From the point of view of this biologist who is a Christian, science is a gift from God—the Creator of all things, including scientific questions and research that makes us uncomfortable. I am not afraid of asking hard questions or living in the tension of not understanding how a scientific discovery fits with my Christian faith. The gospel is strong enough for our questions and lack of understanding. Of course, as with all gifts, humans can find ways to misuse the gift of science. For this reason, people of faith need to be at the table when discussions like these are happening early on. Too often Christians are unaware of important advances in science until things have progressed so far that their reaction is one of immediate rejection. This further pushes the voices of people of faith to the margins. So, continue to learn about these provocative advances in science and then talk about the important questions the science evokes, doing so through the eyes of faith that is secure in the knowledge that God is God over all truth.