Skin Stem Cells: Purification and Characterization

描述（由申请人提供）：干细胞是组织修复和体内平衡的自然单位。最近的研究表明，干细胞可能比以前认识到的更多才多艺。这一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。我们的全球目标是发展多能成体皮肤干细胞及其转运扩增后代的分离、纯化和表征策略。虽然皮肤角质形成细胞是少数可以在体外维持和繁殖的成体干细胞之一，但由于缺乏将干细胞与其后代区分开来的可靠标记，它们的鉴定和分离一直受到阻碍。如果没有这些信息，将干细胞从它们的生态位中移除的压力就不可能评估这些细胞在什么时候会停止作为多能干细胞的功能。在这里，我们提出了一种新的策略，通过利用这些细胞中缓慢循环的特性和少数已知的优先表达的标记物，从自然生态位中纯化多能成年小鼠皮肤干细胞。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。我们将确定并比较这些细胞群的整体表达模式，并利用这些和已知的标记物来研究皮肤干细胞在胚胎发育、出生后、自然稳态、毛发周期、损伤或肿瘤发生反应以及培养过程中如何改变基因表达程序。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其非干细胞谱系区分开来。综上所述，这些研究有望为理解皮肤干细胞如何拥有和维持其独特的自我更新特性，以及它们如何采用不同的命运（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，而不是目前用于烧伤患者的长期植入。

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.