The last few weeks of my internship at Dr. Miki’s lab were incredibly eventful. Every procedure I had completed, every cell culture plate I maintained, and all of the work I had done had finally come to a head.
During the second week of July, I prepared and commenced definitive endoderm differentiation of my undifferentiated H9 cells. Because the lab I work in is focused on treatments for liver diseases, looking at how this new molecule HT4201 could affect endoderm tissue, the precursor to liver tissue, is a crucial aspect of my project. The differentiation process involves media complete with nutrients designed for growth and differentiation. I was obliged to add certain supplements in very specific concentrations throughout the 5-day process. Thus preparation beforehand included separating the basal medium and supplements into usable aliquots and storing them in the -20 degree freezer. Preparation like this is very common in our lab environment, especially because lab-mates use the same reagents. Aliquotting is a courtesy both expected and appreciated in the lab.
After a successful protein array, I performed PCR on the RNA I had isolated throughout the internship from both my H9 and definitive endoderm cells to be able to look into which genes are affected by the presence of HT4201. Using the concentrations that I measured from my RNA beforehand, I made 20 uL of cDNA from each well. I then used this cDNA for qRT-PCR (quantitative real-time PCR). The difference between PCR and qRT-PCR is that with the former the process just amplifies whatever section of DNA you place in the thermo-cycler. But with qRT-PCR, we use a set of primers that target the genes we want to observe, and the machine gives a report on which primers are being used during the process to figure out which genes have been turned off or on. The process of preparing for qRT-PCR was tedious—I was using a 384-well plate and all colorless reagents, so mistakes were easy to make. The important thing to do when making a mistake, however, is to record exactly what went wrong. This prevents any mal-interpretation and possibly fraudulent data.
These 8 weeks I spent at the USC lab were more eventful than my experience last summer. In fact, this year’s internship felt like an extension of last year’s. Since I needed no training, I was able to go directly into my work for the summer. My PI also trusted me with more important projects, like culturing cells for other lab-mates or running an extremely delicate protein array. However, even though I shadowed and learned my techniques last year, I never stopped learning new things during these past 8 weeks. I found that learning by experience is one of the most effective methods for me personally, and that mistakes are part of the process. My PI never discouraged me by being disappointed in any way, and because of his positive attitude the entire lab strived to be the best we could be without being told. I will undoubtedly be using these learning techniques in any field I enter later on in my life. I feel very fortunate to have been able to spend my summer learning and working with such a talented and dedicated team.
During the past three weeks at my internship, I continued maintaining and culturing mouse embryonic stem cells for my eventual protein array. I had started with one plate of cells measuring 100mm in diameter (called a p100 plate); I needed 2 confluent 6-well plates in order to begin preparation of the array. The general idea has been to add HT4102, a small molecule that has recently shown to have an extremely beneficial effect on cancerous cells, to these 6-well plates. After 24 hours these molecules theoretically will have been absorbed through the membrane of the cells and begun expressing protein specific to the molecules. My plan was to differentiate my ES cells into definitive endoderm cells, add HT4102 to them, and use the protein thereafter expressed in a protein array to understand exactly how this small molecule works.
The next week of my internship was dedicated to further practicing proper cell culturing. I also learned a new cell passaging technique: cell cutting. Because I work with stem cells, it is important that I keep my cell colonies as undifferentiated as possible until I purposely differentiate them. Eventually some cells begin differentiating on their own (I somehow even ended up with neurons, which was fascinating but they were really not what I needed). Because of this spontaneous differentiation, researchers “cut colonies,” or manually remove undifferentiated colonies from the original plate and place them on a new plate. The new plate would theoretically have only undifferentiated stem cells, and the researcher would be able to proceed with her/his project. Towards the end of June, I began preparing for definitive endoderm differentiation by ordering the reagents that I needed. These reagents come in large stock volumes, so I must aliquot them as necessary and with the proper concentrations. Aliquotting will take place the day before I intend to use the reagent, because the smaller volumes can only keep for about a week before they are considered unusable.
One of the most intriguing characteristics of the research field is its astonishing rate of advancement, which is something that I learned hands-on during this second portion of my internship. Our lab received a newly developed sample of a reagent for cell passaging from StemCell Technologies that eliminated the need for colony cutting, a technique that I had learned just a week prior. This incredible reagent gently detaches only undifferentiatied cells and leaves differentiated cells on the plate. I must admit the arrival of this reagent provided some relief, because I was still unsure of my skill level in cutting.
Unfortunately, another thing I learned hands-on was the fact that in research all progress can disappear in one day. Two weeks ago,I began my day as usual, by observing my plates before changing their media. I was shocked to find that I could barely see my colonies through the suddenly cloudy media. Upon aspirating the old media and observing the colonies under the microscope again, I saw what seemed to be billions of small blue particles twitching under the lens. My P.I. explained to me that those particles were yeast, and my plates had been contaminated somehow. Even my backup plate was contaminated. I was forced to toss all of my progress in the biohazard bin and start from scratch. As annoying as it was to go through that, though, it was a necessary lesson to learn in the field.
I found during this internship that much of research requires a good deal of time management and multitasking. For example, plates need to be coated with gelatin solutions before plating cells in order for the cells to properly adhere and remain viable. This coating can take 30 minutes to over 2 hours to prepare, depending on the cell line and concentration/composition of the gelatin. I must then plan my work for the day accordingly—if there is a part of the project that can be done in an hour, I would plan to do that while the gelatin is setting. Waiting for tubes to be centrifuged can also take time; I usually take advantage of that time to count cells and do my calculations so that I can continue my work smoothly. Otherwise time is wasted, something that is not an option for an eight-week-long internship.
I am excited to see what else I can learn in the last three weeks of my internship. My newly plated ES cells are almost ready for definitive endoderm differentiation, and I am determined to reach the end of my internship with significant data from my completed protein array in my hands.
I began my internship with Dr. Miki’s lab at USC on Tuesday, June 3rd. Upon arrival, I discussed with Dr. Miki his plans for my stay as a member of his research team. Dr. Miki’s project, in an overarching sense, looks into the use of stem cell research to find a viable, more efficient, and more effective treatment for metabolic congenital liver diseases. My main project for the duration of the eight weeks is to prepare and run an array of a small molecule that has recently proven to have a positive effect on cancer: it seeks only the cancerous cells, and does not harm those that are healthy. The goal of the array is to observe the specific effect of this protein on certain antibodies, which could lead to insight on how it functions in vivo. I am to cultivate ES cells (called H9 cells) until they are confluent (the population has grown to the point of covering the entirety of the cell culture plate), induce differentiation into definitive endoderm cells, and extract the protein created by these cells to use in the array. I am also to extract RNA and sequence it in the hopes of learning more about the nature of this protein.