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904 Adipose multilineage-differentiating stress enduring (Muse) cell maintain pluripotency regardless of donors’ age
ABSTRACTS | Tissue Regeneration and Wound Healing 903
904
iPSC reprogramming rescues epigenetic signature and cellular functions of diabetic foot ulcer fibroblasts to promote healing L Liang1, R Stone1, I Pastar1, I Jozic2, O Stojadinovic1, H Ramirez1, O Kashpur3, B GeramiNaini3, A Maione3, A Smith3, V Yanez3, RS Kirsner1, A Veves4, J Garlick3 and M Tomic-Canic1 1 University of Miami, Miami, FL, 2 University of Miami, Mimi, FL, 3 Tufts University, Boston, MA and 4 Beth Israel Deaconess Medical Center, Boston, MA Diabetic foot ulcers (DFUs) are a debilitating type of non-healing wounds. Fibroblasts are an important cell type involved in wound healing, however their role in DFU pathophysiology is not fully understood. We isolated primary fibroblasts from non-healing DFUs (DFUFs) and evaluated their properties compared to non-diabetic foot fibroblasts on genomic and epigenetic levels. Altered transcriptional/epigenetic profiles in DFUFs revealed impaired cellular migration and proliferation, stimulation of senescence and dysregulated differentiation. Next, we utilized induced pluripotent stem cell (iPSC) reprograming to shift DFUFs to reacquire wound healing functions and healthy miRNA profiles. In response to reprogramming, DFUFs displayed accelerated cell migration and proliferation, along with a reversal of epigenetic, miRNA profiles, including miR-374a-5p expression from induction in DFUFs pre-reprogramming to a suppression in post-reprogrammed fibroblasts. Suppression of miR-374a-5p triggered up-regulation of targets (RPL34, ADAM9) that may contribute to recovered cellular functions. Moreover, reprograming modified DFUF miRNA profile to induce miR-197-3p, which, coupled with a suppression of target gene CAV1, also lead to rescued cell migration upon DFUF reprogramming and subsequent differentiation. Our findings demonstrate effectiveness of an iPSC approach for therapeutic reprogramming, whereby the epigenetic, miRNA profile of healing-incapable fibroblasts may be modified to generate pro-healing cellular phenotype.
Adipose multilineage-differentiating stress enduring (Muse) cell maintain pluripotency regardless of donors’ age T Yamauchi1, K Yamasaki2, K Tsutiyama1, S Koike1 and S Aiba3 1 Tohoku University Graduate School of Medicine, Sendai,, Japan, 2 Department of Dermatology, Tohoku University, Sendai, Japan and 3 Department of Dermatology Tohoku University Graduate School of Medicine, Sendai, Japan Multilineage-differentiating stress enduring (Muse) cell, which can be isolated with a pluripotent marker SSEA3 by FACS sorting, is pluripotent stem cell existing mesenchymal tissues such as human dermis and adipose tissues. We previously reported that Muse cells isolated from human fibroblast and adipose-stem cells (hASC) can differentiate into melanocytes (SID 2014 and 2015). In this study, we asked if the pluripotency of Muse cells and the potency of Muse cell differentiation into somatic cells are affected by donors’ ages. We collected 11 adipose tissue samples from surgical specimens obtained from individuals of various ages, and counted Muse cells included in these adipose tissues. Adipose tissues obtained from individuals under age 40 (n ¼ 5) had more Muse cells than that from over 40 (n ¼6) (3.2% SSEA3 positive cells in hASC from under 40 and 1.3% from over 40, p < 0.01). Both groups of Muse cells are positive for alkaline phosphatase staining and could differentiate into the representative triploblastic cells (neuron, muscle and hepatocyte). We induced these Muse cells differentiation into melanocytes by culturing in melanocyte inducing medium (MIM) contained 10 factors for 6 week as previously reported (JID 2014). Muse cells from both age groups produced similar numbers of Muse cell derived melanocytes (Muse-MC). Both groups of Muse-MC expressed similar levels of melanocyte related genes such as TYR and DCT as well as proteins of TYR, TYRP-1 and PMEL at 6-week culture. Both groups of Muse-MC localized in basal layer in reconstituted skin model and expressed melanocyte related proteins tyrosinase and S100. These data show that aging may reduce the number of Muse cells, but Muse cells maintain pluripotency regardless of the donors’ age.
