“…filled with learning the technicalities and maneuvers of different procedures, equipment, and computer software.”

By: Emma Friedenberg

This week in the Mattis Lab at Cedars-Sinai Board of Governors Regenerative Medicine Institute was filled with learning the technicalities and maneuvers of different procedures, equipment, and computer software. The most interesting and hands-on experience was RNA isolation, the purification of RNA from biological samples. The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiments.

Ribonucleic acid is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases (adenine, guanine, cytosine, and uracil), which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.

A rapid method of analysis for determining the identity of neural stem cells (NSCs) and their sublineages involves the early detection of differentiation markers tracked at the RNA level. This week, I learned the procedure of RNA isolation from cell lines. The first round of RNA extraction was performed on a small set of three cell pellets – one each for my two mentors and one for me. Each step was rotated between the three of us for learning practices. The next day, a total of 18 cell pellets of our cell line underwent RNA isolation which was executed by myself and my post-doctoral mentor. By the end of the procedure, 1 microliter of RNA was transferred to the computer analysis system. Fortunately, all of the cell lines achieved an RNA concentration above the contamination or error mark.

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“….there is more to researching than pipetting and extracting RNA-I say this because it has been big this week but rather it is a lifestyle for many individuals.”

By: Dennis Portillo

As the third week of the CIRM/SPARK program nears its end, the days that have passed have almost been a blur. The routine work of being in a lab has now been established, but still each day a little bit different than the previous. That statement sums up the lives of most scientists, who do not follow the standard procedure of simply working five days a week and taking holidays off. The life of a researcher, in particular, is not one that is necessarily easy, rather but be a journey of ups and downs, sprinkled in with blood, sweat, and tears (just not in the experiment hopefully). From this week, the task of being a researcher goes far and beyond more than just the occupation in its name, with experiments at times taking longer hours than most are accustomed to, and any mishap having the possibility of setting it back experiments. Perhaps learning this lesson in itself is more memorable than any sort of hands-on bench work, or anything that could be attained from an experiment. Talking with my mentors this week has helped me realize that there is more to the questions of simply why and how, using these to stem into other more valuable questions as well as accepting the fact that experiments are not as straightforward as one may think. While it may take a few minutes to a few hours for all the procedures to be done if lucky, it takes much longer to even analyze what has been done and gained from it.

While the past two weeks have mostly consisted of collecting and analyzing data, it has something that I’ve come to enjoy. For many, the summertime has become one of relaxing and enjoying, but the same is not necessarily true for scientists. Earlier in the week, I was discussing how many are eager to go into the field, yet not prepared for much of what they have to give up, with sometimes the future exchanged little in success. While saying this is by no means an effort to be cynical, it gives hindsight to have a realistic point of view and how the main motivation as to why you get up in the morning and power through the day being passion and drive. Perhaps a few cups of coffee may also do the trick but that’s not the point. For many scientists, their work is their everything, and even if the original hypothesis may turn out wrong, or the data provide very little, many falls in love with their work (and rightfully so). So, while counting cells and looking at neurons under the microscope may be some notable highlights of the week, the real takeaways that come from this week are the ones that stay with you forever. As cliché as that may sound, there is more to researching than pipetting and extracting RNA-I say this because it has been big this week but rather it is a lifestyle for many individuals.

“…the knowledge I have acquired and the friendships I have already developed are unlike anything I’ve ever experienced, and I cannot wait to continue my work in the weeks to come!”

By: Candler Cusato 

I can’t believe I’ve already been in the lab for two weeks; it sounds cliché, but time certainly does fly when you’re spending almost seven hours a day learning, experimenting, and meeting new friends. Overall, this week has been filled with simultaneous challenges and triumphs, stretching me as both a scientist and an individual, which I can’t wait to share.

