The 64th annual Montagna Symposium on the Biology of the Skin, “Harnessing Stem Cells to Reveal Novel Skin Biology and Disease Treatments,” was held from 15–19 October 2015 in Gleneden Beach, Oregon. The meeting brought together scientists in academia and industry working on mechanisms of skin homeostasis, regeneration, and tumorigenesis and trainees who wanted to learn more about stem cells in the skin. The Symposium was chaired by Xiao-Jing Wang and Valerie Horsley with Session Chairs Mayumi Ito, Rui Yi, and John McGrath.
The meeting opened with a Keynote presentation by Haifan Lin, Director of Stem Cell Center at Yale University, who unfolded the history of the discovery of RNAs that regulate gene expression and control stem cell activity. In particular, Dr. Lin discussed the intergenic regions within eukaryotic genomes that contain multiple DNA sequences that are not transcribed into protein-coding genes, including transposons, pseudogenes, long noncoding RNAs (lncRNAs), and P-element induced wimpy testis-interacting RNAs (piRNAs). Data from Dr. Lin’s laboratory have revealed that piRNAs can be derived from transposons and pseudogenes and play a major role in degrading mRNAs and lncRNAs in spermatocytes in mice. These data place piRNAs in a core RNA regulatory pathway modulating other RNA species to influence gene expression during meiosis. These exciting new paradigms provide new avenues for exploration of gene regulatory networks in development and stem cell biology in the skin.
“Stem Cells in Development and Diseases” Session Chair Mayumi Ito discussed how melanocyte stem cells function in the hair follicle. Her laboratory showed that Wnt signaling is essential for melanocyte stem cell differentiation; however, lineage tracing of Wnt-active differentiated melanocyte stem cell progeny shows that they can revert back to their undifferentiated state. This plasticity occurs after multiple differentiation stimuli, including UVB irradiation. In aged mice, defective Wnt signaling leads to ectopic differentiation and failure to revert, resulting in melanocyte stem cell loss in the tissue. This novel step in melanocyte self-renewal may be related to melanoma progression, because melanocyte stem cells can generate melanoma in a Wnt-dependent manner.
Marcus Bosenberg continued the discussion of melanoma by describing the current thoughts on cancer stem cells (CSCs) in melanoma. Whether melanoma contains a population of cells that display stem cell properties has been debated in the literature. Dr. Bosenberg’s laboratory has generated several mouse models that develop aggressive melanoma and has begun characterizing heterogeneous populations of cells within these tumors to define how intratumor heterogeneity can influence tumor progression.
Tudorita Tumbar described her work on characterizing the dynamic behavior of epithelial stem cells in the skin. She used a transgenic mouse model in which pulse and chase with a histone, H2B–green fluorescent protein (GFP), effectively labeled epithelial cells based on divisional frequency. She showed lineage tracing data testing the classical stem cell model, in which infrequently dividing cells are stem cells and frequently dividing cells are transit amplifying cells. She presented models of dynamic behavior of epithelial stem cells during adult homeostasis and discussed her work on the molecular markers and mechanisms regulating the behavior of epithelial lineages in the skin.
The molecular mechanisms that regulate epidermal differentiation and self-renewal were explained by George Sen’s talk on the role of RNA helicase DDX6 in the epidermis. Careful molecular analysis of DDX6’s function in keratinocytes defined its role in preventing differentiation through binding to stem loops of mRNAs involved in proliferation and self-renewal to facilitate translation. DDX6 also degrades mRNA transcripts for genes involved in epidermal differentiation, including KLF4. These functions reveal novel regulation of translational control in stem cells in the epidermis.
The “Genetic and Epigenetic Regulation of Stem Cells” session began with Session Chair Rui Yi presenting analyses of the mechanisms that regulate self-renewal and proliferation in hair follicle stem cells. Using modern genetic and molecular analyses for transcription factor binding within genomes and mathematical modeling of mRNA profiling data, Dr. Yi’s laboratory has identified an adaptive transcription factor network that coordinately controls quiescence in hair follicle stem cells, revealing pathways that can be modulated to regulate hair follicle growth.
