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836 Applying FACS-based single cell RNA-seq to study neonatal mouse skin
ABSTRACTS | Skin and Hair Developmental Biology 836
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Applying FACS-based single cell RNA-seq to study neonatal mouse skin L Miao1, M Kelly2, S Barkdull1, H Sherrill3 and I Brownell1 1 Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 2 NIDCD, NIH, Bethesda, MD and 3 Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD Single cell RNA sequencing (scRNA-seq) has been recently developed to study the gene expression profile of individual cells. This high-resolution transcriptomic analysis is enabling deep insights into biological systems and disease mechanisms. Multiple techniques have been used to capture single cells, including microfluidics platforms, microtiter plates, and droplet-based technologies. These are followed by cell lysis and reverse-transcription (RT) of RNA from the individual cells. Barcoded and multiplexed cDNA are then subjected to high throughput sequencing. To apply this powerful approach for the study of skin biology, we have developed a protocol for the isolation of labeled single cells from neonatal mouse skin. Dispase is used to separate the epidermis from the dermis, with hair follicles remaining in the dermis. Enzymatic digestion then generates single cell suspension, and fluorescently-labeled single cells are sorted into microtiter plates using fluorescence-activated cell sorting (FACS). We have optimized this approach to maximize post-sort cell viability and cDNA purification for scRNA-seq library preparation. Taken together, this approach allows for the collection of high quality scRNA-seq data to analyze the biology of lineage specific cells from neonatal mouse skin.
Histologic and immunohistochemical evaluation of Lichen Planopilaris and correlation with clinical disease severity M Porter1, M Yasuda1, R Foreman1, M Hoang1, A Manatis-Lornell1 and M Senna2 1 Massachusetts General Hospital, Boston, MA and 2 Massachusetts General Hospital, Boston, ME Lichen planopilaris (LPP) is a primary cicatricial (scarring) alopecia of the scalp that is difficult to diagnose and treat and can result in permanent hair loss. The diagnosis of LPP is made through evaluation of the clinical presentation, histologic findings, and trichoscopy findings. This study seeks to correlate clinical severity of disease with histologic findings and to identify a potential immunohistochemical scoring system that may be useful in the grading of disease activity. A small pilot study with ten patients with previously diagnosed, biopsy-confirmed lichen planopilaris was performed. Clinical disease at time of biopsy was graded using a new clinical severity scoring system being developed at Massachusetts General Hospital. Biopsy specimens were evaluated with immunohistochemistry markers, including CD8, CD123, and CK15. Further, evaluation of sebaceous glands, presence of mast cells, and location of inflammation was also performed. An immunohistochemical scoring system was developed incorporating these markers to grade histologic severity and activity. Our study found a correlation between the two respective scoring systems used to grade clinical severity and histologic severity. Positive staining for CD8, CD123, and CK15 was observed in specimens. A lack of validated assessment tools and markers of disease activity limit the evaluation of new potential treatments for LPP in clinical trials. In future studies, we plan to validate a new clinical assessment tool for LPP, incorporating this immunohistochemical disease severity scoring system, with the hopes that it can be utilized to assess LPP severity and activity and response to treatment. This would enable better evaluation of treatment options for LPP in the future.
Early and late matrix progenitors form the terminally differentiated cell layers of the hair follicle A Mesler, N Veniaminova, M Lull and S Wong University of Michigan, Ann Arbor, MI During morphogenesis and regeneration, matrix progenitors undergo terminal differentiation to form the concentric layers of the hair follicle. These differentiation events are thought to depend on signals from the mesenchymal dermal papilla (DP); however, it remains unclear how DP-progenitor cell interactions govern specific cell fate decisions. Here, we provide evidence that the matrix progenitor pool can be divided into early and late phases based on distinct temporal, molecular and functional characteristics. Surprisingly, we find that early matrix progenitors initiate terminal differentiation prior to enveloping the DP. These early differentiation events specify Keratin 79+ cells, which form the companion layer, but do not require Shh, BMP or mature DP-derived signals. Using a transient lineage tracing system where only matrix progenitors and their most immediate descendants are labeled, we show that specification of the hair follicle terminally differentiated layers both commences and terminates asynchronously, with early matrix progenitors forming the companion layer, and later matrix populations progressively generating the inner root sheath and hair shaft. By tracing the companion layer into next telogen, we also show that these cells are mostly lost during catagen; however, a subset of cells are retained in the upper region of the inner bulge. Finally, we demonstrate that Keratin 79 null mice exhibit normal hair growth. Together, these findings provide several novel insights into the complex process of terminal differentiation within the hair follicle.