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906
Suppression of DNA repair and lack of cellular migration are major contributors of diabetic foot ulcer pathophysiology H Ramirez1, I Pastar1, R Stone1, T Cao Wikramanayake2, I Jozic3, O Stojadinovic1, N Ojeh4, RS Kirsner1 and M Tomic-Canic1 1 University of Miami, Miami, FL, 2 University of Miami Miller School of Medicine, Miami, FL, 3 University of Miami, Mimi, FL and 4 University Of The West Indies, Bridgetown, Saint Michael, Barbados Diabetic foot ulcers (DFU) are debilitating cutaneous condition with high morbidity and associated mortality. Despite the health burden molecular pathophysiology of DFU is poorly understood. We aimed to identify cellular functions that contribute to non-healing by comparative genomics, miRNA, and protein analyses of DFU and healing acute wounds (AW). Decreased DNA repair mechanisms and suppression of cellular migration were uniquely enriched in DFU, whereas immune cell activation, differentiation and chemotaxis were strikingly absent, but highly induced in AW. We validated mRNA/protein expression of deregulated genes in the DFU-specific network including WEE1, MSH2S, IKBKB, FGF2, RAD50, PIK3R1, and TP53. The inhibition of DNA repair and accumulation of DNA damage was confirmed in patients’ samples by the presence of p-H2AX. We identified miR-15b-5p and miR-193 as potential major regulators responsible for regulation of DFU-specific genes. Indeed, both miR-15b-5p and -193 were found induced in DFU, but not in AW. Inhibitory effects between miRs and their targets were confirmed using 3’-UTR luciferase reporter assays. Functional conformation of miR15b-5p and -193 mediated inhibition of healing was performed by miR overexpression in vitro and murine full thickness wounds in vivo. Overexpression of miR-15b-5p resulted in suppression of DNA repair mechanisms and dampened inflammatory response mainly through suppression of WEE1 and IKBKB. Induction of miR193b-3p dominantly inhibited keratinocyte migration and wound closure in vivo. In summary we identified miR-15b-5p and -193 as major regulators responsible for healing impairment in DFU through suppression of cell migration, DNA repair, and acute immune response.
Systemic HMGB1 administration ameliorates cutaneous and non-cutaneous manifestations in a dystrophic epidermolysis bullosa model mouse T Shimbo1, S Yamazaki2, X Wang3, Y Kikuchi4, L Bruckner-Tuderman5, Y Kaneda4 and K Tamai4 1 Osaka University, Suita, Japan, 2 Genomix Co., Ltd., Osaka, Japan, 3 Genomix Co., Ltd., Suita, Japan, 4 Osaka University, Suita, Japan and 5 Freiburg Institute for Advanced Studies School of Life Sciences, Freiburg im Breisgau, Germany High mobility group box 1 (HMGB1) mainly localizes in the nucleus and regulates transcription by modulating chromatin structure. In a necrotic condition, HMGB1 is released into the extracellular milieu and plays pleiotropic roles in tissue regeneration and immunological processes. Previously, we have reported that the released HMGB1 mobilizes platelet-derived growth factor receptor alpha (PDGFRa) positive bone marrow cells into the damaged tissues using SDF-1a/CXCR4 axis. We have also shown that the HMGB1-induced PDGFRa positive cells can differentiate into both fibroblasts and keratinocytes, and provide type VII collagen (Col7) at the cutaneous basement membrane zone. Based on these results, we hypothesized that HMGB1 can serve as a potential drug for moderate form of dystrophic epidermolysis bullosa (DEB), which possess amino acid substitution mutations at least in one allele of Col7a1 gene to produce less-functional anchoring fibrils and induce dermal-epidermal separation. To test this, we systemically administered HMGB1 to Col7-hypomorphic mice. The treatment with HMGB1 significantly extended the survival with restoring the expression of Col7 at the basement membrane of the skin and the mucosa, while most of the untreated mice died within 20 weeks due to severe malnutrition. Correlated with the recovery of Col7 expression, the DEB symptoms were ameliorated. Collectively, these data position HMGB1 as a promising drug at least for moderate forms of DEB. In addition, our study provides a proofof-concept for a drug which mobilizes multipotent cells from bone marrow, as a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.
