How do we differentiate iPSCs in-vitro? A focus on neural stem cell fates
Written by Liliane Kreuder
Regenerative medicine and the clinical application of induced pluripotent stem cells (iPSCs) is currently a strong research field due to its potential therapeutic benefit for a wide variety of diseases, including arthritis, traumatic brain injury, wound repair, and spinal cord injury. iPSCs come from adult cells, such as skin or blood cells, that have been reprogrammed back to a pluripotent state so that they now have the capacity to differentiate into any specialized cell type. The proper differentiation of iPSCs in a laboratory setting into desired cell types that can be introduced into patients is an important topic of research. How do scientists differentiate the iPSCs in vitro (in a dish) compared to what happens in vivo (in the body)?
In vivo, the differentiation of embryonic stem cells is an autonomous system, where the embryo expresses a variety of growth and signaling factors that result in differentiation to cell types of the three germ layers: mesoderm, ectoderm, and endoderm. The germ layers then continue to differentiate to terminal cell fates creating tissues and organs. In the lab, scientists supplement iPSCs with externally provided growth factors and signaling factors, carefully determining experimentally when to introduce what factor, and for how long. But, how is this done?
Depending on the desired cell type, scientists introduce appropriate factors to iPSCs.These factors were determined by scientists originally studying the development of differentiated cell types in vivo and discovering what transcription factors or growth factors are important. For example, when creating neural cells, iPSCs are first exposed to Noggin (a BMP inhibitor) to prevent the differentiation of non-neural ectoderm and SB4131542 (an Activin and TGFbeta inhibitor) to prevent the differentiation of mesoderm. By inhibiting the differentiation of non-neural ectoderm and mesoderm, the iPSCs are primed towards a neural ectoderm cell fate.
After being exposed to Noggin and SB4131542 for six days, iPSCs begin to express neuroectodermal markers such as SOX1 and PAX6. Subsequently, additional factors can be added to further differentiate the neuroectodermal cells. For instance, adding SHH and FGF between days 6 and 9, followed by BDNF, ascorbic acid, and GDNF between days 9 and 19 leads to the production of dopaminergic neurons. Another example is adding BDNF, ascorbic acid, and retinoic acid between days 6 and 19 to create motor neurons. (Please see the table below for more information regarding the different factors if interested).
Once scientists obtain the desired cell type, they can proceed to the next stage of experiments. While the use of induced neural stem cells has not been approved for humans, there are various animal studies where neural stem cells and other differentiated neural cells have been used to treat models of human brain diseases such as Alzheimer’s and Parkinson’s (reviewed in Zhao et al., 2021). Although it may take some time before these therapies can be introduced in the clinic, it is crucial for scientists to continue to work to better understand the risks and optimize treatment strategies of stem-cell based therapeutics.
| Name | Function |
| Noggin | BMP inhibitor, prevents the differentiation of non-neural ectoderm |
| SB4131542 | Activin and TGFbeta inhibitor, prevents the differentiation of mesoderm |
| SOX1 | Neuroectodermal cell lineage marker |
| PAX6 | Neuroectodermal cell lineage marker |
| SHH | Plays a role in dopaminergic development |
| FGF | Plays a role in dopaminergic development |
| BDNF | Common growth factor for neurons |
| Ascorbic Acid | Helps differentiation of neural cells |
| GDNF | Common growth factor for neurons |
| Retinoic Acid | Induces neural motor cell differentiation |
Citations/Links:
https://www.nature.com/articles/nbt.1529
https://www.frontiersin.org/articles/10.3389/fcell.2020.00815/full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8474718/
Dr. Amy Wong: Modeling human lung development and Cystic Fibrosis using pluripotent cells
Written by Nuzhat Namiha
Cystic fibrosis (CF) is the most lethal recessive genetic disease affecting roughly 4000 Canadians today. Dr. Amy Wong was the first to develop a method for modeling CF in-vitro using airway epithelia derived from induced pluripotent stem cells (iPSC). The CF gene encodes for cystic fibrosis conductance regulator (CFTR). CFTR is a chloride ion channel responsible for regulating fluid flow across all epithelium. △F508 is the most common mutation in the CF gene which prevents CFTR from maintaining salt and water balance. This can lead to the accumulation of mucus in the airways which can promote bacterial colonization and chronic inflammation.
