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 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

Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. Continue reading

Using CellTiter-Glo® Luminescent Cell Viability Assay to Assess Cell Viability in Cancer Cells Treated with Silver Nanoparticles and DNA-PKcs Inhibitor

Silver nanoparticles (Ag-np) are commonly used in many consumer products, including cosmetics, textiles, electronics and medicine, largely due to their antimicrobial properties. More recently, Ag-np are being used to target and kill cancer cells. It has been known for years that silver nanoparticles (Ag-np) can induce cell death and DNA damage. Studies have also shown that Ag-np inhibit cell proliferation and induce apoptosis in cancer cells. However, cancer cells are able to fight back with DNA repair mechanisms such as non-homologous end joining repair (NHEJ). The NHEJ pathway requires the activation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus DNA-PKcs may protect against the Ag-np-induced DNA damage in cancer cells.

Could inhibition of DNA-PKcs increase the ability of Ag-np to kill cancer cells? In a 2017 study, Lim et al. wanted to test whether inhibition of DNA-PKcs can increase the cytotoxic effect of Ag-np in breast cancer and glioblastoma cell lines. To effectively determine cell viability in these cancer cell lines, the authors used the CellTiter-Glo® Luminescent Cell Viability Assay. The CellTiter-Glo® Assay determines the number of viable cells in culture based on quantitation of ATP, an indicator of metabolically active cells. A major advantage of this assay is its simplicity. This plate-based assay involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. This generates a luminescent signal proportional to the amount of ATP present, which is detected using a luminometer. Cell washing, removal of medium and multiple pipetting steps are not required. Another advantage of the CellTiter-Glo® Assay is its high sensitivity. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing, making it ideal for automated high-throughput screening, cell proliferation and cytotoxicity assays.

The authors first confirmed that Ag-np treatment reduced proliferation and induced cell death/DNA damage in two breast cancer cell lines and two glioblastoma cell lines. The cytotoxic effect of Ag-np is specific to cancer cells, as minimal cytotoxicity was observed in non-cancerous human lung fibroblasts used as control. Next, they pre-treated the cancer cells with a DNA-PKcs inhibitor for 1 hour before adding Ag-np. Inhibition of DNA-PKcs increased Ag-np-mediated cell death in all four cancer cell lines. This suggests that DNA-PKcs may be protecting the cells from Ag-np cytotoxicity. The authors further showed that DNA-PKcs may repair Ag-np induced DNA damage by NHEJ and JNK1 pathways. In addition, DNA-PKcs may help recruit DNA repair machinery to damaged telomeres.

This study suggests that a combination of Ag-np treatment and DNA-PKcs inhibition may be a potential strategy to enhance the anticancer effect of Ag-np.

Reference: Hande M.P., et.al. (2017) DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells. Mutat Res Gen Tox En. 824, 32

Evaluating DNA Quantity and Quality in FFPE Tumor Samples After Prolonged Storage Using the ProNex® DNA QC Assay

When tumors are surgically removed from cancer patients, the tumor samples are often stored as formalin-fixed and paraffin-embedded (FFPE) tissue blocks. In many cases, tumor samples need to be analyzed several years after diagnosis in order to develop target treatments. But what happens to the DNA after years of storage in FFPE blocks? How well is the DNA preserved?

Scientists in France tried to answer this question in a recent study published in Virchows Arch. The authors extracted DNA from 46 FFPE tumor samples of lung, colon and the urothelial tract, all stored between 4–6 years at room temperature. They then compared the quantity and quality of the DNA to DNA that had been extracted before storage. Using common fluorimetry and qPCR methods, the authors found that the total amount of DNA extracted decreased by half. In addition, the percentage of amplifiable DNA decreased from 56% to only 15% after prolonged storage. Continue reading

Your Kid Can Become a Citizen Scientist with These 7 Apps

Has your kid ever asked you what you do in the lab all day? (“Hmm…good question, what am I doing all day?”) A simplified answer might sound something like this: I observe, ask a question, collect data, and use those data to answer the question (or at least try!). The scientific method may be difficult to explain to a kindergartner, but you can always start by encouraging them to observe the world around them, ask lots of questions—and even help collect data. In fact, with the help of technology, your child can become a scientist without 7 long years in graduate school or ever setting foot in a lab. A “citizen scientist”, that is. All you need is a smart phone. Here is a list of apps that can make your kid, grandma, neighbor, anyone, become a citizen scientist by helping professional scientists collect data for their research. The apps are all free to download, easy to use and have a real impact on the scientific community.

1. iNaturalist:

Are you sick of not knowing the answer when your child asks “What’s this bug?” on a nature walk? You need the iNaturalist app. Here’s how it works: You observe an interesting plant/insect/animal, take a photo, and the app identifies the name of the species and some basic information about it. There is also the option to share your finding with other users and they can suggest an identification. iNaturalist shares your findings with scientific data repositories like the Global Biodiversity Information Facility to help scientists understand biodiversity. You can even set up scavenger hunt-like activities: iNaturalist birthday party anyone? Continue reading

Measuring Metabolic Changes in T cells with the Lactate-Glo™ Assay

Immunometabolism

Welcome to the emerging frontier of immunometabolism. A decade ago, immunology and metabolism were seen as two distinct areas of study. However, we now know that specific metabolic activities are required for proper immune cell differentiation and function. In tumor microenvironments, immune cells may even alter their metabolism to compete with tumor cells for limiting nutrients.

Glucose metabolism in Naïve vs Effector T cells

What does your car and T cells have in common? They both shift gears! You can shift gears on your car to change the way the engine’s power is used to match driving conditions; when you’re going uphill, you switch to a higher gear. Similarly, when T cells are activated, they change the way they generate energy to match functional needs. This makes sense because activated T cells (known as effector T cells) require more energy and biomass to support growth, proliferation and effector functions.

While cars run on gas, the main fuel for T cells is glucose. Each glucose molecule is broken down into pyruvate while generating 2 ATP molecules. Naïve T cells completely oxidize pyruvate through oxidative phosphorylation to generate 36 ATPs per glucose molecule. However, when T cells are activated and become effector T cells, glycolysis is used to produce 2 ATPs per glucose molecule. Continue reading

iGEM: Saving the World with Science

The University of Chicago 2016 iGEM team group photo (Photo credit: Julia Byeon)

Every year, groups of teenagers gather together and brainstorm ways to save the world—with science. The International Genetically Engineered Machine (iGEM) Foundation is a non-profit organization that is dedicated to educating young scientists and enhancing open community and collaboration in the field of synthetic biology. They hold a competition every year with hundreds of teams participating from around the world.

Last year, Promega provided cloning reagents to the University of Chicago iGEM team, and they received a bronze medal for their work. We asked two of the team members, Steve Dvorkin and Julia Byeon, about their experience. Steve is a junior and majors in biology; he is co-president of the team this year. Julia recently graduated and works in public policy. Continue reading