S144 Journal of Investigative Dermatology (2017), Volume 137
From genotoxic stresses to skin damage: Distinct, novel mechanisms of skin and hair damage in chemo/radiation therapy Z Yue Fuzhou University, Fuzhou, Fujian, China Genotoxic stresses induced by ionizing radiation (IR) and chemotherapeutic agents often damage the skin and hair follicle. Although the details of the DNA damage responses (DDR) have been extensively studied, it remains unclear how the actual damage occur at the tissue/ organ level. My laboratory has focused on this aspect of skin/hair biology, and made several interesting discoveries: (1) In the mammalian hair and avian feather follicles, we found that down-regulation of sonic hedgehog (Shh) gene expression is a critical early event in chemotherapy-induced tissue damage. A notch in the feather morphology was induced, which recorded the reversible impact of chemotherapy on its development. Consistently, cell proliferation in the feather rachis, which is independent of Shh signaling, remains undisrupted by chemotherapy (Xie et al., JID 2015). (2) IR-induced skin damage is a common side effect in cancer radiotherapy. We found that there are significant keratin gene expression changes in skin, Keratin 17 in particular (Liao et al., JID 2016). Furthermore, IR-induced ROS lead to the disruption of cytoskeleton, and degradation of E-cadherin/b-Catenin. These then accumulate to the disruption of adherens junctions (AJs) in the epidermis. Subsequently, Wnt and Hippo signaling are activated, followed by compensatory cell proliferation and desquamation (Xie et al., submitted). (3) The hair follicle has been utilized as a model to investigate how chemoand radiation therapy work together. A synergism between colchicine and IR was reported about fifty years ago (Malkinson et al., JID 1961). We recently tested some common chemo drugs, and indeed found that 5-flurouracil and taxol work best in combination with IR to damage the hair. These agents are widely used in chemo-radiation therapy of various cancer types. Our work thus offers an exciting opportunity to explore the molecular mechanism of this synergism, and promotes the test of novel chemicals that will enhance the efficacy of cancer therapy.
Spatiotemporal antagonism in mesenchymal-epithelial Signaling in sweat versus hair fate decision CP Lu1, L Polak1, B Keyes2 and E Fuchs1 1 Rockefeller University, New York, NY and 2 Calico Life Sciences, South San Francisco, CA The gain of eccrine sweat glands in hairy body skin has uniquely empowered humans to run marathons and tolerate temperature extremes. Epithelial-mesenchymal crosstalk is integral to the diverse patterning of skin appendages, but the molecular events underlying their specification remain largely unknown. Employing genome-wide analyses and functional studies, we show that sweat glands are specified by mesenchymal-derived BMPs and FGFs that signal to epithelial buds and suppress epithelial-derived SHH-production. Conversely, hair follicles are specified when mesenchymal BMP signaling is blocked, permitting SHH-production. Fate determination is confined to a critical developmental window, and is regionally specified in mice. In contrast, a shift from hair to gland fates is achieved in humans when a spike in BMP silences SHH during the final embryonic wave(s) of bud morphogenesis.
Minoxidil dose response study in female pattern hair loss patients determined to be non-responders to 5% topical Minoxidil A Goren1, M Kovacevic2, J McCoy1 and J Shapiro3 1 Applied Biology, Inc., Irvine, CA, 2 University of Rome, Rome, Italy and 3 New York University Langone Medical Center, New York, NY Topical minoxidil is the only US FDA approved drug for the treatment of female pattern hair loss (FPHL). 5% minoxidil foam is only effective at re-growing hair in a minority of women (approximately 40%). Thus, the majority of FPHL patients remain untreated. Previously, we have demonstrated that non-responders to 5% minoxidil have low metabolism of minoxidil in hair follicles. As such, we hypothesized that increasing the dosage of topical minoxidil to low metabolizers would increase the number of responders without increasing the incidence of adverse events. In this study, we recruited FPHL subjects that were identified as non-responders to 5% topical minoxidil utilizing the previously validated assay for minoxidil response. Subjects were treated for 12 weeks with a novel 15% topical minoxidil solution. At 12 weeks, 60% of subjects achieved a clinically significant response based on target area hair counts (>13.7% from baseline), as well as, significant improvement in global photographic assessment. None of the subjects experienced significant hemodynamics changes or any other adverse events. To the best of our knowledge, this is the first study to demonstrate the potentially beneficial effect of a higher dosage of minoxidil in FPHL subjects that fail to respond to 5% minoxidil.