After I toured more of the Cedars campus on Monday and Tuesday, I attempted the most challenging scientific technique I’ve ever been exposed to: retinal whole mounting. Over what seemed like days (but was probably only an hour and a half), I removed the cornea and lens from a rat eye under a microscope and detached the retina from the rest of the eye casing to mount it on a slide. This technique allows you to view the entire retina while preserving vascular and retinal structure in your specimen, which cannot be accomplished by other sectioning techniques that involve cutting tissue with a machine. To understand the difficulty of removing a PBS-soaked retina from the remains of an eye, I would compare it to trying to turn the pages of a soaked, 1000-year-old book without ripping the pages. In short, it is VERY difficult, and it will definitely take me much more tries to improve my technique.

Outside of the lab, I was thoroughly enthralled during my lunchtime learning sessions this week, which is humorously deemed “Knowledge Noshes,” particularly during the presentation by Dr. Robert Baloh, MD, Ph.D. His progress in creating gene and stem cell therapies for amyotrophic lateral sclerosis (ALS/Lou Gehrig’s Disease) is truly fascinating, and his passion for the subject was palpable during his lighthearted but educational presentation. He peaked my curiosity in neurology so much that I am intrigued to explore more about the subject of neurodegenerative diseases and possibly pursue it in my future studies.

Also, this week, I gained a new laboratory companion, as Amanda, an intern from Brown University, arrived on Tuesday to join Dr. Wang’s lab. In just a few days, we’ve braved whole mounts together and suffered through the slow-loading microscope camera software while waiting to take photographs of retinas. With Dr. Wang’s help, we are on our way to perfecting our labeled retinal images in Photoshop and learning the various eye histology techniques and the intricate system of blood flow throughout the eye.

As this second week of CIRM Spark comes to a close, I feel even more fortunate that I was selected for this prestigious opportunity, as the knowledge I have acquired and the friendships I have already developed are unlike anything I’ve ever experienced, and I cannot wait to continue my work in the weeks to come!

“I am slowly yet surely unraveling and participating in, and I look forward to all that is to come.”

By: Kevin Kim

It’s week two already, and I cannot believe how quickly the lab experience has turned up its pace! I’ve finally participated in some hands-on laboratory procedures while reading lots of journals and papers on corneal epithelial cells and wound healing – the life of a researcher has many facets, it seems.
One of the highlights of this week was thoroughly learning about and performing the ICC, or immunocytochemistry, staining, which localizes antigen expression and is dependent on specific epitope-antibody interactions. ICC uses cell cultures and generates a strong and specific signal for each antigen of interest. For example, detection of an abundant protein in cultured cells may require a short fixation period, minimal blocking, and may be compatible with direct visualization using a fluorochrome-conjugated primary antibody. In contrast, detection of a phosphorylation-dependent epitope in a section of archival tissue may require antigen retrieval and be dependent on amplified chromogenic visualization.
The procedure is a five-hour process, so I spent morning to afternoon working on this part of the lab. Although a seemingly daunting and arduous task, the ICC was quite simple with the help of the Research Associate, who carefully and expertly explained each step while having me simulate and perform the exact actions and procedures.
I never really understood why there are so many individual lab procedures involved in the grand scheme of things, and I didn’t quite grasp how all of these facets related to the lab’s focus on gene therapy and corneal diabetes. However, that is a question that I am slowly yet surely unraveling and participating in, and I look forward to all that is to come.

Candler Cusato

Blog 1 Entry:

Hi, my name is Candler Cusato, and I am rising senior at Los Alamitos High School in Orange County. But, more importantly, I am one of eight high school research interns selected to participate in seven weeks of stem cell research funded by the California Institute for Regenerative Medicine at Cedars-Sinai Medical Center. For the next two months, I will be exploring the progression of retinal degeneration and stem cell-based therapies to combat blindness in the millions of patients suffering from retinitis pigmentosa and age related macular dystrophy, under the direction of Dr. Shaomei Wang of the Regenerative Medicine Institute, and this is the account of my first few days as an intern in a state-of-the-art scientific lab!

