Stem-Cell Properties of Human Corneal Keratocytes

描述（由申请人提供）：我在寻求K 08职业发展奖的目标是双重的：1）检查出生后的人角膜细胞和人神经嵴干细胞在卵细胞的多潜能性; 2）通过实践研究经验，教学和指导，发展我作为干细胞生物学独立研究者的职业生涯。人类角膜失明的最常见原因是视觉上显著的基质瘢痕和内皮细胞功能障碍。在美国，据预测，随着屈光手术的出现，适用于移植的供体角膜的供应将显著减少。由于这些挑战，在角膜病症的治疗中，对追求使用具有自我更新、分化成多个细胞谱系和体内重塑组织的能力的细胞存在显著的兴趣。虽然最近有报道称，人角膜干细胞可以被诱导表达与细胞培养中的多潜能性一致的标志物，但对分化的角膜基质细胞的多潜能性知之甚少。我们的初步数据表明，从出生后的角膜分离的人角膜细胞有能力在鸡胚环境中分化为神经嵴衍生物。这是第一个证据，尽管是早期的，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的多潜能性，它们作为部分限制性祖细胞而衍生。该提议的工作假设是，人出生后的角膜细胞保留了其神经嵴前体的多能性，并且具有分化成神经嵴衍生物（包括其他眼组织）的能力。此外，鸡胚胎微环境可能含有将人神经嵴干细胞分化成眼组织以及其他神经嵴衍生结构所需的足够信号。三个具体的目标将被解决;目标1;表征人出生后角膜细胞的多潜能性，使用鸡胚胎环境作为测定系统。目的2：探讨年龄和分化状态对人角膜基质细胞多能性的影响。目的3：探讨人神经嵴干细胞在卵内形成神经嵴眼衍生物的可能性。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，角膜瘢痕是全球第二大常见的致盲原因。我们的目标是了解出生后的人类角膜细胞和神经嵴干细胞在胚胎环境中分化为眼组织的能力。这将作为确定创造用于再生医学的专门细胞的可行性的基础。

DESCRIPTION (provided by applicant): My objectives in seeking a K08 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.