|Ryan Fong working in the Good Manufacturing Facility as part of Gerhard Bauer's lab at UC Davis. He submitted this photo through Instagram to CIRM's #CIRMStemCellLab collection|
This entire experience has been beyond amazing. In this world, it’s too easy for even the most ambitious dreamer to lose sight of their goals and fall off the path that would have realized their full potential. The long road to a professional career can seem long and uncertain at times. Amidst the pitfalls of life, new and old responsibilities, seemingly never-ending work, and immediate trials and tribulations, it’s not hard to see how one might lose their way. This program showed me that hard work really will be recognized and rewarded, leading to great things. It’s treated me like the mature individual I’ve always strived to be. Receiving a stipend has allowed this program to be financially feasible and making it all the more real. And finally, it has reassured any doubts my mind might have fostered that I was anything but on the right path to success.
Now, on to my project. Embryonic stem cells (ESCs) were a breakthrough discovery for regenerative medicine, because of their ability to differentiate into all three germ layers, including tissues that won’t normally be repaired in the body, like cardiac or nerve tissue. However, ESCs require the disassembly of a human blastocyst. Induced pluripotent stem cells (iPSCs) are derived from adult cells, without the involvement of an embryo, and also allow future treatments to be made using a patient’s own cells. iPSCs are extremely difficult to obtain, even with careful coddling, babysitting, prayers, etc. (≤0.1% viability) - only a handful of colonies are viable out of millions of cells.
The iPSC induction method I used was based on a HIV-derived lentiviral vector containing four transcription factors, Sox2, Oct4, Klf4, and c-Myc, which upregulate the expression of pluripotency genes still present in a cell’s genome. With these factors, the cell will express these downregulated pluripotency genes - in effect, a slate wiped clean to be differentiated into a cell type of choice.
I worked in the GMP facility here, which meant donning a full coverall gown, gloves which were taped to the cuffs, and surgical mask for hours at a time (you could only see my eyes) all the while practicing special technique for working in this environment. An outside environment has ~35,000,000 particles ≥ 0.5 µm per cubic foot per minute, but in the GMP facility, there are ~100,000 particles ≥ 0.5 µm per cubic foot per minute. Additionally, all work was conducted within a biosafety cabinet, which permits a mere ~100 particles ≥ 0.5 µm per cubic foot per minute. I cultured HEK-293T cells, which were transfected using lisosomes (a technique called lipofection) with DNA plasmids so they could serve as producer cells to manufacture my vector. That same vector was used to transduce human fibroblasts, and after two weeks, several iPSC colonies had been created.
I will remember this experience forever. I’m entering this next school year with my passion for my studies reinvigorated, after seeing how that knowledge translates to the practical, and I really hope that many more students will be able to experience what I experienced in the future through this Creativity program. Thank you CIRM!