Yali Dou’s talk initiated a discussion of histone methyltransferase regulation of stem cell biology. Her laboratory has developed a small molecule inhibitor of mixed lineage leukemia protein 1, a lysine 4 (K4) histone methyltransferase. Using this mixed lineage leukemia protein 1 inhibitor, Dr. Dou’s laboratory identified the importance of histone methylation at distinct stages of development in embryonic stem cells. Future experiments analyzing how this pharmacological inhibitor alters skin development and regeneration may reveal the importance of K4 methylation in skin biology.
William Lowry presented work on the regulation of tumorigenesis in the skin by stem cells in the hair follicle. Lineage tracing of cell populations in the hair follicle has revealed the capacity of follicular bulge stem cells to form squamous cell carcinomas upon genetic activation of KRAS, and shown how stem cell quiescence protects these cells from contributing to squamous tumors. Dr. Lowry’s group is now defining novel mechanisms that regulate squamous cell carcinoma initiation and determining how tumor cell heterogeneity arises in response to different cell origins, genetic alterations, and molecular changes during tumorigenesis.
The regulation of histone methylation in skin progenitor cells continued with the talk of Elena Ezhkova, who analyzed the function of the polycomb repressive complex (PRC) 2, which generates trimethylation of histone H3K27, leading to chromatin compaction. Deletion of components of the PRC2 complex showed that polycomb is not required for generation of epidermal specification. However, mice lacking PRC2 display increased numbers of Merkel cells, the sensory cells involved in the tactile response of skin. Molecular and genetic analysis of keratinocytes lacking PRC2 showed that polycomb is required for activation of transcription factors essential for Merkel cell differentiation. These studies indicate that Merkel cell lineage specification requires chromatin regulation by the polycomb complex.
Session Chair Valerie Horsley introduced the “Microenvironment of Stem Cells in the Skin” session by discussing how dermal adipocytes are regulated during hair cycling and wound healing. Her laboratory previously defined a role of these cells in regulating the hair follicle cycle and fibroblast function during skin wound healing. Her recent data implicate platelet-derived growth factor signaling in the regulation of the dermal adipocyte niche and identified a novel interaction between dermal adipocytes and monocyte-derived cells after skin injury.
Fiona Watt continued the discussion of mesenchymal cells and epidermal cells during skin homeostasis. Her laboratory has studied heterogeneity in the dermal fibroblasts and currently is analyzing the role of signaling pathways that differentially regulate these populations of cells. In addition, her laboratory has continued to explore the heterogeneity of epidermal stem cell populations using lineage tracing in mouse models.
An intriguing presentation by Melissa Wong focused on a unique role of macrophages in controlling CSC properties. Using mouse models of melanoma (as well as colorectal and mammary cancer), Dr. Wong’s laboratory has demonstrated that macrophages can fuse with CSCs to form macrophage-tumor cell fusion hybrids that harbor properties of the parental macrophage cell, effectively providing metastatic behaviors to the cancer cell, including the ability to survive as a circulating tumor cell (in humans and mice) and seed distant metastatic sites. Her group’s data provide a new paradigm for understanding how cancer cells acquire metastatic traits and have implications for novel approaches to therapeutic targeting of this unique tumor population.
Matthew Rodeheffer presented data on the regulation of adipocyte precursor cells in response to diet. Dr. Rodeheffer’s laboratory identified and characterized a population of adipocyte precursor cells in adipose tissue in the mouse that respond rapidly to high-fat diet and contribute to increased fat mass. His talk provided potential targets for obesity that regulate adipocyte precursor cells.
In the Banquet Keynote address, George Cotsarelis outlined his contributions to the hair follicle stem cell field since localizing these cells to the mouse hair follicle bulge in 1990 and the human bulge in 1996. His Nature Biotechnology
paper in 2004 described the isolation, lineage tracing, molecular characterization, and multipotency of the bulge cells (
Morris et al., 2004
- Morris R.J.
- Liu Y.
- Marles L.
- Yang Z.
- Trempus C.
- Li S.
- et al.
Capturing and profiling adult hair follicle stem cells.
