Skin renewal is characterised by progenitor cells at the epidermal basal layer dividing and differentiating, yet the identity and properties of these progenitors remain poorly understood. In a new study, Tudorita Tumbar and colleagues identify, for the first time in vivo in the mouse epidermis, a distinct population of non-self-renewing progenitor cells that exhibit biphasic transit-amplifying behaviour. To find out more about the story behind the paper, we caught up with first author Sangeeta Ghuwalewala, penultimate author David Shalloway and corresponding author Tudorita (Doina) Tumbar, Professor of Molecular Biology and Genetics at Cornell University, USA.
Sangeeta Ghuwalewala (left), David Shalloway (centre) and Tudorita (Doina) Tumbar (right)
Doina and David, can you give us your scientific biographies?
DT: I have been fascinated with stem cells since I was a senior graduate student looking for a new field to study for my post-doc. I read the beautiful paper by James Thompson on the isolation of human embryonic stem cells and fell in love with these intriguing cells that can both self-renew and differentiate for extended periods of time. Tissue stem cells were hard to isolate and study, due to lack of markers to identify them. Decades-old models suggested that stem cells are more infrequently dividing than transit amplifying cells. I joined the skin stem cell field and developed a pulse-chase system in mice that allowed me to isolate infrequently dividing cells as putative stem cells and more frequently dividing cells as putative transit-amplifying non-self-renewing progenitors. Isolation and transcriptional profiling led to the identification of putative markers for stem and transit-amplifying cells. Later, with new markers identified, I was able to prove by lineage tracing that this approach worked well for characterising stem and transit-amplifying cells in the hair follicle, but this did not seem to be the case in the epidermis.
DS: I received my PhD in theoretical physics and did a post-doc in that field. But, motivated by my childhood experience with cancer, I did another extended post-doc to change my research direction to experimental studies of the activation and regulation of oncogenes. Later, as mathematical modelling and computational analysis gained importance in biology, I was delighted to put my mathematical training back to use by adding a computational biology group to my program. Beyond our own studies, we've enjoyed collaborating with experimental groups studying interesting problems requiring computational modelling.
Sangeeta, how did you come to work in Doina's lab?
SG: I am a stem cell enthusiast with a PhD focused on exploring the mechanisms of stemness in cancer stem cells of oral squamous cell carcinoma (OSCC). My interest in understanding normal stem cells led me to discover the Tumbar lab, which focuses on skin stem cells and aligned perfectly with my previous research. I met Doina at a stem cell training workshop and was inspired by her work and energy, which motivated me to apply for a post-doc position in her lab. During my tenure in her lab, I have investigated lineage organisation in the epidermis. My research included clonal lineage tracing, which revealed intriguing details about stem cell behaviour in the skin.
David, how did you come to collaborate with the Tumbar group?
DS: I was originally introduced to the field of skin development by a seminar presented by Prof. Tumbar and became intrigued by the possibility that my group's expertise in mathematical modelling and computational biology could be applied to the experimental data being generated by her group. Our groups have enjoyed a long and fruitful collaboration covering a variety of topics. Over time, I have even learned quite a bit about development.
What is the background that inspired this work?
DT: A series of high-profile articles that started in 2007 and span into today's age proposed that there is a single basal type of progenitor cell and that, in fact, stem and transit-amplifying cells do not exist in the epidermis or in many other epithelial tissues. This was disappointing because this meant that epidermal stem cells were not the unique fascinating cell type I hoped to understand. We began to lean into this model too when our work on the epidermis using the pulse-chase approach identified two self-renewing cell populations (one faster dividing than the other) and no transit-amplifying population (Sada et al., 2016). Working with two motivated undergraduate students (who are both middle co-authors in this study), we went on through many years of painstaking clonal analysis counting cells on confocal images to study in detail stem/progenitor cell behaviour in the epidermis. A large amount of clonal data began to accumulate that Sangeeta, the first author of the paper, then assembled and completed by 2021.
Can you give us the key results of the paper?
DT: We observe that unlike all other populations characterised to date, which have a continuous behaviour, the Aspm-CreER marked population was initially robustly contributing to homeostasis but was abruptly switching gears at the latter stages, when it was completely lost from the tissue. This was the first biphasic transit-amplifying progenitor identified to date in the epidermis, and we were extremely excited to find it. I still remember showing David the raw plotted data and pointing to the lack of growth in colony size and their complete disappearance by 1 year of chase.
