“Is this a real human brain?” I asked. The answer was yes. The liver, lungs, spleen and stomach that were on display were also real—all from donated human bodies. My 3-year-old daughter put on a latex glove and eagerly touched each of the organs, while my 6-year-old son stood back at a distance, wide-eyed. We were at the Discovery Expo on the University of Wisconsin-Madison campus, a free kid-friendly science event featuring dozens of interactive exploration stations. Continue reading
Author: Johanna Lee
My City Flooded, and There’s More to Come
It seemed like the rain was never going to stop. It started in the morning, and when I left work around 5pm, it was still coming down hard. I took my normal route home through a back country road. As I turned right onto Fitchrona Road, a long line of cars came into view. There’s usually some congestion leading to the stop sign ahead. Except today, something was different. About 20 yards of the road ahead was submerged in water. Continue reading
Autophagy: The Poem
Roberta A. Gottlieb, MD, is the Director of Molecular Cardiobiology at Cedar-Sinai, a nonprofit academic healthcare organization. She is interested in the role of autophagy in myocardial ischemia, a kind of heart disease in which blood flow to the heart is blocked. (Studies have shown that autophagy is upregulated during myocardial ischemia, but why this happens is not entirely clear.) Her ultimate goal is to understand and mitigate ischemic injury, with the hope of developing therapeutics for humans.
And—she’s a poet. Continue reading
Using Environmental DNA to Find Sharks
Sharks are often known as one of the fiercest predators in the ocean. Yet they are also one of the most threatened marine species—largely because they are hunted by humans for their highly valuable fins. How do we know they are being threatened? Continue reading
High-Throughput Drug Screening Using 3D Cell Cultures
For a long time, the drug industry has relied on flat 2D cell cultures grown on a plate to screen for potential drugs. However, 2D models do not accurately reflect the native environment of cells in vivo. 3D cell cultures, on the other hand, better represent the numerous cell-cell and cell-matrix interactions and hypoxic conditions that have a profound effect on the behavior of cells. In a 2018 study published in Oncogene, Kota et al. developed a high-throughput 3D spheroid-based screening assay to identify drug candidates that target RAS proteins.
RAS proteins are GTPases that transmit extracellular signals into cellular signaling pathways, which could activate cell proliferation, differentiation and survival mechanisms. Oncogene mutation in the three human RAS genes (HRAS, NRAS and KRAS) are found in 30% of all cancers, making RAS proteins the most common oncogene. In fact, mutations in KRAS are found in >90% of pancreatic cancers. Despite the prevalence of RAS mutations, targeting RAS proteins with drugs is extremely challenging due to the complex nature of the protein.
The authors in this study wanted to test a new approach using a 3D spheroid-based screening assay to find drugs that target RAS proteins. They first harvested 2D monolayer cultures of pancreatic epithelial tumor cells that express either wild-type KRAS or mutant oncogenic KRAS, and tested their ability to form 3D spheroids. They confirmed spheroid growth using the CellTiter-Glo® 3D Cell Viability Assay with linearity of detection in the range of 1,000–10,000 cells seeded.
The 3D spheroids were then treated with a library of 1,280 known drugs. From the high-throughput screen, they identified one compound with the greatest selective inhibition against oncogenic KRAS. The compound is called Proscillaridin A, a cardiac glycoside that is known for treating congestive heart failure and cardiac arrhythmia. In 3D spheroids, Proscillardin A inhibited oncogenic KRAS at a >90% inhibition rate, with <10% inhibition of wild-type KRAS. In 2D cultures, however, there was no selective inhibition of oncogenic KRAS (inhibition rates for both oncogenic and wild-type KRAS were about 50%). This means that Proscillaridin A would not have been identified as a candidate if the screen was done using only 2D cultures.
Next, the authors wanted to determine how Proscillaridin A impacts tumor cell viability. Could it induce apoptosis in tumor cells? To test this, they used the RealTime-Glo™ Annexin V Apoptosis Assay. This bioluminescent assay is able to detect apoptosis in real time, based on the exposure of phosphatidylserine on the outer leaflet of the cell membrane when apoptosis occurs. Using this assay, they found that Proscillaridin A induced apoptosis at earlier time points and higher rates in 3D spheroids expressing oncogenic KRAS compared with wild-type KRAS. In 2D cultures, there was no difference in the rate of apoptosis.
