Skin Stem Cells: Purification and Characterization

描述（申请人提供）：干细胞是组织修复和体内平衡的天然单位。最近的研究表明，干细胞可能比以前认识到的更多才多艺。这种特性为组织再生带来了希望，特别是对于争议较少的成体干细胞。我们的全球目标是开发多能成体皮肤干细胞及其transitamplifying后代的分离、纯化和表征策略。虽然皮肤角质形成细胞是可以在体外维持和繁殖的少数成体干细胞之一，但由于缺乏区分干细胞与其后代的可靠标记物，它们的鉴定和分离受到阻碍。如果没有这些信息，将干细胞从它们的小生境中移除的压力使得不可能评估这些细胞在什么时候可能停止作为多能干细胞发挥作用。在这里，我们提出了一种新的策略，从他们的自然生态位纯化多能成年小鼠皮肤干细胞，通过利用慢循环特性和少数已知的标记优先在这些细胞中表达。我们还设计了纯化这些细胞的三种不同的转运扩增后代的策略。我们将确定和比较这些细胞群体的全球表达模式，并采用这些和已知的标记物来研究皮肤干细胞如何改变胚胎发育过程中的基因表达程序，以及出生后，在自然的体内平衡中，随着毛发周期，在对损伤或肿瘤发生的反应中，以及当放置在培养中时。最后，我们将利用这些信息来探索改变关键基因表达程序的功能后果，这些基因将皮肤干细胞与非干细胞谱系区分开来。总之，这些研究有望为理解皮肤干细胞如何拥有和保持其独特的自我更新特性以及它们如何采取不同的命运（包括表皮和毛囊分化）提供新的重要见解。这项研究是确定皮肤干细胞用于治疗目的的潜力的基本先决条件，这些目的超出了目前在烧伤患者中提供长期移植的用途。

DESCRIPTION (provided by applicant): Stem cells are natural units of tissue repair and homeostasis. Recent studies suggest that stem cells may be more versatile than previously appreciated. This property holds promise for tissue regeneration, particularly for less controversial adult stem cells. Our global objective is to develop strategies for the isolation, purification and characterization of multipotent adult skin stem cells and their transitamplifying progeny. While skin keratinocytes are one of the few adult stem cells that can be maintained and propagated in vitro, their identification and isolation have been hampered by the lack of reliable markers that distinguish stem cells from their progeny. Without this information, the stress of removing stem cells from their niche make it impossible to assess at what point these cells might cease to function as multipotent stem cells. Here, we propose a novel strategy for purification of multipotent adult murine skin stem cells from their natural niche, by taking advantage of the slow-cycling properties and the few known markers preferentially expressed in these cells. We also devise strategies for purifying three different transit-amplifying progeny of these cells. We will determine and compare the global expression patterns of these cell populations and employ these and known markers to examine how skin stem cells change programs of gene expression during both embryonic development, and postnatally, in natural homeostasis, with the hair cycle, in response to injury or tumorigenesis, and when placed in culture. Finally, we will use this information to explore the functional consequences of altering the expression programs of key genes, which distinguish skin stem cells from their non-stem lineages. Taken together, these studies are expected to provide new and important insights into understanding how skin stem cells possess and maintain their unique self-renewing properties, and how they adopt different fates, including epidermal and hair follicle differentiation. This research is a fundamental prerequisite to ascertaining the potential of skin stem cells for therapeutic purposes that go beyond their present use in providing long-term engraftment on burn patients.