). These discoveries ignited the field of hair follicle stem cell biology, leading to a multitude of studies from his and other laboratories to solidify the importance of these cells in hair follicle cycling and to identify mechanisms that control their activity. His laboratory also described the preservation of the bulge cells in androgenetic alopecia and the role of prostaglandin D2 in inhibiting stem cells in this disorder.
The “Stem Cell-Based Therapies and Innovative Reprogramming Technologies” session was introduced by John McGrath, who presented the urgent need for therapies for patients with an inherited blistering skin disease, recessive dystrophic epidermolysis bullosa (RDEB). His work has identified the ability of mesenchymal stromal cells to be recruited to skin-damaged areas. Clinical trials using bone marrow transplantation and injection of allogeneic fibroblasts in patients with RDEB have shown some clinical benefits, including reductions in inflammation, pain, and itching. Future cell therapies from other sources were discussed.
This clinical theme was continued by Jakub Tolar, who has performed clinical trials for patients with RDEB after hematopoietic cell transplantation. Previous work from Dr. Tolar’s laboratory suggested that hematopoietic cell transplantation increased expression of collagen VII in patients. Dr. Tolar expanded this study, suggesting that modulation of transplanted cells can improve collagen VII expression and reduce blistering, while emphasizing the need for future studies and analysis of cell populations that are contributing, so as to meet the dire clinical need for new treatments.
Angela Christiano presented fascinating progress in the development of three-dimensional skin equivalents from human cells. Combining differentiation protocols for fibroblasts, keratinocytes, and melanocytes from induced pluripotent cells (iPS) and bioengineering techniques, Dr. Christiano’s laboratory has developed complex three-dimensional skin constructs that more faithfully model human skin. These models will be a unique tool for pharmaceutical screening and as therapeutic grafts for skin diseases.
Markus Frank presented work on a dermal cell population that expresses adenosine triphosphate–binding cassette member B5. Recent work from his laboratory suggests that these cells have unique immunoregulatory functions and can be used in preclinical allotransplantation models as well as for improving wound healing. His talk revealed novel molecular mechanisms underlying the function of this cell population and its relationship to melanoma stem cells.
The utility of iPS cells for therapeutic reprogramming and treatment of monogenic human diseases was further discussed by Anthony Oro. His laboratory has genetically corrected iPS cells from epidermolysis bullosa patients and provided proof of concept for disease-modifying activity with corrected human keratinocyte sheets. Given the low efficiency of generating human keratinocytes, his laboratory has analyzed the transcriptional and chromatin dynamics underlying human embryonic stem cell differentiation into keratinocytes. This strategy has revealed transcription factors, lncRNAs, and chromatin regulatory elements that are altered during keratinocyte specification in human embryonic stem cells. These advances in genetic manipulation and keratinocyte specification show potential for efficient therapeutic strategies for patients with skin diseases.
Session Chair Xiao-Jing Wang introduced the “Normal Stem Cell to Cancer Stem Cell Conversion” session by introducing her work on CSCs in squamous cell carcinomas. Using models of active Kras12D and Smad4 deletion in K15+ stem cells in mice, Dr. Wang’s laboratory has shown that although CSCs have the capacity to form multiple types of tumors, isolated CSCs can only generate malignant squamous cell carcinomas. Characterization of CSC properties revealed specific markers of CSCs with metastatic potential. By manipulating specific properties of CSCs, the metastatic potential of CSCs was attenuated, providing leads for exciting targeted therapies for metastatic skin cancer.
The heterogeneity of cancer cells within skin tumors was the topic of Markus Schober’s presentation, which revealed that tumor cells have distinct proliferative rates, containing quiescent and rapidly dividing cells. Molecular analysis of different populations of CSCs showed that quiescent tumor propagating cells are enriched in transforming growth factor-β1 (TGF-β1) target genes. These data support including TGF-β1 inhibitors in chemotherapeutic targeting strategies for tumor remission.