907
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Predictors of wound healing in lower extremity wounds status post Mohs micrographic surgery J Honaker1, K Cooper2, E Madigan3 and JS Bordeaux4 1 Bolton School of Nursing Case Western Reserve University, Twinsburg, OH, 2 Department of Dermatology School of Medicine Case Western Reserve University, Cleveland, OH, 3 Bolton School of Nursing Case Western Reserve University, Cleveland, OH and 4 University Hospitals Cleveland Medical Center and Case Western Reserve University School of Medicine, Cleveland, OH The purpose of the study is to determine relationships between delayed wound healing (wound total surface area), symptoms (depressive symptoms [DS], pain, sleep disturbance [SD]), and health related quality of life (HRQoL) in older adults undergoing Mohs micrographic surgery [MMS] of lower extremity non melanoma skin cancer (NMSC) healing by secondary intention. This is a prospective exploratory study of patients undergoing MMS for NMSC in a single academic dermatological surgery clinic. Subjects were enrolled on the day of surgery and followed weekly for 4 weeks, except 4 subjects were followed weekly until healed. Descriptive statistics and T-tests were used to explore differences between those with and without wound expansion at week 1 among these variables: demographics, co-morbidities, laboratory values, and NIH PROMIS DS, pain, SD, and HRQoL surveys. Associations were performed between the variables at enrollment and week 4 to determine relationships. A preliminary analysis of 12 subjects revealed 4 with wounds greater than -15% expansion and 8 without expansion at week 1. Of the variables evaluated, age was trending toward significance with the wound expansion group averaging 10 years older (p ¼ 0.11). Of the 4 subjects followed until healed, 2 subject’s wounds expanded and required 10-12 weeks to heal compared to 6-8 weeks in 2 subjects without wound expansion. When evaluating associations between the variables, people with higher DS scores had significantly higher pain (p¼ < 0.05) and SD (p¼ < 0.05). However, no variables were associated with wound total surface area. In conclusion, this preliminary report reveals that older adults may be more likely to have wound expansion, which may result in delayed wound healing.
S156 Journal of Investigative Dermatology (2017), Volume 137
Keratinocytes and fibroblasts facilitate maturation of sensory neurons differentiated from human induced pluripotent stem cells Z Guo1, C Tong1, Y Doucet1, H Abaci1, J Jackow1, E Lumpkin2 and AM Christiano1 1 Columbia University, New York, NY and 2 Columbia University Dept. of Physiology & Cellular Biophysics& of Dermatology, New York, NY Human skin is a complex structure containing many different cell types, including sensory neurons which transduce sensations such as itch, pain, heat, cold and touch. It is very difficult to obtain human sensory neurons for regenerative applications and pharmaceutical development. Induced pluripotent stem cells (iPSCs) have enormous potential for cell therapy and disease modeling by providing an unlimited source of different cell types. However, the efficient generation of desired cell lineages remains challenging. Several protocols have been developed to differentiate sensory neurons from human iPSCs, but there is variability between different iPSC lines to obtain mature sensory neurons from human iPSCs. Previously, we manipulated the Wnt, transforming growth factor b (TGF-b), bone morphogenetic protein 4 (BMP 4) and Notch signaling pathways to enhance neuronal differentiation. We differentiated one iPSC line into neurons (iNCs), and these iNCs displayed neural markers and action potentials. Calcium imaging analysis demonstrated that the iNCs responded specifically to itchrelated stimuli (proteinase-activated receptor-2 activating (PAR2) agonist peptide or histamine). Here, we modified the media formulation and differentiated iNCs from 3 different iPSC lines and one embryonic cell line (H9). Importantly, co-culture with keratinocytes rendered the iNCs capable of continuously firing, whereas fibroblasts instead promoted neurite outgrowth. We have established a robust protocol to differentiate iPSCs into sensory neurons, and discovered that keratinocytes and fibroblasts play distinct functional roles in neuronal maturation. The availability of these specialized iNCs will allow us to study their function and screen for small molecules that can be used for the treatment of itch.