This study shows that high-throughput screening in 3D spheroids can identify potential drugs that would not have been discovered in a 2D format. This provides hope for finding drugs against difficult target proteins such as RAS.
Wonders of the Conscious and Unconscious Mind—What I Learned from the International Forum on Consciousness
When Heather Berlin was 5 years old, she realized that, at some point in the future, she was going to die. This disturbed her so much that she couldn’t sleep all night. The next morning, she asked her father where she could store all her thoughts so they could live on after she died. There’s no way to do that, said her physician father. “What can I do to make this happen?” she asked. “Maybe become a psychiatrist?” said her dad. Decades later, she became an Assistant Professor of Psychiatry at Icahn School of Medicine at Mount Sinai. Her research focuses on interactions of the brain and mind, with the goal of treating and preventing psychiatric and neurological disorders.
Dr. Heather Berlin told this story at the International Forum on Consciousness held at the BioPharmaceutical Technology Center Institute in Madison, Wisconsin last week. This annual forum gathers scientists from around the world, all interested in understanding how our conscious and unconscious minds work. This year, the forum focused on the newest research and technology for detecting and measuring consciousness. As someone with limited knowledge in this field, my mind was blown by how much researchers have learned so far about consciousness. (No, we can’t store our thoughts in a box…yet.) Here are a few takeaways: Continue reading
20 Years of Human Embryonic Stem Cells
Dr. David Russell presenting at the 13th Wisconsin Stem Cell Symposium. The session was moderated by Dr. James Thomson.
“20 years ago, when I first heard about the creation of human embryonic stem cells, I knew that this was the future. I immediately requested the cells from Dr. Thomson and dropped almost everything else we were doing in our lab. It has been my focus to this day.” The person presenting is Dr. David Russell, a professor at the University of Washington. He is just one of the hundreds of researchers gathered at the BioPharmaceutical Technology Center Institute (a nonprofit supported by Promega) in Madison, Wisconsin for the 13th Annual Wisconsin Stem Cell Symposium that happened this week. This year, it’s not just a symposium, but also a celebration—it’s the 20-year anniversary of the first-ever isolation and culture of human embryonic stem (ES) cells.
In 1998, Dr. James Thomson, at the University of Wisconsin-Madison, created the first ES-cell line using donated (unused) embryos from a fertility clinic. The study sent a shockwave through the scientific community and general public. We now had the technology to grow human pluripotent ES cells—with the potential to develop into every cell type in the human body—in a dish! Thomson quickly became a celebrity scientist. (Thomson’s headshot was on the cover of the August 20, 2001 issue of Time Magazine, next to big text that read: “The Man Who Brought You Stem Cells”.)
However, not all were excited about the news. Backlash from conservative communities, who opposed the use of human embryos, resulted in a temporary ban on developing new ES cell lines with government funding. Nonetheless, the ban did not deter researchers from studying ES cells using private or state funding. By 2001, human ES cells have been successfully derived into neural, cardiac, hematopoietic, endothelial, and insulin-producing cells. In 2010, the first in-human clinical trial was initiated; which used human ES cell-derived materials to treat spinal cord injury.
2006 marked another milestone in stem cell research: the discovery of induced pluripotent stem (iPS) cells. Dr. Shinya Yamanaka at Kyoto University successfully reprogrammed adult fibroblasts (common cells in connective tissue that form the extracellular matrix and collagen) to revert back into an embryonic-like pluripotent state—simply by expressing four specific genes. He named these reprogrammed cells “induced pluripotent stem cells” or iPS cells. A year later, human iPS cells were made in a similar fashion by both Thomson and Yamanaka. Yamanaka later received the 2012 Nobel Prize (some argue that Thomson deserved to share the prize).
Photo credit: 123rf stock photos.