Dr. Amy Wong’s lab published their first iPSC differentiation protocol to create lung airway cells in 2012. The recent development of new technologies has allowed Dr.Wong’s group to revamp their original method of generating lung airway cells from differentiated iPSC to now producing renewable fetal cells. These renewable fetal lung tissues make up the same cell types and cell fates observed in the 16th week of gestation in fetal lung tissue. Thus, for the first time, they can developmentally match fetal lung epithelial cells to late pseudo glandular lung tissue. The significance of their new differentiation model is that it has allowed them to generate canonical cell types found in adult stages from fetal lung cells. These canonical cell types include basal cells, ciliated cells and goblet cells.
There are no cures for cystic fibrosis. Drug treatment efficacies vary in different individuals and there are long-term side effects of these drugs. Using one of the fetal cell models for iPS cells from the Wong lab, Janet Jiang, from the Dr. Christine Bear lab, screens for small molecule modulators. In this study, they tested several CFTR modulators and small molecules that restore CFTR protein production in iPS cell lines harbouring a rare mutation called W1282X. W1282X is a mutation that results in no CFTR protein production because a premature stop codon is causing activation of nonsense-mediated decay resulting in mRNA degradation. The Non-CF plate consisting of a culture containing wild-type CFTR showed robust activation of CFTR channel activity by forskolin. The CF plate contained iPSCs that were differentiated into lung progenitor epithelium. The iPSCs are from a donor who is homozygous for W1282X. In the CF plate, they used different combinations of small molecules to test CFTR channel activity. They found that different combinations of small molecules resulted in different CFTR responses. Some combinations showed higher CFTR activity than others. Results from this experiment prove that iPSC-derived based models have the potential to be used as pre-screening for primary patient cells and to inform patients about different treatment options.
Work from the Wong Lab is crucial for the development of the CF Individualized Therapy program. This program consists of over 100 cell lines from individuals with various CF disease-causing mutations. The program also contains whole-genome sequencing data and RNA sequencing data from each individual in the program to support the ongoing research on cystic fibrosis.
Citations:
https://pubmed.ncbi.nlm.nih.gov/22922672/
https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpz1.341
https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(21)00497-5
Medicine By Design Conference
Senior students in our focus group attended the Medicine by Design (MBD) annual conference in December 2019. The MBD conference displayed the expertise of international researchers, as well as those at The University of Toronto and affiliated hospitals engaged in cutting-edge science across a number of regenerative medicine disciplines. The theme of the conference was: Technology Advancing Biological Insights and Driving Innovation.
Nancy Allbritton: Micro-engineered Intestinal Architecture
Written by Erika McCartney
Dr. Nancy Allbritton, based out of the University of Washington, presented her work in the field of Biomedical Engineering; her current research focuses on the development of in vitro assays that better model the intestinal epithelium while comparing against current organoid models. The mammalian large intestine is composed of many organized gradients (i.e. oxygen, stem cell factors, inflammatory factors, single chain fatty acids, etc.) along crypts that produce specific niches supporting different cell types along the epithelial sheet. While the current model for in vitro study of the intestinal epithelium, the organoid assay, provides researchers with all cell types of the large intestine epithelium, it does so in a way that is disorganized and does not account for any chemical gradients.
Dr. Allbritton sought out to design an in vitro assay that would allow researchers to better model the intestinal epithelium that accounts for the patterning cell compartments and different gradients that exist in the gut. To do this, she decided to produce a monolayer of gut epithelial cells that was folded into microwells, mimicking the existence of crypts. By adding the known chemical gradients to this monolayer in a manner reflecting those of the intestine, the patterning of cells along the microwells were maintained in an identical pattern to the intestine producing an in vitro model that almost identically mimics the in vivo conditions.
As this assay is typically built with human cells, experiments can accurately model the effect of different drug treatments in disease, nutrient and drug uptake and metabolism, the effects of the microbiome, etc. Dr. Allbritton was successfully able to design a model that allows study of the intestine in a way that presents results similar to in vivo studies that is less intrusive and easier to maintain, allowing future opportunities for studies of the intestine and its many functions in human health.