904
iPSC reprogramming rescues epigenetic signature and cellular functions of diabetic foot ulcer fibroblasts to promote healing L Liang1, R Stone1, I Pastar1, I Jozic2, O Stojadinovic1, H Ramirez1, O Kashpur3, B GeramiNaini3, A Maione3, A Smith3, V Yanez3, RS Kirsner1, A Veves4, J Garlick3 and M Tomic-Canic1 1 University of Miami, Miami, FL, 2 University of Miami, Mimi, FL, 3 Tufts University, Boston, MA and 4 Beth Israel Deaconess Medical Center, Boston, MA Diabetic foot ulcers (DFUs) are a debilitating type of non-healing wounds. Fibroblasts are an important cell type involved in wound healing, however their role in DFU pathophysiology is not fully understood. We isolated primary fibroblasts from non-healing DFUs (DFUFs) and evaluated their properties compared to non-diabetic foot fibroblasts on genomic and epigenetic levels. Altered transcriptional/epigenetic profiles in DFUFs revealed impaired cellular migration and proliferation, stimulation of senescence and dysregulated differentiation. Next, we utilized induced pluripotent stem cell (iPSC) reprograming to shift DFUFs to reacquire wound healing functions and healthy miRNA profiles. In response to reprogramming, DFUFs displayed accelerated cell migration and proliferation, along with a reversal of epigenetic, miRNA profiles, including miR-374a-5p expression from induction in DFUFs pre-reprogramming to a suppression in post-reprogrammed fibroblasts. Suppression of miR-374a-5p triggered up-regulation of targets (RPL34, ADAM9) that may contribute to recovered cellular functions. Moreover, reprograming modified DFUF miRNA profile to induce miR-197-3p, which, coupled with a suppression of target gene CAV1, also lead to rescued cell migration upon DFUF reprogramming and subsequent differentiation. Our findings demonstrate effectiveness of an iPSC approach for therapeutic reprogramming, whereby the epigenetic, miRNA profile of healing-incapable fibroblasts may be modified to generate pro-healing cellular phenotype.
Adipose multilineage-differentiating stress enduring (Muse) cell maintain pluripotency regardless of donors’ age T Yamauchi1, K Yamasaki2, K Tsutiyama1, S Koike1 and S Aiba3 1 Tohoku University Graduate School of Medicine, Sendai,, Japan, 2 Department of Dermatology, Tohoku University, Sendai, Japan and 3 Department of Dermatology Tohoku University Graduate School of Medicine, Sendai, Japan Multilineage-differentiating stress enduring (Muse) cell, which can be isolated with a pluripotent marker SSEA3 by FACS sorting, is pluripotent stem cell existing mesenchymal tissues such as human dermis and adipose tissues. We previously reported that Muse cells isolated from human fibroblast and adipose-stem cells (hASC) can differentiate into melanocytes (SID 2014 and 2015). In this study, we asked if the pluripotency of Muse cells and the potency of Muse cell differentiation into somatic cells are affected by donors’ ages. We collected 11 adipose tissue samples from surgical specimens obtained from individuals of various ages, and counted Muse cells included in these adipose tissues. Adipose tissues obtained from individuals under age 40 (n ¼ 5) had more Muse cells than that from over 40 (n ¼6) (3.2% SSEA3 positive cells in hASC from under 40 and 1.3% from over 40, p < 0.01). Both groups of Muse cells are positive for alkaline phosphatase staining and could differentiate into the representative triploblastic cells (neuron, muscle and hepatocyte). We induced these Muse cells differentiation into melanocytes by culturing in melanocyte inducing medium (MIM) contained 10 factors for 6 week as previously reported (JID 2014). Muse cells from both age groups produced similar numbers of Muse cell derived melanocytes (Muse-MC). Both groups of Muse-MC expressed similar levels of melanocyte related genes such as TYR and DCT as well as proteins of TYR, TYRP-1 and PMEL at 6-week culture. Both groups of Muse-MC localized in basal layer in reconstituted skin model and expressed melanocyte related proteins tyrosinase and S100. These data show that aging may reduce the number of Muse cells, but Muse cells maintain pluripotency regardless of the donors’ age.
