Stem-Cell Properties of Human Corneal Keratocytes

个人描述(申请人提供)：我寻求K08职业发展奖的目标有两个：1)研究出生后人类角质形成细胞和人类神经嵴干细胞在卵细胞中的多潜能；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员的职业生涯。人类角膜失明的最常见原因是视觉上显著的基质瘢痕形成和内皮细胞功能障碍。在美国，据预测，随着屈光手术的到来，适合移植的供体角膜供应将显著减少。由于这些挑战，人们对利用具有自我更新能力、分化为多个细胞系并在体内重建组织的细胞在治疗角膜疾病方面表现出极大的兴趣。虽然最近有报道称人角膜干细胞可以在细胞培养中被诱导表达与多潜能一致的标记，但对分化的角膜基质细胞的多潜能知之甚少。我们的初步数据表明，从出生后的角膜中分离的人角膜基质细胞在鸡胚胎环境中具有分化为神经脊衍生物的能力。这是第一个证据，尽管还很早，人类角质形成细胞和出生后(相对于胚胎)的角质形成细胞保留了神经脊前体细胞的多重潜能，它们是作为部分受限的前体细胞产生的。这一建议的工作假设是，人类出生后的角膜基质细胞保留了其神经脊前体细胞的多潜能，并具有分化为神经脊衍生物的能力，包括其他眼组织。此外，鸡胚胎微环境可能包含将人类神经脊干细胞分化为眼组织以及其他神经脊衍生结构所需的足够信号。将讨论三个具体目标：目标1；使用鸡胚胎环境作为测试系统，表征人类出生后角质形成细胞的多潜能。目的2：研究年龄和分化状态对人角膜基质细胞多能性的影响。目的3：探讨人神经脊干细胞在卵子内形成神经脊眼部衍生物的可能性。相关性(见说明)；供体角膜的可获得性往往限制了治疗角膜疤痕的能力，角膜疤痕是全球第二大最常见的致盲原因。我们的目标是了解出生后的人类角质形成细胞和神经脊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.