After waking at 5:45 AM and braving the daily 405 shuffle to get to Cedars from Long Beach, I arrived on Monday morning and immediately fell back into my usual pattern of rapid note taking during biology lectures, as program director Dr. Virginia Mattis gave us an introductory lecture on stem cells. With her help, my eyes only slightly widened when I reached the lab later that afternoon and everyone uttered acronyms, such as IPSC’s, IVF, FDSC’s, with practically every breath. Luckily, they fed us pizza to keep our heads from spinning, and I went home feeling proud but slightly cross-eyed, after I spent my afternoon gazing into the microscope and successfully removing rat lenses and corneas.

Tuesday morning, after the slight hiccup of waiting for the wrong elevator for approximately 10 minutes, I was immediately whisked into a meeting between Dr. Wang and her outgoing intern, and I listened attentively to their conversation on how human cell injections influenced rat RPE production in the retina. Searching, as all scientists do, for more data, we all journeyed into the lab and examined the retinal slides tagged with various fluorescents to show the distinction between cell types, seeing first hand how the injection of foreign cells significantly decreased retinal pigment epithelium. By the end of the day, I had also helped feed and change the medium in the lab’s neurosphere cultures, which I learned will soon become my full responsibility, and journeyed down to Third Street to discover the wonders of Somma Somma, a Chipotle-style Mediterranean restaurant.

Wednesday morning, after a long conversation with my mentor about my life and college plans, I went to work preparing slides and examining them under the microscope to search for folds in our eye section samples, easily able to be ruined since they are only millimeters thick. With help from the post-baccalaureate intern, Alice, I was able to identify the layers of the retina, as she walked me through their usual structures and appearances under the microscope after being stained with cresyl violet. Thursday, I continued my work in staining, learning the complex and ten-step process that ensures my hard work does not go to waste and my slide contents can be seen under the microscope. Although my first batch was too light, I eventually was able to view my specimens, and to my immense relief, they were identifiable as retinal cells. Score!

In days at Cedars so far, I have been surrounded by unparalleled friendliness and pursuit of knowledge for the overall benefit of mankind. My time here, although brief, has already yielded a new understanding of eye structure, disorders, and laboratory techniques crucial to my success in my future undergraduate studies. If this week is any indication, I am confident that I will leave in August with a newfound appreciation and understanding of scientific research and the preparedness to undertake a career in the scientific field.

 

 

Soah Franklin

Blog Entry 1: 

Hi! My name is Soah and I am an incoming junior at Crossroads School. This was my first week in the Cedars-Sinai CIRM SPARK Program. I am working in the lab of Dr. Mehrnoosh Ghiam, who is studying the role of miRNA in corneal homeostasis. This week, I got to observe the process of harvesting and growing the limbal (peripheral cornea) epithelial cells, prepare buffer solution, and learn about Western blotting. On day one, I watched Mangesh (Dr. Ghiam’s research fellow) analyze the Western blot of the LESC proteins that he had grown. I learned about the reactions between an antigen and the primary and secondary antibodies, and how Dr. Ghiam was using antibodies to evaluate their reaction with human proteins. On day two, I studied ocular anatomy and the layers of the cornea. I learned about the differences between limbal and corneal epithelial cells and read a research article about how micro-RNA’s like miR-146a affected wound healing and cell migration in diabetic patients. Diabetes can lead to ocular complications like keratopathy and can cause problems with epithelial erosions, microbial infections, and delayed wound healing. Dr. Ghiam is researching and trying to understand the role miRNA plays in the regulation of the eye’s stem cells, specifically those in the limbus.

On day three, I got to observe the incredible process of harvesting the epithelial stem cells. It was amazing watching Mangesh dissect and study the primary human eye tissue, which was donated post-mortem from a diabetic patient. I had never seen a dissection of a human organ, so this was a new and exciting experience for me! On day four, I attended my first Knowledge Nosh with my CIRM cohort. Our first speaker was Dr. Kimberly Gregory, and OB-GYN, who gave an inspiring talk on how she overcame adversity to become a doctor. I want to work in obstetrics when I am older, so hearing Dr. Gregory’s talk was especially informative.