This was the first biphasic transit-amplifying progenitor identified to date in the epidermis, and we were extremely excited to find it
The difficulty was now that all the mathematical modelling done to date was on progenitors with continuous behaviour, not biphasic. The latter change their parameters as they ‘age’ and the homeostatic population is a mixture of cells at different clonal ages, making modelling much more difficult. What I thought it'd be a quick calculation to provide quantitative parameters for our clonal data, turned into years of insulating work by David to adapt the critical birth-death model into the generalised birth-death model. This mathematical apparatus will now apply to lineage tracing data for any type of progenitors in any type of tissue where lineage tracing is performed.
Maximum projection through confocal stitched z-stack images of whole-mount mouse tail epidermis stained for B1 integrin (green) and DNA (blue). Red represents tdTomato marked epidermal clones of a newly identified transit-amplifying epidermal progenitor 1 week after genetic labelling induced by Aspm-CreER recombinase activation.
Maximum projection through confocal stitched z-stack images of whole-mount mouse tail epidermis stained for B1 integrin (green) and DNA (blue). Red represents tdTomato marked epidermal clones of a newly identified transit-amplifying epidermal progenitor 1 week after genetic labelling induced by Aspm-CreER recombinase activation.
Were there any particular result or eureka moment that has stuck with you?
SG: Absolutely! Throughout my lineage-tracing studies, I was potentially researching for a stem cell type region that was specific to the tail skin epidermis. While other populations demonstrated long-term persistence and clonal expansion, indicating a stem cell presence in a specified manner, the clones of the Aspm lineage exhibited a perplexing outcome. After an initial increase, they disappeared completely within 1 year. This discovery was a true eureka moment for me, which I also confirmed in many experimental repeats. To our satisfaction, it was further confirmed to be a transit-amplifying cell through mathematical modelling, thanks to years of hard work by Prof. David Shalloway.
Were there any moments of frustration or despair during this project?
SG: Given the sensitivity of lineage tracing by tamoxifen injection, I experienced numerous moments of frustration when the CreER recombination failed to work, or the dose wasn't accurately captured. This often resulted in an insufficient number of clones or required scanning large areas to confirm my findings. Countless experimental repeats were necessary to ensure that our conclusions were unambiguously correct.
DT: A very low point in this project was when, for one version of a submitted manuscript (reviewed before submission to Development), we were required to identify the mechanisms of phase transition of this population. We expect this endeavour to be the focus of another decade of searches by many labs, so the paper remained stranded in bioRxiv from 2022. This restricted funding on my grant and Sangeeta had to move on to a different lab and project without publishing her paper. Compounded with the unexpected difficulties in implementing the sophisticated generalised birth death model to lineage-tracing data, this was a serious low moment for me and the project. However, Sangeeta and David both stuck with me, despite the incredible difficulties, and they never gave up. Sangeeta kept working on the paper as much as she could at night and weekends, while working full time in another lab, commuting over 1 hour each way and caring for her young daughter. David had retired in 2019 due to health issues but continued to work for free (since I lacked funding support until late 2023) full time from his treehouse in Ithaca (NY, USA) to complete this study. David had been a great role model for me and for Sangeeta; with his integrity and rigor, he has been a great inspiration for both of us.
Sangeeta and David both stuck with me, despite the incredible difficulties, and they never gave up
How was your experience submitting your manuscript to Development?
DT: We are very impressed with the professionalism of editors, the quality and depth of reviews, and the feedback we received at Development. We are so thrilled and feel rewarded that our paper has been picked to be featured as a Research Highlight.
Sangeeta, what is next for you after this paper?
SG: Currently, I have transitioned to a lab specialising in the hair follicle stem cell niche, where I am specifically investigating the role of dermal papillae (DP) in the hair cycle. While my primary focus is on the signalling mechanisms underlying DP-mediated hair induction, our lab is also dedicated to identifying the ‘cell of origin’ for DP progenitors. This research aims to enhance our understanding of DP maintenance and propagation throughout the hair cycle.
Doina, where will this story take your lab next?
DT: We are very excited about the prospect of continuing this study. We believe it opens a new era in epidermis stem cell biology. It puts stem and transit-amplifying cells back on the map, demonstrates that all basal progenitors are not functionally equivalent, and elicits new and very intriguing questions about mechanisms of long-term self-renewal and of phase transition for different classes of progenitors. These mechanisms are now more feasible to address directly using our system and we are very excited to pursue some of these directions. This study is a completion of over two decades of hard work and focus to find these unusual cells. To the young scientists I will tell: it was worth it.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA.
E-mail: [email protected]