The ability to reprogram adult cells back into a pluripotent state suggested we could create an unlimited supply of pluripotent cells that genetically matched a specific individual—without the ethical baggage of using human embryos. This meant, in theory, you could take fibroblasts from a patient with a neurological disorder, such as Parkinson’s disease, revert the fibroblasts into iPS cells, edit the “faulty genes” in those cells, then redifferentiate the healthy iPS cells into neural stem cells that can be introduced back into the same patient to produce healthy neurons. Of course, this is easier said than done. The technical difficulties and high cost of generating and editing iPS cells from individual patients have complicated the development of iPS-based treatments. Currently, there is only one human clinical trial using cells derived from iPS cells, which treats macular degeneration (an incurable eye disease that leads to blindness).
Despite the emergence of iPS cells, ES cells have continued to dominate in the clinical realm. To this date, there are 18 clinical trials using ES cells to treat various disorders, including macular degeneration, Parkinson’s disease, spinal cord injury, heart disease and diabetes. The future is bright, but there is still one major problem in ES cell-based therapies. Because ES cell treatments use donor cells from other healthy individuals—not the patients’ own cells—there is a high risk of immune rejection. But no fear, scientists have a plan.
In 2017, Dr. David Russell (mentioned in the beginning of this blog) re-engineered human ES cells to remove specific proteins—human leukocyte antigens (HLA)—from the cell surface. HLA proteins allow the immune system to determine whether the presenting cell is “self” or “foreign”. Removing HLA proteins is like wrapping the foreign cell with an invisible cloak, rendering it unnoticeable by the immune system. In his talk at the Stem Cell Symposium, Russell discussed the many advantages of using these “universal donor cells (UDCs)” to treat diseases. Only one cell line is needed, which reduces the cost, complexity and time required for clinical trials. Also, it does not require immunosuppression, which weakens the patient’s immune system. Russell and many others believe that UDCs are the future of regenerative medicine. In fact, UDC-based therapies to treat cancer, macular degeneration, skin wounds and type 1 diabetes are already being developed.
It is amazing to see how far we have come over the last 20 years. Thanks to visionary scientists like James Thomson, Shinya Yamanaka, David Russell—and countless other principal investigators, post-docs and grad students who work tirelessly in the lab every day—treatments for many life-threatening diseases may be available in the near future. Nonetheless, there is still much more to learn and many more challenges to overcome. Who knows where the next 20 years will take us?
How to Reduce Cell Culture Variability
Scenario 1: Jake needs a flask of MCF-7 cells for an assay, so he sends an email to the graduate student listserv asking for cells. Melissa replies that she has an extra flask of cells that she could share. Jake happily accepts the cells and begins his experiment.
Scenario 2: Michael passaged his cells yesterday and, according to the protocol, was supposed to plate cells today for treatment. However, his previous experiments were delayed, so he decides to plate them tomorrow instead. The cells look healthy, so it should be ok.
What is wrong with the above scenarios? These actions may seem harmless, but they could be the cause of variability, leading to irreproducible results. Continue reading
Weird samples? Contact Tech Serv to find the right DNA purification kit for you.
“Dear Tech Serv,
We would like to detect DNA collected from swabs rubbed on the inside thighs of frogs. What would be the best DNA extraction kit to use for this?”
“Hi Tech Serv,
I need to find out a suitable kit for extracting DNA from bird fecal samples. Can I use ReliaPrep™ gDNA Tissue Miniprep System for that?”
These are just some examples of unconventional sample type inquiries that the Promega Technical Services Team receives regularly from scientists around the world. Many of these inquiries land in the hands of Technical Services Scientist, Paraj Mandrekar (a.k.a. “sample type guru”). Continue reading
A Virus-like Neural Pathway Hints at the Origins of the Mammalian Brain
The mammalian brain is extremely complex. We know that it processes and stores information through synaptic connections within a complicated neural network. But how exactly do neurons communicate with each other? And how did this neural network come to exist? A recent paper published in Cell may provide some answers. It describes a previously unknown signaling pathway–with surprising origins–that transports RNA between neurons. Continue reading