You can read more about this work here: https://doi.org/10.1016/j.tibtech.2018.12.001
Dr. Shana Kelley: Population Bottlenecks
Written by Foram Vyas
Dr. Shana Kelley, a professor at the University of Toronto, focused her talk on the transition from cycle one within the Medicine by Design projects to a more collaborative cycle two.
The main topic of her project in cycle one was to investigate population bottlenecks arising from mesoderm differentiation and how to target it towards regenerative therapies. Dr. Kelley chose to address heterogeneity in subpopulations seen within the early events of hematopoietic stem cell differentiation, and also focused on identifying genetic drivers, generating reagents and creating a strong platform for single cell-omics and high throughput functional genomics.
Dr. Kelley highlighted the importance of collaboration within the Medicine by Design community, emphasizing that the efforts within cycle one from not only her lab, but also all of the participating labs, have led to a very promising set of cycle two projects. As the list of projects and topics is quite long, the following is simply one example of how incredible the collaborations have been. The translation from cycle one to cycle two of a genome-scale loss of function screening project for inducible Cas9 human pluripotent stem cells allows for an investigation into phenotypic screens for driver identification of heterogeneity within those human pluripotent stem cells.
You can read more about this work here: https://www.kelleylaboratory.com/
Dr. Jeff Wrana: Next Generation Organoid Models of Human Brain Development and Disease
Written by By Komal Parmar
Dr. Jeff Wrana is a senior investigator in the Sinai Health system. His talk discussed some of his recent work on next generation organoid models of human brain development and disease. He highlighted the transition from cells to tissue in 4D models. Specifically, Wrana stated that it is important to understand how single cell behaviour drives both tissue degeneration and regeneration.
The Wrana team used single cell technology to generate their organoid models. They aimed to figure out how stem cells function to generate organized structures as well as aiming to understand how tissue complexity can be achieved. During the talk, the hippo pathway was emphasized to provide a potential answer to these questions. Through the use of various stem cell tests, they were able to determine that the hippo pathway was an essential regulator of tissue size control.
The talk also highlighted some of the issues that surrounded their organoid model development. Initially, when they first started to develop organoids, the biggest challenge that they faced was a lack of vascularization in the organoids which led to short culture life. This was due to limited nutrient and oxygen delivery. To overcome this challenge, Wrana and his team utilized de novo vascularization in the human organoid system.
In conclusion, Wrana and his team were able to generate vascularized organoid models of the brain that can provide a platform to study human brain development and disease.
You can read more about this work here: http://attisanowranalabs.science/wrana-publications
Dr. Markus Grompe: Drug selection for the enhancement of liver cell and gene therapy
Written by Iyeh Mohammadi
Dr. Markus Grompe, scientist and the director of the Oregon Stem Cell Center, presented his research on using drugs to enhance liver-directed gene and cell therapies. Innovative new treatments like liver-directed gene repair therapies are becoming more and more popular, but unfortunately, they are not efficient and not many cells within the liver can be successfully treated. Rather than attempting to increase the efficiency of the therapy to treat more cells, Dr. Grompe’s lab instead chose to tackle this problem by using chemoselection to select for the gene-edited hepatocytes and allow them to expand their populations.
His team used non-toxic metabolites that are hepatotoxic precursors, taking advantage of the cells’ biochemical pathways to cell-autonomously kill the unedited hepatocytes while the gene-edited hepatocytes proliferate. Proliferation in edited cells is due to a mutation, introduced along with the treatment. This approach has been proven to be incredibly effective using the tyrosine catabolic pathway in mice, so they opted to use acetaminophen, or Tylenol, a drug that has already been approved for use by the FDA.
Acetaminophen can be metabolized into the toxin NAPQI using CYPOR proteins. Using a CRISPR/Cas9 construct, and later self-cleaving RNAs, to knock out the CYPOR proteins in mice, Dr. Grompe showed the effectiveness of this drug in expanding the gene-edited hepatocyte populations in mice, along with the results of experiments indicating that CYPOR-deficient hepatocytes do not adversely affect liver function in mice. Proof of principle experiments have been successfully done using other gene-integration methods: transposons and lentiviruses. While more research can be done, his work shows just how effective chemoselection with drugs can be in enhancing and improving the effectiveness of gene and cell therapies for liver diseases.