Overall, I had a great first week in the CIRM SPARK program. At first, I was intimidated, because I am a little younger than most of my fellow interns, and I didn’t think that I could keep up. But as the week progressed, I felt more confident. My lab supervisors were really understanding and made me feel comfortable about asking questions. This was my first time doing research, and I already know that I want to continue into my adult career. My friends think I’m crazy because I willingly gave up my summer to work in a lab, but I am so happy. I found something that I am truly passionate about – something that makes me excited to wake up in the morning, something that inspires me. Stem cells research is incredible – I am witnessing the future of medicine unfold, and I feel so proud and lucky to be a part of it!

Maya Golob

Blog Entry 1:

My name is Maya Golob, and I am a rising senior at Harvard-Westlake School in Los Angeles, California. I am one of eight research interns in the seven week CIRM SPARK summer program at Cedars-Sinai. I am working in the Regenerative Medicine Institute under my mentor, Gianni Carraro, in Dr. Barry Stripp’s lab.

I’ve nearly completed my first week in the CIRM SPARK research internship at Cedars-Sinai. With my assigned mentor out of town, I have had many lectures and from the Research Associates about what they are working on and other things that go on in the lab. In four days, I have already learned so much! I want to explain the three main topics I’ve learned: flow cytometry, gel electrophoresis, and CF cells.

Flow cytometry: Each cell has different surface markers. We can select fluorochrome antibodies to bind to a specific surface marker that we want to study, which is essentially labeling the cells because they create a fluorescence that we can track. We then put the cells into the machine and lasers excite them, making the electrons jump to a higher energy state. When the electron falls, it releases energy called a photon (light energy). This allows us to see the colors the cells emit and the machine sends data to the computer for each cell. We can then track which cells glowed certain colors (meaning they are positive for a certain gene), and check to see if the experiment is working all in a matter of seconds.

Gel electrophoresis: First, we isolate the DNA from bacteria following the steps in a kit. Then we add restriction enzyme digestion. With an Nkx2-1 plasmid, we use HindIII as the enzyme to produce cuts with sizes 7001, 1001, and 1011 base pairs. We run the DNA through the PCR machine to make copies of the DNA. Then we stain the DNA and insert it into the 1% Agrose gel we made. The results of the gel electrophoresis tell us that the plasmid was successfully inserted into the DNA if the bands line up with the 7000 band and the 1000 band.

CF cells: These cells are a certain type of cystic fibrosis stem cells. SOX2 is an essential transcription factor for maintaining self-renewal (pluripotency) for stem cells. We can insert this into the cells by creating a plasmid with the SOX2 gene via bacteria. We can then extract the plasmid from the bacteria through a series of steps. Once we have the plasmids, we can insert them into the cells and check it with gel electrophoresis. The cells will first produce GFP, meaning that they glow green. Then the guide RNA, which we made to find the gene of interest, tells CRISPR where to cut. Bacteria use CRISPR as a defense mechanism against viruses because it cuts the DNA of the virus. Scientists found a way to use it to edit the DNA of all cells, including human cells. Then, natural transcription will insert the plasmid loop into the DNA, which takes about three days. If the plasmid is digested successfully, it will leave the SOX2 gene and blue fluorescent protein (BFP). Through the fluorescent microscope and flow cytometry, we can see if the cells glow blue. If they do, we know that they contain the plasmid we wanted.

In these four days, I was also able to practice sterile technique while culturing mesenchymal cells in the tissue culture room that has the fume hoods. I was able to practice my pipetting while making more media, a colored fluid that the cells live in. I look forward to working with my mentor when he returns and I’m eager to start working on my project for the summer!