905
906
Suppression of DNA repair and lack of cellular migration are major contributors of diabetic foot ulcer pathophysiology H Ramirez1, I Pastar1, R Stone1, T Cao Wikramanayake2, I Jozic3, O Stojadinovic1, N Ojeh4, RS Kirsner1 and M Tomic-Canic1 1 University of Miami, Miami, FL, 2 University of Miami Miller School of Medicine, Miami, FL, 3 University of Miami, Mimi, FL and 4 University Of The West Indies, Bridgetown, Saint Michael, Barbados Diabetic foot ulcers (DFU) are debilitating cutaneous condition with high morbidity and associated mortality. Despite the health burden molecular pathophysiology of DFU is poorly understood. We aimed to identify cellular functions that contribute to non-healing by comparative genomics, miRNA, and protein analyses of DFU and healing acute wounds (AW). Decreased DNA repair mechanisms and suppression of cellular migration were uniquely enriched in DFU, whereas immune cell activation, differentiation and chemotaxis were strikingly absent, but highly induced in AW. We validated mRNA/protein expression of deregulated genes in the DFU-specific network including WEE1, MSH2S, IKBKB, FGF2, RAD50, PIK3R1, and TP53. The inhibition of DNA repair and accumulation of DNA damage was confirmed in patients’ samples by the presence of p-H2AX. We identified miR-15b-5p and miR-193 as potential major regulators responsible for regulation of DFU-specific genes. Indeed, both miR-15b-5p and -193 were found induced in DFU, but not in AW. Inhibitory effects between miRs and their targets were confirmed using 3’-UTR luciferase reporter assays. Functional conformation of miR15b-5p and -193 mediated inhibition of healing was performed by miR overexpression in vitro and murine full thickness wounds in vivo. Overexpression of miR-15b-5p resulted in suppression of DNA repair mechanisms and dampened inflammatory response mainly through suppression of WEE1 and IKBKB. Induction of miR193b-3p dominantly inhibited keratinocyte migration and wound closure in vivo. In summary we identified miR-15b-5p and -193 as major regulators responsible for healing impairment in DFU through suppression of cell migration, DNA repair, and acute immune response.
Systemic HMGB1 administration ameliorates cutaneous and non-cutaneous manifestations in a dystrophic epidermolysis bullosa model mouse T Shimbo1, S Yamazaki2, X Wang3, Y Kikuchi4, L Bruckner-Tuderman5, Y Kaneda4 and K Tamai4 1 Osaka University, Suita, Japan, 2 Genomix Co., Ltd., Osaka, Japan, 3 Genomix Co., Ltd., Suita, Japan, 4 Osaka University, Suita, Japan and 5 Freiburg Institute for Advanced Studies School of Life Sciences, Freiburg im Breisgau, Germany High mobility group box 1 (HMGB1) mainly localizes in the nucleus and regulates transcription by modulating chromatin structure. In a necrotic condition, HMGB1 is released into the extracellular milieu and plays pleiotropic roles in tissue regeneration and immunological processes. Previously, we have reported that the released HMGB1 mobilizes platelet-derived growth factor receptor alpha (PDGFRa) positive bone marrow cells into the damaged tissues using SDF-1a/CXCR4 axis. We have also shown that the HMGB1-induced PDGFRa positive cells can differentiate into both fibroblasts and keratinocytes, and provide type VII collagen (Col7) at the cutaneous basement membrane zone. Based on these results, we hypothesized that HMGB1 can serve as a potential drug for moderate form of dystrophic epidermolysis bullosa (DEB), which possess amino acid substitution mutations at least in one allele of Col7a1 gene to produce less-functional anchoring fibrils and induce dermal-epidermal separation. To test this, we systemically administered HMGB1 to Col7-hypomorphic mice. The treatment with HMGB1 significantly extended the survival with restoring the expression of Col7 at the basement membrane of the skin and the mucosa, while most of the untreated mice died within 20 weeks due to severe malnutrition. Correlated with the recovery of Col7 expression, the DEB symptoms were ameliorated. Collectively, these data position HMGB1 as a promising drug at least for moderate forms of DEB. In addition, our study provides a proofof-concept for a drug which mobilizes multipotent cells from bone marrow, as a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.