You can read more about this here: https://www.ohsu.edu/stem-cell-center/markus-grompe-md
Dr. Gordon Keller: Novel cell and tissue therapies to treat liver failures
Written by Echo Jing
Dr. Gordon Keller is a senior scientist at University Health Network and his talk discussed the novel cell and tissue therapies to treat liver failures. As a critical detoxification organ, the liver plays an important role in biochemical reactions and metabolism, through liver cells such as hepatocytes and specialized macrophages. Compromised liver function may lead to a variety of liver diseases, which require effective therapies for tissue regeneration and drug application.
In order to analyze primary liver tissue, they performed molecular profiling and mapping of the transcriptome of the liver tissue that directly came from the patient. This helped them identify liver cell types and also to examine the cellular base of the liver diseases. Many different cell types are needed to make a functional liver. The general goal for generating liver cells from human Pluripotent Stem Cells (h-PSCs) is to direct the differentiation of h-PSCs into endoderm cells, then into hepatoblasts, and finally into hepatocytes and cholangiocytes. Hepatocytes can be induced from hepatoblasts with cAMP signaling. Function of hepatocytes is different according to where they locate in the liver — zone 1 or 2. Differentiation of zone 1 and 2 hepatocytes is determined by the gradient of Wnt signaling. In an in vivo transplantation experiment, both types of hepatocytes could be regenerated in the mouse liver. Cholangiocytes, the epithelial cells that line the bile duct, can also be induced from hepatoblasts with the correct signaling. These are ciliated cells, in which primary cilia can be used as an indicator for mature cholangiocytes. The main application of cholangiocytes is to produce monolayer cysts. Liver Sinusoidal Endothelial Cells (LSECs) are highly specialized liver endothelial cells that are responsible for the initiation and progression of chronic liver diseases. These cells have been a more difficult cell type for directed differentiation in vitro as the process for maintaining these cells in culture has not been determined. The Keller Lab used a new in-vivo approach: transplantation of liver cells into an infant mouse showed an increase in human tissue growth in mouse liver, and engraftment of human LSECs into adult mouse showed a better liver tissue generation. These findings are promising steps towards liver regeneration in human patients.
You can read more about this work here: https://pubmed.ncbi.nlm.nih.gov/26167630/
Dr. Maryam Faiz: Gene therapies to promote neuroregeneration and enhance neuroplasticity
Written by Phoebe Bhagoutie
Dr. Maryam Faiz is an assistant professor at the University of Toronto researching gliosis and NSC reprogramming in the brain after stroke. In response to injury in the brain, proliferating glial cells can be reprogrammed into a neuronal cell fate offering a new regenerative type of therapy. Dr. Faiz presented this novel approach in regenerative medicine to evaluate the ability of specific transcription factors to induce a functional neuronal state from astrocyte build-up in the injured brain.
Using endo-1 mouse stroke models to induce the targeted gliosis in the rodent brain, Dr. Faiz and colleagues were able to infect mouse brains with AAV vectors containing astrocyte specific markers and investigate the functional and persisting effects of transdifferentiation of astrocytes into neurons. After treatment, behavioral changes and the restoration of behavior lost due to stroke further supports this therapeutic approach in post stroke in patients. More investigation is required to determine if the effect on behaviour is due to cellular or acellular factors. In future using AAV vectors and the combination of focused ultrasound Dr. Faiz hopes to implement a less invasive delivery of the treatment. Overall, the conversion of astrocytes into functional neurons has promising therapeutic potential.
You can read more about this work here: https://pubmed.ncbi.nlm.nih.gov/26456685/
Dr. Michael Laflamme: Overcoming barriers to cardiac regeneration using pluripotent stem cells
Written by Gregor McEdwards
Dr. Michael Laflamme, a senior scientist and staff pathologist at The McEwan Stem Cell Institute in Toronto, insightfully presented his research and discussed barriers to cardiac regeneration. His focus on the improvement and development of culture conditions required for the differentiation of human Embryonic Stem Cells (h-ESCs) to viable cardiomyocytes has been groundbreaking. Millions of cardiomyocytes can be differentiated for therapeutic use from comparably few h-ESCs. His team has also demonstrated that these cardiomyocytes can engraft into an injured heart and repair fibrotic tissue after myocardial infarction to improve heart function in guinea pig, macaque, and pig models.
