Somatic Stem Cells in Engineered Human Skin

工程人体皮肤中的体干细胞

基本信息

批准号：
7930984
负责人：
Steven T Boyce
金额：
$ 24.7万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7930984
关键词：
4-Hydroxy-Tamoxifen Acute Address Alopecia Anatomy Area Autologous Basal Cell Basement membrane Biological Preservation Bromodeoxyuridine Burn injury Cell Culture Techniques Cell Density Cell Proliferation Cell physiology Cells Chronic Cicatrix Clinical Clinical Management Clinical Research Collagen Commit Connective Tissue Data Dermal Development Developmental Biology Devices Elements Embryo 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 Nevus Normal tissue morphology Nude Mice Operative Surgical Procedures Outcome Patients Performance Phenotype Physiologic pulse Physiology Pigmentation physiologic function Pliability Population Porifera Pre-Clinical Model Premature aging syndrome Preparation Probability Process Regulation Reporting Research Research Personnel Signal Pathway Signal Transduction Site Skin Skin Substitutes Skin Tissue Skin Transplantation Skin graft Source Stem cells Stratified Epithelium Structure Surface TFRC gene Techniques Testing Therapeutic Thick Tissue Donations Tissue Engineering Tissues Transferrin Transplantation Vascularization Western Blotting Wnt proteins Wnt-3A protein Wound Healing analog base clinical efficacy density disability healing improved in vivo keratinocyte malformation postnatal pre-clinical preclinical study prospective protein expression receptor receptor expression skin regeneration stem success tongue papilla wound

项目摘要

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）：组织工程皮肤移植物的移植为因大面积、全层烧伤引起的急性皮肤伤口患者带来了新的医疗益处，但这些设备在择期手术（即烧伤疤痕）中的应用受到了影响由于它们在体外的寿命有限，以及解剖学上的缺陷，包括缺乏毛发、腺体和指甲。研究人员已经证明，使用自体培养皮肤替代品（CSS）可以稳定烧伤、慢性伤口和巨大痣的伤口闭合，该替代品由填充有培养的人类成纤维细胞和角质形成细胞的胶原蛋白海绵组成。这种工程人体皮肤模型发育出角化上皮、基底膜，并具有真皮替代物，但需要高接种密度，体外寿命有限，并且没有表皮附件结构。研究人员假设：a) CSS 的临床疗效将得到改善，并且通过选择表皮的体细胞干细胞 (SSC) 并在角质形成细胞培养过程中保留 SSC 表型，可以实现择期手术和附件结构； b) CSS中的角质形成细胞在Wnt/p-catenin信号通路的调节下可以被诱导形成毛发。为了解决这些假设，提出了三个具体目标：1）通过06整合素、CD90的高表达、EGF和转铁蛋白（CD71）受体的低表达来选择表皮SSC，并通过低量的TGF-β来保存SSC表型。 01 中等。 SSC 表型将通过 CSS 和愈合的人类皮肤中的标签保留细胞 (LRC) 进行追踪。 2) 通过在角质形成细胞中表达稳定的 p-eatenin 来诱导 CSS 中的毛发形态发生，无论是组成型还是在雌激素响应元件下；或者，通过在成纤维细胞中表达Wnt 3a蛋白。 3) 将具有 SSC 表型的 CSS 移植到切除的全层烧伤，并评估移植率、闭合伤口与供体皮肤的面积比以及温哥华量表的定性结果。这些研究将在细胞培养、皮肤替代品以及将 CSS 移植到无胸腺小鼠全层皮肤伤口的充分表征的临床前模型中进行。将在体外研究皮肤结构（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、柔韧性），以改善细胞增殖和分化的调节以制备 CSS和体内。具体目标 1 的结果预计将促进目标 2 的成功，并将在具体目标 3 中进行临床测试。具体目标 2 的临床前研究预计将开辟替代烧伤皮肤中的毛发和腺体的可能性，并有可能解决烧伤脱发和热调节等情况。这些进展需要从过去的伤口愈合研究到未来的发育生物学研究的范式转变。只有通过这种方法上的改变，皮肤再生的最终目标才能取代导致疤痕和残疾的伤口修复教条。研究人员充分期待CSS的研究能够进一步减少移植所需的组织捐献，初步演示工程皮肤的附件发育，提高CSS治疗烧伤疤痕的功效，并增加挽救患者生命的可能性。危及生命的烧伤患者。