Effect of Smoking on Adult Stem Cell Differentiation

描述（由申请人提供）： 多能干细胞产生各种替代细胞和组织的能力突出了它们在基于细胞的再生疗法中的潜在应用。虽然人胚胎干细胞（ESC）和诱导的多能干细胞（iPS）是具有多分化潜能的干细胞的多用途来源，但是诸如畸胎瘤形成和免疫原性排斥反应的担忧仍然是障碍。多能干细胞存在于成人神经嵴（neural crest，NC）来源的组织中，参与组织的再生或修复，这表明成人患者自身的多能干细胞可以用于疾病的治疗，从而避免ESC移植的潜在问题。 在发育过程中，起源于神经管背侧区域的NC含有多能细胞，其有助于体内各种器官/组织的发育。牙周膜韧带（PDL）来源于颅侧NC。我们最近的研究表明，从成人PDL分离的细胞表达ESC标记物（Oct 4，Nanog，Sox 2）和NC标记物（p75，Nestin，Sox 10），并表现出分化为所有3个胚层的潜力。这表明具有ESC或NC特征的PDL干细胞可能确实表现出多能性。在成体组织中发现具有ESC样表型和多能性行为的细胞群的价值和意义不能被夸大。 吸烟是众所周知的多种疾病的危险因素。显著的组织缺氧主要归因于一氧化碳（CO）和尼古丁。一氧化碳升高羧基血红蛋白水平，而尼古丁引起血管收缩，进一步降低组织氧合.骨折后吸烟会延迟60%的骨骼愈合。尼古丁通过影响骨愈合过程中的基因表达来延缓骨生长。目前还不清楚吸烟是否会影响内源性干细胞。由于口腔组织首先与吸烟直接接触，这是一个独特的机会来检查吸烟对这种独特的多能干细胞群体的活力和再生/分化潜力的影响。 我们的假设是，多能干细胞的同质人口可以从成人牙周膜分离。由吸烟引起的CO和尼古丁引起的组织缺氧大大降低了该干细胞群体的再生潜力和活力。 我们有三个目标。具体目的1：从成人牙周韧带中分离并进一步表征多能干细胞的同质群体。基于我们在心血管疾病、软骨和骨修复的细胞疗法方面的机构优势，具体目标2a：我们将比较干细胞的活力及其再生潜力，例如从年龄匹配的吸烟者和非吸烟者的PDL中分离的成骨作用。具体目标2b：评价吸烟对PDL细胞亚群的影响，这可能对心肌发生很重要。具体目标2c：评估机械诱导PDLSC心肌样分化的机制，并利用这种反应来增强当前的分化方案。具体目的3：评价多能性人PDLSC的体内植入潜力。

DESCRIPTION (provided by applicant): The ability of pluripotent stem cells to generate various replacement cells and tissues highlights their potential application in cell-based regenerative therapies. Although human embryonic stem cells (ESCs) and inducted pluripotent stem cells (iPS) are versatile sources of stem cells with multiple differentiation potential, however concerns such as teratoma formation and immunogenic rejection remain as obstacles. Pluripotent stem cells reside in adult human tissues derived from neural crest (NC) and may participate in tissue regeneration or repair.This suggest that the adult patients' own pluripotent stem cells could be isolated for the treatment of diseases, thus avoiding the potential problems of ESC transplantation. During development, NC originating at the dorsal region of the neural tube contains pluripotent cells that contribute to development of varieties of organs/tissues in the body. Periodontal ligament (PDL) is derived from cranial NC. Our recent study showed that cells isolated from adult human PDL expressed ESC markers ( Oct4, Nanog, Sox2) and NC markers (p75, Nestin, Sox10), and exhibited the potential to differentiate into all 3 germ layers. This suggests that PDL stem cells with ESC or NC characteristics may indeed exhibit pluripotency. The value and significance of discovering cell populations within adult tissues that possess an ESC-like phenotype and pluripotent behavior cannot be overstated. Cigarette smoking is a well-known risk factor for varieties of diseases. Significant tissue hypoxia mainly attributed to carbon monoxide (CO) and nicotine. CO raises carboxyl- hemoglobin levels, whilst nicotine induces vasoconstriction, which further decreases tissue oxygenation. Smoking delays skeletal healing by 60% following fractures. Nicotine delays bone growth by influencing gene expression in bone healing process. It is unclear whether cigarette smoking affects endogenous stem cells. Since oral tissues are first to come in direct contact with smoking, this is a unique opportunity to examine the effect of smoking on the viability and regenerative/differentiation potential of this unique population of pluripotent stem cell population. Our hypothesis is that a homogeneous population of pluripotent stem cells can be isolated from human adult periodontal ligament. Tissue hypoxia caused by CO and nicotine from smoking greatly reduce regenerative potential and viability of this population of stem cells. We have 3 aims. Specific Aim 1: To isolate and further characterize a homogeneous population of pluripotent stem cells from adult Periodontal Ligament. Based on our institutional strengths on cellular therapies on cardiovascular diseases, cartilage and bone repair, Specific Aim 2a: We shall compare the viability of stem cells and their regenerative potential e.g. osteogenesis isolated form PDL from the age-match smokers to nonsmokers. Specific Aim 2b: Evaluate the effect of smoking on the subpopulation of PDL cells that may be important for cardiomyogenesis. Specific Aim 2c: To evaluate the mechanism of cardiomyogenic differentiation of PDLSC to mechanical induction and utilize this response in the enhancement of current differentiation protocols. Specific Aim 3: To evaluate the in vivo engraftment potential of the pluripotent human PDLSC.