Current barriers that would limit the ameliorative benefits of such recellularization therapies include the subsequent prevalence of tachyarrhythmias (fast, irregular heartbeats) after cardiomyocyte engraftment. Although introduced cardiomyocytes fuse with the injured heart, they beat asynchronously. This means that the grafted cells are unable to couple electrophysiologically with the host heart. Elucidating mechanisms underlying this problem remain areas of current research.
You can read more about this work here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524945/
Dr. Freda Miller: Modelling stem cell niches to understand and enhance endogenous repair
Written by Jevithen Nehru
Dr. Freda Miller is a senior scientist at The Hospital for Sick Children, in Toronto who discussed and highlighted some of her recent work in stem cell research. The potential regenerative and therapeutic applications of stem cells are very powerful and in order to harness this, researchers need to get a better understanding of how endogenous repair occurs in normal tissues. These repair mechanisms involve stem cells and the environments or niches of these stem cells play incredibly important roles in their maintenance and regulation.
The Miller lab is working on understanding stem cell niches and environments in an attempt to improve the endogenous mechanisms of neural tissue repair. By understanding the host’s native ability to repair injured tissue, researchers can take strides to manipulate and probe these repair systems to function more efficiently.
Dr. Miller stressed the importance of holistic and systematic approaches to study these challenging and intricate mechanisms of repair. To ensure that they can achieve the most accurate understanding of a native repair mechanism, the team uses cutting-edge technologies including: lineage tracing of neural stem cells, and comparison of single cell transcriptomics via high-throughput sequencing. By doing so the researchers ensure the involved transcription factors, RNA, ligands, and growth factors are appropriately studied. Dr. Freda Miller’s talk highlighted the significance of using systems biology when studying complex mechanisms involved with neural tissue repair.
You can read more about this work here: https://pubmed.ncbi.nlm.nih.gov/30503141/
Lab-Grown Blood Vessels and a Whole Lot of Sugar: How Stem Cell Biology is Helping Unpack the Mysteries of Diabetes
Review by William Shepherd based on:
Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, et al. 2019. Human blood vessel organoids as a model of diabetic vasculopathy. Nature565(7740): 505-510.
What’s new here?
In a piece of ground-breaking research, scientists have been able to harness stem cell biology to create a laboratory model of how high levels of sugar damage blood vessels. Researchers exposed human induced pluripotent stem cells (adult cells that have been engineered back into a stem-like state) to a unique cocktail of signaling molecules in order to guide the cells into forming fully functional, self-assembling networks of mature blood vessels. These blood vessels (which are near-exact replicas of the ones in your body) each consist of a tube of endothelial cells that are wrapped up in a scaffold of molecules that comprise the basement membrane. The basement membrane not only helps anchor the endothelial cells in place but also acts as a seal to prevent the blood vessels from leaking.
The vessels were then flooded with a sugar-rich solution and were carefully watched in real time. Sugar exposure not only caused endothelial cells to die off, but also pushed the basement membrane encasing each vessel to swell uncontrollably until it began to split at a microscopic level—just like a seam bursting in a pair of jeans that don’t quite fit. These splits made the blood vessels leaky and compromised their function.
Why is it important?
This research is interesting because it helps us unpack the mysteries of Type II Diabetes. The bodies of patients with the affliction can’t control the levels of sugar in their blood, and the havoc that sugar wreaks upon blood vessels has been shown to cause kidney failure, blindness, and even limb loss.
Using this laboratory model, though, researchers were able to find two proteins, called DLL4 and NOTCH3, that drive the uncontrolled growth and breakage of the basement membrane in the presence of sugar. When special molecules were used to stop DLL4 and NOTCH3 from functioning, they were able to reverse some of the harmful effects and preserve the sealing function of the basement membrane. Drugs that could block the function of DLL4 and NOTCH3 in humans could help millions of people worldwide.
What’s next?