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Applying FACS-based single cell RNA-seq to study neonatal mouse skin L Miao1, M Kelly2, S Barkdull1, H Sherrill3 and I Brownell1 1 Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 2 NIDCD, NIH, Bethesda, MD and 3 Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD Single cell RNA sequencing (scRNA-seq) has been recently developed to study the gene expression profile of individual cells. This high-resolution transcriptomic analysis is enabling deep insights into biological systems and disease mechanisms. Multiple techniques have been used to capture single cells, including microfluidics platforms, microtiter plates, and droplet-based technologies. These are followed by cell lysis and reverse-transcription (RT) of RNA from the individual cells. Barcoded and multiplexed cDNA are then subjected to high throughput sequencing. To apply this powerful approach for the study of skin biology, we have developed a protocol for the isolation of labeled single cells from neonatal mouse skin. Dispase is used to separate the epidermis from the dermis, with hair follicles remaining in the dermis. Enzymatic digestion then generates single cell suspension, and fluorescently-labeled single cells are sorted into microtiter plates using fluorescence-activated cell sorting (FACS). We have optimized this approach to maximize post-sort cell viability and cDNA purification for scRNA-seq library preparation. Taken together, this approach allows for the collection of high quality scRNA-seq data to analyze the biology of lineage specific cells from neonatal mouse skin.
Histologic and immunohistochemical evaluation of Lichen Planopilaris and correlation with clinical disease severity M Porter1, M Yasuda1, R Foreman1, M Hoang1, A Manatis-Lornell1 and M Senna2 1 Massachusetts General Hospital, Boston, MA and 2 Massachusetts General Hospital, Boston, ME Lichen planopilaris (LPP) is a primary cicatricial (scarring) alopecia of the scalp that is difficult to diagnose and treat and can result in permanent hair loss. The diagnosis of LPP is made through evaluation of the clinical presentation, histologic findings, and trichoscopy findings. This study seeks to correlate clinical severity of disease with histologic findings and to identify a potential immunohistochemical scoring system that may be useful in the grading of disease activity. A small pilot study with ten patients with previously diagnosed, biopsy-confirmed lichen planopilaris was performed. Clinical disease at time of biopsy was graded using a new clinical severity scoring system being developed at Massachusetts General Hospital. Biopsy specimens were evaluated with immunohistochemistry markers, including CD8, CD123, and CK15. Further, evaluation of sebaceous glands, presence of mast cells, and location of inflammation was also performed. An immunohistochemical scoring system was developed incorporating these markers to grade histologic severity and activity. Our study found a correlation between the two respective scoring systems used to grade clinical severity and histologic severity. Positive staining for CD8, CD123, and CK15 was observed in specimens. A lack of validated assessment tools and markers of disease activity limit the evaluation of new potential treatments for LPP in clinical trials. In future studies, we plan to validate a new clinical assessment tool for LPP, incorporating this immunohistochemical disease severity scoring system, with the hopes that it can be utilized to assess LPP severity and activity and response to treatment. This would enable better evaluation of treatment options for LPP in the future.
Early and late matrix progenitors form the terminally differentiated cell layers of the hair follicle A Mesler, N Veniaminova, M Lull and S Wong University of Michigan, Ann Arbor, MI During morphogenesis and regeneration, matrix progenitors undergo terminal differentiation to form the concentric layers of the hair follicle. These differentiation events are thought to depend on signals from the mesenchymal dermal papilla (DP); however, it remains unclear how DP-progenitor cell interactions govern specific cell fate decisions. Here, we provide evidence that the matrix progenitor pool can be divided into early and late phases based on distinct temporal, molecular and functional characteristics. Surprisingly, we find that early matrix progenitors initiate terminal differentiation prior to enveloping the DP. These early differentiation events specify Keratin 79+ cells, which form the companion layer, but do not require Shh, BMP or mature DP-derived signals. Using a transient lineage tracing system where only matrix progenitors and their most immediate descendants are labeled, we show that specification of the hair follicle terminally differentiated layers both commences and terminates asynchronously, with early matrix progenitors forming the companion layer, and later matrix populations progressively generating the inner root sheath and hair shaft. By tracing the companion layer into next telogen, we also show that these cells are mostly lost during catagen; however, a subset of cells are retained in the upper region of the inner bulge. Finally, we demonstrate that Keratin 79 null mice exhibit normal hair growth. Together, these findings provide several novel insights into the complex process of terminal differentiation within the hair follicle.
