2019 Jan 1;9(1):65-76. doi: 10.7150/thno.29766. The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. An aliquot of the culture is infected with the, Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a. doi: 10.1093/burnst/tkaa028. B-actin was run as a loading control. Engineering Bioactive Self-Healing Antibacterial Exosomes Hydrogel for Promoting Chronic Diabetic Wound Healing and Complete Skin Regeneration. Extensive growth potential of recessive…, Figure 2.  |  The epidermal cells perform a barrier function in human body, protecting against invasion of bacteria and foreign particles and regulating … NIH No competing financial interests exist. See this image and copyright information in PMC. 2017;23(24):3455-3482. doi: 10.2174/1381612823666170526094606. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. Determination of proviral integration sites in corrected stem cell. Phase 1/2a clinical trial of gene-corrected autologous cell therapy for recessive dystrophic epidermolysis bullosa. Gene Ther. Canine epidermal neural crest stem cells: characterization and potential as therapy candidate for a … Epidermal stem cells are excellent targets for gene therapy seeking to correct genetic deficiencies permanently. Xu P, Wu Y, Zhou L, Yang Z, Zhang X, Hu X, Yang J, Wang M, Wang B, Luo G, He W, Cheng B. Burns Trauma. Recent Advances: An immense amount of research has uncovered the underlying mechanisms behind the complex and highly regulated wound healing process. Significance: Skin serves as a protective barrier for mammals. This site needs JavaScript to work properly. 2019 May 8;27(5):986-998. doi: 10.1016/j.ymthe.2019.03.007. Strategy to perform ex vivo gene therapy from a single epidermal stem cell, Figure 2. Epidermal stratification in mouse model. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). A review: therapeutic potential of adipose-derived stem cells in cutaneous wound healing and regeneration. -. Five specific signals (red) were detected on five different chromosomes. © 2015 The Authors. N Engl J Med. The keratinocytes at the leading edge migrate along the upper layer of blood clot, while the basal keratinocytes behind the front line start to proliferate and differentiate, restoring the barrier function of the epithelium. This abnormal growth of … A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. Titeux M, Pendaries V, Zanta-Boussif MA, Décha A, Pironon N, Tonasso L, Mejia JE, Brice A, Danos O, Hovnanian A. Mol Ther. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. Gene therapy: pursuing restoration of dermal adhesion in recessive dystrophic epidermolysis bullosa. 2020 Jun 11;11(1):232. doi: 10.1186/s13287-020-01755-y. Preliminary studies of hair follicle regeneration by injections of epidermal stem cells and dermal papilla cells into nude mice. Please enable it to take advantage of the complete set of features! EMBO Mol Med. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. -, Barrandon Y, Green H. Cell-size as a determinant of the clone-forming ability of human keratinocytes. Please enable it to take advantage of the complete set of features! These four stages are spatial–temporal overlapping and can continue up to 1 year after wounding. 1. Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) inhibitors are medicines that bind to certain parts of the EGFR and slow down or stop cell growth. 2015 Apr;7(4):366-7. doi: 10.15252/emmm.201404859. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy. Treatment will be performed 2 times a week. Figure 6. Huang S, Hu Z, Wang P, Zhang Y, Cao X, Dong Y, Cheng P, Xu H, Zhu W, Tang B, Zhu J. Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). Keywords: Significance: Skin serves as a protective barrier for mammals. Stem Cell Res Ther. Critical Issues: We will comprehensively discuss the current clinical practices used on patients with cutaneous wounds, including both traditional skin grafting procedures and advanced grafting techniques with cultured skin stem cells. There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. 2011;18:674–681. HHS Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. 2013;495:418–419. Stem cell therapy is an exciting and active field of biomedical research. Exp Dermatol. A feat in stem-cell therapy highlights what can be achieved when basic and clinical research combine to advance biological understanding and treatment. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. EGFR is a protein that is found on the surface of some cells that causes cells to divide when epidermal … -, Aiuti A, Cattaneo F, Galimberti S, Benninghoff U, Cassani B, Callegaro L, Scaramuzza S, Andolfi G, Mirolo M, Brigida I, et al. Rat epidermal stem cells promote the angiogenesis of full-thickness wounds. eCollection 2020. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Osborn MJ, Newby GA, McElroy AN, Knipping F, Nielsen SC, Riddle MJ, Xia L, Chen W, Eide CR, Webber BR, Wandall HH, Dabelsteen S, Blazar BR, Liu DR, Tolar J. J Invest Dermatol. No ghostwriters were used to write this article. Epub 2014 Jan 17. 