Before we get too excited, though, it’s important to remember that this research is still in the very earliest stages. While it’s easy to stop proteins like DLL4 and NOTCH3 from functioning in a plate in the lab, it can be difficult to design drugs that can do the same thing in humans—finding a molecule that can distribute evenly throughout the body and help cure disease without causing serious, toxic off-target effects can be like trying to find a needle in haystack. That being said, while drug development is still a project for the future, this new model will still allow scientists to uncover new targets that could become the foundation for new therapies. It serves as yet another incredible example of how stem cell biology can move medicine forward.
Vascularized in vivo human brain organoids and their potential
Review by Nikolai Ho based on:
Mansour AA, Goncalves JT, Bloyd CW, Li H, Fernandes S, Quang D, et al. An in vivo model of functional and vascularized human brain organoids. Nat Biotechnol. 2018. 36(5):432-41.
An in vivo model
Human pluripotent stem cells can be used to create artificial miniature brains called brain organoids. These brain organoids contain different cell types and can display some similar functions to a human brain. However, without the support systems that accompany real brains the potential to use organoids in development and disease modelling is quite limited. One of the biggest limitations is the lack of vascularization; without blood vessels delivering oxygen, organoid tissue would die. In addition, simple organoids fail to exhibit processes, such as neuronal migration, that make the brain so unique. A solution employed by Mansour et al. was to engraft or transplant the vascularized organoid into a mouse brain. This approach had the benefit of using the mouse’s circulatory system to supply the vascularized organoid with oxygen. Being the first procedure of its kind, the engraftment was highly successful since the organoid integrated with the host brain. The organoid grafts demonstrated processes normally exhibited in a developing brain: neuronal differentiation, gliogenesis (development of glial cells), further development of the vascular system, and the growth of organoid axons to different parts of the host brain. Remarkably, the organoid and the host brain even developed synaptic connectivity.
Study significance
Previous to this study, brain organoid systems lacked a vascular system–this meant that the central tissues would die limiting the development of the organoid. Also, organoid development in a culture dish, as done previously, does not replicate the complex environment of a human brain. The host system allows for crucial interactions between cells, tissues, within organs, and between organisms. Placing the organoid in a live organism allowed for analysis of how the organoid interacted and behaved in the context of all of these systems, thus achieving the closest possible replication of its real environment. This approach is only useful if the organoid can integrate with the host vasculature, requiring a vascular system within the organoid. This study showed just that, vascularized organoids integrate to a high degree within a mouse brain allowing for vascularization, neuronal differentiation, gliogenesis, and axonal migration within the human organoid tissue. Organoids also integrated well with the organism–most mice responded well to the engraftment. When placed in a special maze designed to test cognitive function, grafted mice showed no significant difference in learning ability and only a slight spatial memory deficit compared to ungrafted mice which could be attributed to the invasive operation. This experiment provides great insight into brain development since these organoids underwent maturation, differentiation, gliogenesis, synaptogenesis, and axonal migration – all processes which a developing brain would undergo. Furthermore, the in vitro organoid most closely represents the embryonic brain, and its ability to form a neuronal network and integrate into a host brain can reveal new aspects of neuronal development. The authors even hypothesize that organoid transplantation may facilitate maturation of neural circuits. The fact that these organoids integrate so well into existing brains opens up the possibility that they can be used to treat brain disorders or injuries. Perhaps one day, it will be possible to implant organoids into damaged or diseased brain regions and restore functionality in human patients. Further studies could reveal the mechanism behind how neurodegenerative disorders arise and how neural cells interact with their environment, and could allow for drug testing at a resolution never before seen. The possibilities are endless.
The limits
All of the above hypothetical applications of this study are tentative, and many more studies would need to be performed to obtain more information. For example, to test for the possibility of using brain organoids to restore lesions caused by Alzheimer’s, animal models would have to show positive results before proceeding cautiously to human trials. In addition immune rejection may pose a very serious threat in humans if embryonic stem cells are used to drive brain organoid development for therapeutic approaches. A solution may be found in the use of induced pluripotent stem cell (iPSC) technology, which reprograms the patient’s own cells, but the feasibility of this option can only be assessed with further studies. Overall, this research opens a window for new possibilities in developmental research and disease treatment. Many of these possibilities used to seem distant, they may now be within our reach.