Takahiro Kunisada presented data on the role of Kit ligand (Kitl) in the establishment of melanocyte niches. His data indicate that epidermal expression of Kitl leads to the recruitment of melanocytes to the interfollicular epidermis. This ligand coordinates with endothelin 3 to provide a niche for melanocytes. This niche activity is altered with x-ray-induced hair graying, inducing the loss of Kitl expression. These data suggest that melanocyte homing and maintenance are controlled by keratinocytes through the secretion of specific ligands.
The session ended with Sarah Millar’s talk on the role of Wnt10a in epithelial tissue maintenance of progenitor cell proliferation. Recent work has identified mutations in Wnt10a in ectodermal defects in humans including thinning hair, smooth tongue and other ectodermal appendage disorders. Dr. Millar’s laboratory has developed mouse models that allow the study of Wnt10Aa’s function in a variety of epithelial tissues, uncovering defects in multiple epithelial tissues similar to human patients with Wnt10a mutations. Combined with lineage tracing of Wnt responsive cells, Dr. Millar’s data indicate that Wnt/β-catenin signaling marks self-renewing stem cells in multiple epithelial tissues and that Wnt10a is required for their proliferation.
In addition, short talks were selected from abstract submissions. For example, Andrew Gladden presented data on the role of the junctional polarity protein Merlin in hair follicle stem cell specification. Ryan Driskell discussed fibroblast heterogeneity in the skin. Pritinder Kaur introduced another class of mesenchymal cells, pericytes, which reside on blood vessels, and their function in regulating keratinocyte organization. Kif Liakath-Ali presented characterization of mice lacking alkaline ceramidase (Acer1), implicating ceramide in homeostatic mechanisms in the skin. The regulation of epidermal CSCs was posed by Naoki Oshimori, who found that TGF-β signaling from perivascular cells can modulate tumor cells, leading to heterogeneity in CSCs within epithelial tumors. The theme of the microenvironment was expanded by Michael Rendl, who presented a thorough transcriptional analysis of eight different cell types in the embryonic skin, already available online as a resource for the community at www.hair-gel.net
. The final short talk by Brett Shook discussed the contribution of adipocytes to skin wound healing.
Xiao-Jing Wang and Valerie Horsley moderated three panels for a session entitled “Beyond the Data: Tips for a Successful Career.” Panelists Carl Baker, Molly Kulesz-Martin, and Jakub Tolar led a discussion on approaches to funding, highlighting the importance of resilience and forging new areas to diversify your research program in the current funding climate. Mayumi Ito, Rui Yi, and Anthony Oro led a discussion on successful strategies for publishing your work. Finally, Mélanie Berta, Robert Binder, and Sarah Millar discussed strategies for successful industry and academic job searches. These lively sessions provided a fruitful discussion and connection for meeting participants and brought the meeting to an inspiring end.
Society for Investigative Dermatology Eugene M. Farber Travel Awards for Young Investigators
Moyassar Al-Shaibani, Newcastle University; Hitomi Aoki, PhD, Gifu University; Chih-Chiang Chen, MD, PhD, Taipen Veterans General Hospital; Kif Liakath-Ali, PhD, King’s College London; Naoki Oshimori, PhD, Rockefeller University; Guillermo Rivera Gonzalez, PhD, Yale University; Brett Shook, PhD, Yale University; Gerline van de Glind, MSc, Leiden University; Iris Verginnen, MS, University of Leuven.
Japanese Society for Investigative Dermatology Travel Award for Young Investigator
Azusa Miyashita, MD, PhD, Kumamoto University.
Lingjie Li, PhD, Stanford University
Montagna Symposium Director’s Award
Angel Morrow, PhD, Duke University
Conflict of Interest
The authors state no conflict of interest.
Montagna Symposium 2016
20–24 October 2016, Salishan Spa & Golf Resort, Gleneden Beach, Oregon
“The Skin: Our Sensory Organ for Itch, Pain, Touch, and Pleasure”
Gil Yosipovitch, MD, Temple University
Diana Bautista, PhD, UC Berkeley
Ethan Lerner, MD, PhD, Harvard University/Massachusets General Hospital
Ellen Lumpkin, PhD, Columbia University
Francis McGlone, PhD, University of Liverpool