S144 Journal of Investigative Dermatology (2017), Volume 137
From genotoxic stresses to skin damage: Distinct, novel mechanisms of skin and hair damage in chemo/radiation therapy Z Yue Fuzhou University, Fuzhou, Fujian, China Genotoxic stresses induced by ionizing radiation (IR) and chemotherapeutic agents often damage the skin and hair follicle. Although the details of the DNA damage responses (DDR) have been extensively studied, it remains unclear how the actual damage occur at the tissue/ organ level. My laboratory has focused on this aspect of skin/hair biology, and made several interesting discoveries: (1) In the mammalian hair and avian feather follicles, we found that down-regulation of sonic hedgehog (Shh) gene expression is a critical early event in chemotherapy-induced tissue damage. A notch in the feather morphology was induced, which recorded the reversible impact of chemotherapy on its development. Consistently, cell proliferation in the feather rachis, which is independent of Shh signaling, remains undisrupted by chemotherapy (Xie et al., JID 2015). (2) IR-induced skin damage is a common side effect in cancer radiotherapy. We found that there are significant keratin gene expression changes in skin, Keratin 17 in particular (Liao et al., JID 2016). Furthermore, IR-induced ROS lead to the disruption of cytoskeleton, and degradation of E-cadherin/b-Catenin. These then accumulate to the disruption of adherens junctions (AJs) in the epidermis. Subsequently, Wnt and Hippo signaling are activated, followed by compensatory cell proliferation and desquamation (Xie et al., submitted). (3) The hair follicle has been utilized as a model to investigate how chemoand radiation therapy work together. A synergism between colchicine and IR was reported about fifty years ago (Malkinson et al., JID 1961). We recently tested some common chemo drugs, and indeed found that 5-flurouracil and taxol work best in combination with IR to damage the hair. These agents are widely used in chemo-radiation therapy of various cancer types. Our work thus offers an exciting opportunity to explore the molecular mechanism of this synergism, and promotes the test of novel chemicals that will enhance the efficacy of cancer therapy.
Spatiotemporal antagonism in mesenchymal-epithelial Signaling in sweat versus hair fate decision CP Lu1, L Polak1, B Keyes2 and E Fuchs1 1 Rockefeller University, New York, NY and 2 Calico Life Sciences, South San Francisco, CA The gain of eccrine sweat glands in hairy body skin has uniquely empowered humans to run marathons and tolerate temperature extremes. Epithelial-mesenchymal crosstalk is integral to the diverse patterning of skin appendages, but the molecular events underlying their specification remain largely unknown. Employing genome-wide analyses and functional studies, we show that sweat glands are specified by mesenchymal-derived BMPs and FGFs that signal to epithelial buds and suppress epithelial-derived SHH-production. Conversely, hair follicles are specified when mesenchymal BMP signaling is blocked, permitting SHH-production. Fate determination is confined to a critical developmental window, and is regionally specified in mice. In contrast, a shift from hair to gland fates is achieved in humans when a spike in BMP silences SHH during the final embryonic wave(s) of bud morphogenesis.
Minoxidil dose response study in female pattern hair loss patients determined to be non-responders to 5% topical Minoxidil A Goren1, M Kovacevic2, J McCoy1 and J Shapiro3 1 Applied Biology, Inc., Irvine, CA, 2 University of Rome, Rome, Italy and 3 New York University Langone Medical Center, New York, NY Topical minoxidil is the only US FDA approved drug for the treatment of female pattern hair loss (FPHL). 5% minoxidil foam is only effective at re-growing hair in a minority of women (approximately 40%). Thus, the majority of FPHL patients remain untreated. Previously, we have demonstrated that non-responders to 5% minoxidil have low metabolism of minoxidil in hair follicles. As such, we hypothesized that increasing the dosage of topical minoxidil to low metabolizers would increase the number of responders without increasing the incidence of adverse events. In this study, we recruited FPHL subjects that were identified as non-responders to 5% topical minoxidil utilizing the previously validated assay for minoxidil response. Subjects were treated for 12 weeks with a novel 15% topical minoxidil solution. At 12 weeks, 60% of subjects achieved a clinically significant response based on target area hair counts (>13.7% from baseline), as well as, significant improvement in global photographic assessment. None of the subjects experienced significant hemodynamics changes or any other adverse events. To the best of our knowledge, this is the first study to demonstrate the potentially beneficial effect of a higher dosage of minoxidil in FPHL subjects that fail to respond to 5% minoxidil.