Oxidative Stress is a Conserved and Necessary Upstream Signal to Embryonic Wound Healing
Review by Jevithen Nehru based on:
Miranda V Hunter, Patrick Morley Willoughby, Ashley E E Bruce, Rodrigo Fernandez-Gonzalez. Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing. Dev Cell. 2018 Nov 5;47 (3): 377-387.e4. doi: 10.1016/j.devcel.2018.10.013.
What’s new here?
The findings presented in this paper by Hunter et al. are instrumental for building a better understanding of conserved mechanisms behind wound healing. Prior to this work, the role of oxidative stress in the embryonic wound healing process was not fully understood. The authors measured levels of oxidative stress using reactive oxygen species (ROS), which can cause damage to DNA and lead to cell death. Using a variety of methods and two different model organisms, the research group was able to elucidate ROS involvement in both invertebrate (fruit fly, Drosophila melanogaster) and vertebrate (zebrafish, Danio rerio) models.
The team used laser ablation to damage fruit fly embryos and used fluorescent microscopy and appropriate tags to observe the wound healing process in real-time. During wound healing in embryos, the actomyosin cable that forms around the wound opening will begin to shift and constrict, closing the wound space. Cell migration is a vital aspect of wound healing and by measuring myosin polarization, local dynamin levels (indicative of endocystosis) and cadherin trafficking, we can better measure wound healing in embryos.
The authors were able to observe a correlation between sites of ROS production and re-arrangements of cytoskeletal proteins and proteins that interact with the cytoskeleton, like the adhesion molecule E-cadherin. Next, a wide range of ROS inhibitors were added to the embryos and the wound closing process was compared to negative control embryos. Inhibitors of ROS led to marked decrease in myosin polarization, cadherin trafficking and dynamin localization to the wound edge. This data showed a clear relationship and highlighted the positive influence that ROS had on embryonic wound healing.
To further understand the molecular mechanisms behind this process, the researchers looked towards src42A, an ortholog to the human src gene. This gene and its family were implicated in embryonic wound healing and Src protein can be post-translationally modified by ROS via redox-sensitive cysteine residues. The authors hypothesized that through this modification, ROS might direct Src42A to manipulate cytoskeletal elements to promote wound healing. To test this hypothesis, the group used an embryo line with an “oxidation-resistant” mutant form of the Src42A protein which lacked the critical cysteine residue. Supporting their proposed mechanism of action, fluorescent microscopy showed a clear reduction in wound healing in animals with the mutant Src42A protein vs control animals. The cysteine residue of Src42A, and its interaction with ROS were shown to be necessary for the characteristic embryonic wound healing action.
To corroborate their findings, the researchers performed similar experiments with a vertebrate species, zebrafish. They found that effective embryonic wound healing in zebrafish was dependent on both Src and ROS. This finding tells us that ROS’ upstream signalling in embryonic wound healing is conserved in both invertebrate and vertebrate species.
Why is it important?
The findings of this paper have profound implications to the field of development and clear up the role of oxidative stress in the embryonic wound healing process. Embryos have a strong regenerative potential and learning more about this can help us improve adult wound and injury healing processes. We now have a better understanding of oxidative stress and how it can act as an upstream signal and direct thorough wound healing in embryos. The finding that this mechanism and interaction is conserved is also important. If present in humans, perhaps these molecular components can be manipulated in ways to strengthen the potential of adult wound healing. As we learn more about the molecular mechanisms behind wound healing, we can attempt to develop treatment protocols and strategies aimed at helping those with weaker healing abilities and improve adult healing as well.
What’s next?
This paper presents a potential mechanism of oxidative stress promoting wound healing in embryos. Many questions can now be asked here to further our understandings of wound healing. For one, in humans, Src is one protein in a family of several other similar proteins. We have to take a look at identifying all of the components involved in this process. It would be interesting to study whether this mechanism is also conserved in both human embryos and adult humans. Perhaps adults lack certain components in this pathway, and this is the cause of our reduced healing abilities, which often lead to scarring. If we can identify these differences, then through treatments and interventions we might be able to re-create the scarless embryonic wound healing ability in adult systems.