Stem-Cell Properties of Human Corneal Keratocytes

我申请克格勃职业发展奖的目的有两个：1）考察 出生后的人类角膜细胞和人类神经嵴干细胞在卵细胞和; 2）发展我的职业生涯， 通过实践研究经验、教学法和指导，成为干细胞生物学的独立研究者。 人类角膜失明的最常见原因是视觉上显著的基质瘢痕形成， 内皮细胞功能障碍在美国，据预测，随着屈光手术的出现， 适合移植的供体角膜将显著减少。由于这些挑战， 研究人员对使用具有自我更新、分化成多种细胞的能力感兴趣， 细胞谱系和在体内重塑组织，用于治疗角膜疾病。尽管已经 最近有报道称，人角膜干细胞可以被诱导表达与多细胞性角膜炎一致的标志物， 尽管在细胞培养中的细胞分化潜能有限，但对分化的角膜基质细胞的多潜能性知之甚少。我们 初步数据表明从出生后角膜分离的人角膜细胞具有 在鸡胚环境中分化为神经嵴衍生物。这是第一个证据，尽管 早期，人类角膜细胞和出生后（相对于胚胎）角膜细胞保留了 神经嵴前体，它们作为部分限制性祖细胞而衍生。工作 该建议假设人出生后的角膜细胞保持其神经嵴的多能性 前体，并具有分化成神经嵴衍生物，包括其他眼组织的能力。 此外，鸡胚微环境可能包含分化所需的足够信号， 将人神经嵴干细胞植入眼组织以及其他神经嵴衍生的结构。三 将提出具体的目的;目的1;表征人出生后角膜细胞的多潜能性， 使用鸡胚环境作为测定系统。目标2：检查年龄和 分化状态对人角膜基质细胞多能性的影响。目标3：探索人类的潜力 神经嵴干细胞在卵内形成神经嵴眼衍生物。 相关性（参见说明）; 供体角膜的可用性往往限制了治疗角膜瘢痕的能力， 全世界失明的原因。我们的目标是了解出生后人类角膜细胞的能力， 神经嵴干细胞在胚胎环境中分化成眼组织。这将作为一个 这是确定创造用于再生医学的专门细胞的可行性的基础。

My objectives in seeking a KGB career development award are two-fold: 1) to examine the multipotentiality of post-natal human keratocytes and human neural crest stem cells in ovo and; 2) to develop my career as an independent investigator in stem cell biology by hands-on research experience, didactics and mentorship. The most common causes of human corneal blindness are visually significant stromal scarring and endothelial cell dysfunction. In the US, it is predicted that with the advent of refractive surgery, the supply of donor corneas suitable for transplantation will be significantly reduced. Because of these challenges, there is significant interest in pursuing the use of cells that have the ability to self-renew, differentiate into multiple cell lineages, and remodel tissues in vivo, in the treatment of corneal disorders. While there have been recent reports of human cornea stem cells that can be induced to express markers consistent with multi- potency in cell culture, little is known about the multi-potentiality of differentiated cornea stromal cells. Our preliminary data indicate that human keratocytes isolated from postnatal corneas have the ability to differentiate into neural crest derivatives in the chick embryonic environment. This is the first evidence, albeit early, that human keratocytes and postnatal (versus embryonic) keratocytes retain the multi-potentiality of the neural crest precursors from which they are derived as partially restricted progenitors. The working hypothesis of this proposal is that human postnatal keratocytes retain the multi-potency of their neural crest precursors and have the ability to differentiate into neural crest derivatives, including other ocular tissues. Further, the chick embryonic microenvironment likely contains the adequate signals required to differentiate human neural crest stem cells into ocular tissues, as well as other neural crest-derived structures. Three specific aims will be addressed; Aim 1; characterize the multipotentiality of human post-natal keratocytes, using the chick embryonic environment as an assay system. Aim 2: examine the effects of age and differentiation status on the multipotentiality of human keratocytes. Aim 3; explore the potential for human neural crest stem cells to form neural crest ocular derivatives in ovo. RELEVANCE (See instructions); The availability of donor corneas often limits the ability to treat corneal scarring, the second most common cause of blindness worldwide. Our goal is to understand the ability of post-natal human keratocytes and neural crest stem cells to differentiate into ocular tissues in an embryonic environment. This will serve as a basis for determining the feasibility of creating specialized cells for use in regenerative medicine.