Hector Medrano

Blog Entry 1:

My name is Hector Medrano, and I am an Intern for 2017 California Institute of Regenerative Medicine, Summer Program to Accelerate Regenerative Knowledge at Cedars-Sinai Medical Center. This summer, I will be working under the guidance of Dr. Vaithi Arumugaswami, Director of the Liver Program in the Board of Governors Regenerative Medicine Institute. In his laboratory, Dr. Arumugaswami’s research delves into the regeneration of liver and bile duct cells using Induced Pluripotent Stem Cells (iPSc) and Pluripotent Stem Cells (PSc), as well as the different effects Zika Virus has on specific organs, viewing different modes of infection.

This being my first week in the laboratory, however, my experiences were not as tactile as expected. Who knew one had to learn to perform the techniques and protocols needed for one’s experiments and conduct research on the research one is going to conduct, before conducting their research? Nonetheless, whether observing a post-doctoral research fellow change a cell culture’s medium, or undergo a flow cytometry analysis, every second spent in the lab seized to amaze me. What was most exciting was observing the iPSc and PSc’s under the microscope, where one was able to see the intricate morphology of each cell line. Aside from observing such, the remainder of my time in the laboratory was spent reviewing articles and past experiments conducted on pluripotent stem cells, and overwhelming my post-doctoral mentors with numerous questions.

Better-acquainting myself with the methodology of a “wet-laboratory,” I am very excited/eager to witness what the remainder of the summer has in store. I would like to thank the California Institute of Regenerative Medicine for offering such astounding programs, allowing high school students as myself to obtain hands-on experiences working in state-of-the-art laboratories. I would also like to thank Cedars-Sinai Medical Center for hosting the program and providing us all with wonderful mentors, as Dr. Vaithi

Dennis Portillo

Blog Entry 1:

As I write this entry, the first week of the CIRM/ SPARK internship is rearing its head and is drawing towards a close. It’s amazing at how seven days ago prior to today, I was in bed confronting self-induced butterflies seemingly sprouting in my stomach. While that statement is nowhere near scientifically correct, it was a clash of nerves and excitement that were playing a game of seesaw internally. Flash toward to today, and while part of me is looking forward to sleeping in for the first time this week (yes, it’s summeritus), the other half of me is astounded at how fast this week has gone by. The expression of time flies when you’re having fun seems applicable here, with that being appropriate in regards to learning and meeting new faces.

Doing research outside of school has always been something that I’ve been looking forward to since the beginning of high school. While most are enjoying their summer by traveling or going out, my summer consists of spending a little over six hours in a laboratory, learning about topics that I barely knew existed. In school, my favorite subject has always been biology simply because it has been the one subject I have enjoyed reading further beyond the given course material. However, one thing that I never really got to experience was doing actual lab work. Going to Hamilton High School, the science courses that I have taken were generally more orientated toward lectures. Sure, some dissections came around once a year, but being the little nerd that I am, I wanted more. However, I still didn’t know what to expect in terms of what I was going to be doing, let alone what any of these published papers meant. With the help of Nur and Veronica, however, those nerves have been quelled, with them making every step of the way easier. Both so far have been patient while teaching, as well as being very nice, with the plan to learn as much from them, as well as get to know them more throughout the coming weeks. The entire Svendsen lab has treated me the same way, whether it be other post-docs or interns. Under Nur so far, I have learned more about a gene called BRCA1 and its role in DNA repair, whereas, under Veronica, it was geared more towards neuron activity and how electrodes can be used to record activity. Playing the role of data analysis, they both have emphasized many points, such as the stressing of asking questions, as well as the importance of communicating, while putting the “re” in research. On top of that, they’ve also taken a bunch of measures to make sure I still have all ten digits.

The CIRM/ SPARK program so far has been different from other internships that I have done, granted this being the only research one. With what I have learned so far, whether it be working with waveforms and sorting spikes to capturing pictures of cells with different fluorescent markers, it has only made me enjoy research more. Obviously, I can’t wait to work more hands on, but those days will be here sooner than expected, and by the end of the program, the main goal is to make sure I don’t lose Veronica’s very expensive program key, as well as learn as much as I can in regards to information on a topic and lab protocol/techniques.