Somatic Stem Cells in Engineered Human Skin

工程人体皮肤中的体干细胞

• 批准号: 7492881
• 负责人: Steven T Boyce
• 金额: $ 29.64万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-04 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7492881
• 关键词: 4-Hydroxy-Tamoxifen; Acute; Address; Alopecia; Anatomy; Area; Autologous; Basal Cell; Basement membrane; Biological Preservation; Bromodeoxyuridine; Burn injury; Cell Density; Cell Proliferation; Cell physiology; Cells; Chronic; Cicatrix; Clinical; Clinical Management; Clinical Research; Closure; Collagen; Commit; Condition; Connective Tissue; Cultured Cells; Data; Dermal; Development; Developmental Biology; Devices; Elements; Embryo; End Point; Engineering; Engraftment; Epidermal Growth Factor Receptor; Epidermis; Epithelium; Erythema; Estrogens; Evaluation; Extracellular Matrix; Failure; Fibroblasts; Future; Gland; Goals; Growth; Growth Factor Receptors; Hair; Hair follicle structure; Harvest; Healed; Height; Histology; Human; Human Engineering; Hydration status; Immunohistochemistry; In Situ; In Vitro; Incubated; Inflammation; Integrin Binding; Integrins; Investigation; Knowledge; Label; Life; Ligands; Longevity; Measures; Medical; Messenger RNA; Microscopy; Modeling; Molecular; Morphogenesis; Mus; Nail plate; Natural regeneration; Nevus; Normal tissue morphology; Nude Mice; Operative Surgical Procedures; Outcome; Patients; Performance; Phenotype; Physiologic pulse; Physiology; Pigmentation physiologic function; Pliability; Polymerase Chain Reaction; Population; Porifera; Pre-Clinical Model; Premature aging syndrome; Preparation; Probability; Process; Pulse taking; Rate; Regulation; Reporting; Research; Research Personnel; Score; Signal Pathway; Signal Transduction; Site; Skin; Skin Substitutes; Skin Tissue; Skin graft; Source; Stem cells; Stratified Epithelium; Structure; Surface; TFRC gene; Techniques; Testing; Therapeutic; Thick; Tissue Donations; Tissue Engineering; Tissues; Transferrin; Transplantation; Vascularization; Week; Western Blotting; Wnt proteins; Wnt-3A protein; Wound Healing; analog; base; clinical efficacy; day; density; disability; epidermis cell; healing; improved; in vivo; keratinocyte; malformation; postnatal; pre-clinical; preclinical study; prospective; protein expression; receptor; receptor expression; stem; success; tongue papilla; wound

DESCRIPTION (provided by applicant): Transplantation of tissue-engineered skin grafts has delivered new medical benefits to patients with acute skin wounds arising from extensive, full-thickness burns, but applications of these devices to elective surgery (i.e., burn scars) is compromised by their limited lifespan in vitro, and by anatomic deficiencies, including absence of hair, glands and nails. The investigators have demonstrated stable wound closure in burns, chronic wounds and giant nevi with autologous cultured skin substitutes (CSS) consisting of a collagen-based sponge populated with cultured human fibroblasts and keratinocytes. This model of engineered human skin develops a keratinized epithelium, basement membrane, and has a dermal substitute, but requires high inoculation densities, has a limited lifespan in vitro, and has no epidermal adnexal structures. The investigators hypothesize that: a) the clinical efficacy of CSS will improve, and elective surgery and adnexal structures will be enabled by selection for the somatic stem cells (SSC) of the epidermis, and preservation of the SSC phenotype during keratinocyte culture; and, b) keratinocytes in CSS can be induced to form hair under regulation of the Wnt/p-catenin signaling pathway. To address these hypotheses, three specific aims are proposed: 1) Selection of epidermal SSCs by high expression of 06 integrin, CD90, low expression of receptors for EGF and transferrin (CD71), and preservation of the SSC phenotype by low amounts of TGF-01 in medium. The SSC phenotype will be tracked by label-retaining cells (LRCs) in CSS and healed human skin. 2) Induction of hair morphogenesis in CSS by expression in keratinocytes of stabilized p-eatenin, either constitutively, or under an estrogen-responsive element; or, by expression in fibroblasts of Wnt 3a proteins. 3) Grafting of CSS with a SSC phenotype to excised, full-thickness burns, and evaluations of rates of engraftment, area ratios of closed wounds to donor skin, and qualitative outcome by the Vancouver Scale. These studies will be performed in well-characterized, preclinical models of cell cultures, skin substitutes, and grafting of CSS to full-thickness skin wounds in athymic mice. Improvements in regulation of the cellular proliferation and differentiation for preparation of CSS will be studied in skin structure (e.g., histology, expression of protein & mRNA), physiology (LRCs in situ), and function (e.g., epidermal barrier, pliability) in vitro and in vivo. Results from specific Aim 1 are expected to promote the success of Aim 2, and will be tested clinically in Specific Aim 3. Preclinical studies in Specific Aim 2 are expected to open possibilities for replacement of hair and glands in burned skin with the potential to address conditions such as burn alopecia and thermal regulation. These advances require a paradigm shift from past studies of wound healing to future studies of developmental biology. Only by making this change in approach will the ultimate goal of skin regeneration replace the dogma of wound repair which leads to scar and disability. The investigators expect fully that the proposed studies of CSS can further reduce the donation of tissue required for grafting, make initial demonstrations of adnexal development in engineered skin, improve efficacy of CSS for treatment of burn scars, and increase the probability of saving the lives of patients with life-threatening burns.

