This month’s research spotlight shines on an article published in the journal Experimental Dermatology by John Wiley & Sons Ltd. Entitled “Genome-wide profiling reveals pervasive transcriptional alterations in fibroblasts derived from lesional skin in vitiligo including a reduced potential to proliferate”, this study was carried out in the lab of Principal Scientist Dr Archana Singh with a PhD student Rohit Gupta as the lead author. We interviewed Rohit about his experience in this journey of publication:
Pulse: Congratulations on your publication, Rohit! Let’s start with an introduction to Vitiligo and then about what exactly is genome wide profiling?
Rohit Gupta: Thank you! Sure. Human skin is majorly comprised of 3 main cellular entities epidermal melanocytes, keratinocytes, and dermal fibroblasts. These 3 cell types talk to each other via secretory molecules to maintain skin homeostasis, which is important to the health of the skin. Loss of the melanocytes from the basal epidermis leads to depigmentation conditions in the skin which is known as vitiligo. This is an acquired depigmentation disease with 1-2% prevalence worldwide. Depigmented skin is commonly called Lesional (L) skin, however adjacent pigmented skin is commonly known as peri-lesional (PL) skin. Despite the loss of melanocytes, that are central to maintenance of skin homeostasis, both the L and PL skin seem to have their homeostasis state maintained. Scientists have found that in vitiligo patients, in the absence of melanocytes, keratinocytes are also getting perturbed at cellular, molecular as well as on transcriptomics levels. But, a clear picture of alteration in the third cell type – dermal fibroblasts – remained unknown.
So, we decided to do the genome-wide profiling (total RNA sequence) of L and PL skin-isolated fibroblasts from a similar anatomical site. Total RNA was extracted and sequenced to understand the alteration in the molecular and biological pathways of L and PL skin-derived fibroblasts.
Pulse: What is the gist/summary of your work?
Rohit: We found that despite the similar anatomical site PL and L skin-isolated fibroblasts are altered at the transcriptomic level, with different signatures. L skin-isolated fibroblasts exhibit lower proliferation and higher differentiation potential. When we clustered the top-downregulated genes we found that other than the proliferative pathway most of the pathways were related to keratinocyte biology, which indicates the cross-talk between both cellular entities.
Pulse: That’s interesting. What was the most difficult moment while working on this paper?
Rohit: As you might have noticed, this study was dependent on the patient’s skin samples. Recruitment of patients for this study was one of the most challenging tasks during the initial phase of this study. Most of the patients were not ready to give the skin biopsy from PL skin, even if they agreed to the L skin. Although that was the job of our collaborator, Dr. Dr Rachita Mishri, it was the starting material for our experiments. The next challenge we faced was the standardization of in-vitro culture conditions for the isolation and propagation of fibroblast cells from the very small size of the biopsy sample. We usually got a 2-3mm punch biopsy from the skin (both PL and L), plus dermal fibroblasts are very less in number in the skin dermis. Anyway, by the time we figured out how to adhere fibroblast cells to the surface, the Covid pandemic hits us. So, for at least 1.5 years we did not get any skin samples since vitiligo is not a life-threatening disease (that is, patients would live even without regular visits to the doctor) and thus, patients were not visiting hospitals.
Pulse: Kudos to you and the team for tiding over these difficulties! Would you like to share some of your memorable moments while working on this paper?
Rohit: The most memorable time was when we finally published this study after several efforts not just made by me, but also by my mentor Dr Archana Singh and my collaborator Dr Rachita Mishri who recruits vitiligo patients for biopsies, and Manish Chowdhary who help me during revision work.
Pulse: How much time do you usually spend in the lab and outside the lab? How did that change while the paper was in revision?
Rohit: I usually spend 10-11 hours in the lab, however during revision work it didn’t change much as Manish my colleague was helping me in the experiments.
Pulse: How did the reviewers treat your paper?
Rohit: I felt that the suggestions from the reviewers were very critical of my work. In the initial draft, we had 2 or 3 experiments which suggests the viability and the proliferation, however, addressing the changes in the phospho-protein level was entirely the idea/suggestion of the reviewers. But then I feel sometimes reviewers’ suggestions and suggested experiments make your manuscript clearer.
Pulse: How did you feel when your paper got accepted?
Rohit: Yeah, I was happy when I read the manuscript acceptance mail. Not just me but my whole team was happy!
You, our readers might agree that this was quite an adventurous ride in research! Our key takeaway is that patience and perseverance pays off at the end. What was your takeaway from Rohit’s journey? Share with us in the comments (or drop us an email to get featured in the next editorial!).