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治疗中的潜在应用。尽管人类胚胎干细胞(ESCs)和诱导型多能干细胞(IPS)是具有多向分化潜能的干细胞的多种来源，但诸如畸胎瘤的形成和免疫原性排斥反应等问题仍然是障碍。多能干细胞存在于成人神经脊区组织中，可能参与组织再生或修复，提示可以分离出成年患者自身的多能干细胞用于治疗疾病，从而避免胚胎干细胞移植的潜在问题。在发育过程中，起源于神经管背侧的NC含有多能细胞，这些细胞有助于体内各种器官/组织的发育。牙周膜(PDL)来源于颅骨NC。我们最近的研究表明，从成人PDL分离的细胞表达ESC标记(Oct4，Nanog，Sox2)和NC标记(p75，Nestin，Sox10)，并显示出分化为所有3个生殖层的潜力。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。在成人组织中发现具有ESC样表型和多能性行为的细胞群体的价值和意义怎么强调都不为过。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由一氧化碳(CO)和尼古丁引起。一氧化碳会提高羧基血红蛋白水平，而尼古丁则会引起血管收缩，从而进一步降低组织的氧合。吸烟会使骨折后的骨骼愈合延迟60%。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。由于口腔组织是第一个与吸烟直接接触的组织，这是一个独特的机会来研究吸烟对这一独特的多潜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，可以从成人牙周膜中分离出同质的多能干细胞群体。由CO和吸烟产生的尼古丁引起的组织缺氧极大地降低了这类干细胞的再生潜力和活性。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鉴定同质多能干细胞群体。基于我们在心血管疾病、软骨和骨修复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性和它们的再生潜力，例如从年龄匹配的吸烟者和非吸烟者的PDL中分离出的成骨。特定目的2b：评估吸烟对PDL细胞亚群的影响，这些细胞亚群可能对心肌发生有重要作用。具体目的2c：评估PDLSC对机械诱导的心肌细胞分化的机制，并利用这种反应来加强现有的分化方案。具体目的3：评价多能人PDLSC的体内植入潜能。

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.