描述（由申请人提供）：组织工程的皮肤移植物的移植已为急性皮肤伤口的患者带来了新的医疗益处，这是由于广泛的，全厚度的烧伤而引起的，但这些设备对选举手术的应用（即烧伤疤痕）遭到了有限的自由度，包括自由度和象征性的毛发，包括有限的寿命，包括有限的寿命，包括hart弱的植被，包括hard虫的毛发。研究人员已经证明，烧伤，慢性伤口和巨大的NEVI的伤口闭合，其自体培养的皮肤替代物（CSS）由基于胶原蛋白的海绵组成，该海绵由培养的人类成纤维细胞和角质形成细胞组成。这种工程的人体皮肤模型开发了角膜上皮，地下膜，具有真皮替代品，但需要高的接种密度，体外寿命有限，并且没有表皮辅助结构。研究人员假设：a）CSS的临床功效将提高，选举手术和附加结构将通过选择表皮的体细胞（SSC）以及在角质形成型培养过程中的SSC表型的保留来启用。并且，b）CSS中的角质形成细胞可以在Wnt/p-catenin信号通路的调节下诱导形成头发。为了解决这些假设，提出了三个特定目的：1）通过高表达06整合素，CD90，EGF和转铁蛋白的受体低表达（CD71）（CD71）选择表皮SSC，以及通过培养基中TGF-01的低量来保留SSC表型。 SSC表型将通过CSS中的标签细胞（LRC）跟踪并治愈了人体皮肤。 2）通过组成性稳定的p-雌激素的角质形成细胞中的表达诱导CSS的头发形态发生，无论是组成性的，还是在雌激素反应元件下；或者，通过在Wnt 3a蛋白的成纤维细胞中表达。 3）用SSC表型嫁接CSS，以切除，全厚度燃烧，以及植入率的评估，封闭伤口与供体皮肤的面积比以及通过温哥华量表进行定性结果。这些研究将在良好的细胞培养物，皮肤替代品和CSS嫁接到无胸腺小鼠的全厚性皮肤伤口的临床前模型中进行。将在皮肤结构（例如，组织学，蛋白质和mRNA的表达），生理学（原位LRC）和功能（例如，表皮屏障，pllc，pllc，pllc，pllc，pllc，蛋白质和mRNA的表达）中研究细胞增殖和分化的调节和分化的调节。预计特定目标1的结果将促进AIM 2的成功，并将在特定的目标3中进行临床测试。特定AIM 2中的临床前研究有望开放以替换燃烧的皮肤中的头发和腺体的可能性，并有可能解决诸如燃烧脱发和热调节等条件。这些进步需要从过去对伤口愈合的研究转变为未来的发育生物学研究。只有通过进行这种变化，皮肤再生的最终目标才能取代伤口修复的教条，从而导致疤痕和残疾。研究人员预计，对CSS的拟议研究可以进一步减少嫁接所需的组织的捐赠，对工程皮肤中的附加性发育进行初步证明，提高CSS治疗烧伤疤痕的疗效，并增加挽救生命危害生命的烧伤患者的生命的可能性。