2010 Aug;18(8):1509-18. doi: 10.1038/mt.2010.91. When cutaneous injuries occur, skin homeostasis and integrity are damaged, leading to dire consequences such as acute, chronic, or infected wounds. Epidermal stem cells in wound healing and their clinical applications. Identification of stem cells producing COLVII. In this review, we will dissect the biological process of adult skin wound healing and emphasize the importance of epidermal stem cells during the wound healing. Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa. GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling. Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Clipboard, Search History, and several other advanced features are temporarily unavailable. Small insertion–deletions and SNP calling were performed and compared to GWAS databases. Curr Pharm Des. Epidermal stem cells are responsible for maintaining skin homeostasis. Indications for Use: The RECELL ® Autologous Cell Harvesting Device is indicated for the treatment of acute thermal burn wounds in patients 18 years of age and older. -, Almarza D, Bussadori G, Navarro M, Mavilio F, Larcher F, Murillas R. Risk assessment in skin gene therapy: viral-cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. The grafts grew, thanks to included epidermal stem cells. Scale bar: 50 μm. See this image and copyright information in PMC. JAMA. Epub 2019 Mar 15. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. NLM Characterisation of proviral integrations in…, Figure 5. It is the next chapter in organ … Gene therapy for immunodeficiency due to adenosine deaminase deficiency. The indels were not associated with disease and the SNP was synonymous (CCU–CCG both codons correspond to proline). Proc Natl Acad Sci USA. Wang C, Wang M, Xu T, Zhang X, Lin C, Gao W, Xu H, Lei B, Mao C. Theranostics. Keywords: COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Basal keratinocytes at the wound edges are activated and initiate changes in cell morphology, as well as migration ability. Epub 2011 Jun 8. Adv Wound Care (New Rochelle). 2014 Mar;3(3):334-45. doi: 10.5966/sctm.2013-0129. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro. This site needs JavaScript to work properly. "It's very similar to what we've been using for full-thickness burns: Take a biopsy and isolate cells. Human epidermal stem cells … Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Psoriasis is a chronic, relapsing inflammatory disease affecting 2–3% of the population, and is considered to be a systemic disease rather than one limited to the skin. Figure 1. Basal-cell carcinoma cells appear similar to epidermal basal cells, and are usually well differentiated. The reads were mapped to the hg19 reference sequence. DARS was not significantly changed in clone 6 compared to the cells before transduction. Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Stem Cell Res Ther. doi: 10.1016/j.jid.2019.07.701. A Single Epidermal Stem Cell Strategy for Safe Ex Vivo Gene Therapy - PubMed There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance… 2011 Oct;138(10):710-1. doi: 10.1016/j.annder.2011.05.002. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Platelet-rich plasma accelerates skin wound healing by promoting re-epithelialization. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. 2019 Oct 3;4(19):e130554. Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing. Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion–deletion in COL7A1 (Hilal et al, 1993). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. 2020 Aug 14;8:tkaa028. Epidermal stem cells are one of the few types of stem cell already used to treat patients. The latter corresponded to proviruses. 2013;341:1233151. To achieve HF regeneration in grafted epidermal sheets coming from cultured epidermal SC, DP cells will need to be transplanted together with epidermal cells in order to stimulate epidermal SC to adopt hair fol… The RECELL ® device is used by an appropriately-licensed healthcare professional at the patient’s point of care to prepare autologous RES® Regenerative Epidermal … Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs. As the injury happens, wound healing process initiates immediately and is classically divided into four stages: hemostasis, inflammation, reepithelialization, and remodeling. In this case, the researchers created so-called induced pluripotent stem cells, or iPSCs. Cell Tissue Bank. Under the slow cell phase, epidermal stem cells are quiescent. Determination of proviral rearrangements in transduced clones.  |  The epidermal cells form the walls of the cyst and then secrete the protein keratin into the interior. Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. 2021 Jan 7;12(1):42. doi: 10.1186/s13287-020-02106-7. 2015 Oct 23;16(10):25476-501. doi: 10.3390/ijms161025476. Transmission electron microscopy (TEM) demonstrated the presence of anchoring fibrils (arrows) at the DEJ of the corrected epidermis (middle right panel) and in a normal human skin biopsy (left panel). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. Stem Cell Res Ther. … 2020 Aug 8;11(1):344. doi: 10.1186/s13287-020-01844-y. Published under the terms of the CC BY 4.0 license. Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT–PCR. doi: 10.1172/jci.insight.126243. Long-term restoration of COLVII expression, generation of epidermis and anchoring fibrils by the…. The mapping highlighted two indels and one SNP in the DARS genomic sequence. Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells. RDEB cells were used as a negative control and healthy donor cells as a positive control. Lwin SM, Syed F, Di WL, Kadiyirire T, Liu L, Guy A, Petrova A, Abdul-Wahab A, Reid F, Phillips R, Elstad M, Georgiadis C, Aristodemou S, Lovell PA, McMillan JR, Mee J, Miskinyte S, Titeux M, Ozoemena L, Pramanik R, Serrano S, Rowles R, Maurin C, Orrin E, Martinez-Queipo M, Rashidghamat E, Tziotzios C, Onoufriadis A, Chen M, Chan L, Farzaneh F, Del Rio M, Tolar J, Bauer JW, Larcher F, Antoniou MN, Hovnanian A, Thrasher AJ, Mellerio JE, Qasim W, McGrath JA. Diagram of wound healing stages. COVID-19 is an emerging, rapidly evolving situation. Error bars represent the standard deviation of three replicates. Figure 4. 2020 Feb;140(2):338-347.e5. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Hair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Epub 2010 May 18. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis (right panel). Note the absence of anchoring fibrils in the RDEB epidermis (middle left panel). Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). Single stem cell gene therapy for genetic skin disease. Condorelli AG, Dellambra E, Logli E, Zambruno G, Castiglia D. Int J Mol Sci. In uncertain cases, immunohistochemistry using BerEP4 can be used, having a high sensitivity and … Epidermal stem cells contribute to cutaneous wound healing. 2014 Jan;23(1):1-6. doi: 10.1111/exd.12246. Extensive growth potential of recessive dystrophic epidermolysis bullosa (RDEB) epidermal keratinocytes.  |  Published on Nature the first life-saving epidermal stem cell-based gene therapy which has allowed international team coordinated by EuroStemCell Partner Prof. Michele De Luca of the University of Modena and Reggio Emilia (Unimore) to save the life of a child suffering from Epidermolysis Bullosa and achieve fundamental knowledge in the field of stem cell … National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error, Schematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control. Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. 1 The treatment varies, … Background: The epidermis is maintained throughout adult life by pluripotential stem cells that give rise, via daughter cells of restricted self-renewal capacity and high differentiation probability (transit-amplifying cells), to interfollicular epidermis… The upper band corresponded to the endogenous signal. To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. While entering transit amplifying cell phase, they are quickly divided and the number of skin cells is amplified for the … JCI Insight. Stem Cell Therapy For Anti Aging. As the injury happens, wound healing process initiates…, Epidermal stem cells contribute to cutaneous wound healing. JCI Insight. Reads were mapped to the hg19 reference sequence. HE staining is performed in WT mouse embryonic 18.5-day skin section. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. Primers used annealed downstream of the proviral integration site. Figure 5. Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified. Siprashvili Z, Nguyen NT, Gorell ES, Loutit K, Khuu P, Furukawa LK, Lorenz HP, Leung TH, Keene DR, Rieger KE, Khavari P, Lane AT, Tang JY, Marinkovich MP. HE staining is performed in WT mouse embryonic…, Diagram of wound healing stages. Smaller bands corresponded to rearranged proviruses marked with an asterisk. Thanks to a discovery made in 1970 by Professor Howard Green in the USA, epidermal stem cells can be taken from a patient, multiplied and used to grow sheets of epidermis … 2009;360:447–458. Skin wound healing is an intrinsic self-saving chain reaction, which is crucial to facilitating the replacement of damaged or lost tissue. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. 2019 Jun 6;4(11):e126243. When cutaneous injuries occur, skin homeostasis and integrity are … 1985;82:5390–5394. To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. by Junying Yu * and James A. Thomson **. Basal keratinocytes at the wound…, NLM Bonafont J, Mencía Á, García M, Torres R, Rodríguez S, Carretero M, Chacón-Solano E, Modamio-Høybjør S, Marinas L, León C, Escamez MJ, Hausser I, Del Río M, Murillas R, Larcher F. Mol Ther. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Narrow-band UVB treatment … 1.1: How … Hu MS, Rennert RC, McArdle A, Chung MT, Walmsley GG, Longaker MT, Lorenz HP. Future Directions: Understanding epidermal stem cells and their essential functions during skin wound healing are fundamental components behind the development of clinical treatment on patients with cutaneous wounds. 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