907
908
Predictors of wound healing in lower extremity wounds status post Mohs micrographic surgery J Honaker1, K Cooper2, E Madigan3 and JS Bordeaux4 1 Bolton School of Nursing Case Western Reserve University, Twinsburg, OH, 2 Department of Dermatology School of Medicine Case Western Reserve University, Cleveland, OH, 3 Bolton School of Nursing Case Western Reserve University, Cleveland, OH and 4 University Hospitals Cleveland Medical Center and Case Western Reserve University School of Medicine, Cleveland, OH The purpose of the study is to determine relationships between delayed wound healing (wound total surface area), symptoms (depressive symptoms [DS], pain, sleep disturbance [SD]), and health related quality of life (HRQoL) in older adults undergoing Mohs micrographic surgery [MMS] of lower extremity non melanoma skin cancer (NMSC) healing by secondary intention. This is a prospective exploratory study of patients undergoing MMS for NMSC in a single academic dermatological surgery clinic. Subjects were enrolled on the day of surgery and followed weekly for 4 weeks, except 4 subjects were followed weekly until healed. Descriptive statistics and T-tests were used to explore differences between those with and without wound expansion at week 1 among these variables: demographics, co-morbidities, laboratory values, and NIH PROMIS DS, pain, SD, and HRQoL surveys. Associations were performed between the variables at enrollment and week 4 to determine relationships. A preliminary analysis of 12 subjects revealed 4 with wounds greater than -15% expansion and 8 without expansion at week 1. Of the variables evaluated, age was trending toward significance with the wound expansion group averaging 10 years older (p ¼ 0.11). Of the 4 subjects followed until healed, 2 subject’s wounds expanded and required 10-12 weeks to heal compared to 6-8 weeks in 2 subjects without wound expansion. When evaluating associations between the variables, people with higher DS scores had significantly higher pain (p¼ < 0.05) and SD (p¼ < 0.05). However, no variables were associated with wound total surface area. In conclusion, this preliminary report reveals that older adults may be more likely to have wound expansion, which may result in delayed wound healing.
S156 Journal of Investigative Dermatology (2017), Volume 137
Keratinocytes and fibroblasts facilitate maturation of sensory neurons differentiated from human induced pluripotent stem cells Z Guo1, C Tong1, Y Doucet1, H Abaci1, J Jackow1, E Lumpkin2 and AM Christiano1 1 Columbia University, New York, NY and 2 Columbia University Dept. of Physiology & Cellular Biophysics& of Dermatology, New York, NY Human skin is a complex structure containing many different cell types, including sensory neurons which transduce sensations such as itch, pain, heat, cold and touch. It is very difficult to obtain human sensory neurons for regenerative applications and pharmaceutical development. Induced pluripotent stem cells (iPSCs) have enormous potential for cell therapy and disease modeling by providing an unlimited source of different cell types. However, the efficient generation of desired cell lineages remains challenging. Several protocols have been developed to differentiate sensory neurons from human iPSCs, but there is variability between different iPSC lines to obtain mature sensory neurons from human iPSCs. Previously, we manipulated the Wnt, transforming growth factor b (TGF-b), bone morphogenetic protein 4 (BMP 4) and Notch signaling pathways to enhance neuronal differentiation. We differentiated one iPSC line into neurons (iNCs), and these iNCs displayed neural markers and action potentials. Calcium imaging analysis demonstrated that the iNCs responded specifically to itchrelated stimuli (proteinase-activated receptor-2 activating (PAR2) agonist peptide or histamine). Here, we modified the media formulation and differentiated iNCs from 3 different iPSC lines and one embryonic cell line (H9). Importantly, co-culture with keratinocytes rendered the iNCs capable of continuously firing, whereas fibroblasts instead promoted neurite outgrowth. We have established a robust protocol to differentiate iPSCs into sensory neurons, and discovered that keratinocytes and fibroblasts play distinct functional roles in neuronal maturation. The availability of these specialized iNCs will allow us to study their function and screen for small molecules that can be used for the treatment of itch.