Skin Stem Cells: Purification and Characterization

描述(申请人提供)：这项研究的全球目标是阐明哺乳动物皮肤组织动态平衡和再生的潜在机制，并了解这一过程在包括癌症在内的人类疾病中是如何出错的。实现这一目标的核心是对皮肤内不同的干细胞(SC)群体进行纯化和表征，并确定它们对组织稳态和伤口修复的相对贡献。过去的AR050452研究导致了毛囊(HF)凸起细胞的纯化，并建立了它们作为自我更新的干细胞，在毛发周期和伤口修复中发挥作用。与毛囊间表皮干细胞(IFE-SCs)相比，HF-SCs表现出更强的自我更新能力和更广泛的组织再生潜能。几个不同之处可能解释了这一点。一种是，与人类IFE-SCs一样，小鼠HF-SCs长期处于静止状态。另一种是它们独特地表达转录因子，如NFATc1，当NFATc1缺失时，会导致HF-SCs失去静止并持续循环HFs。过去的AR050452研究已经为解决以下关键问题奠定了基础：(1)。HF-SC的静止状态对其长期维护至关重要吗？当HFs持续循环时会发生什么？随着时间的推移，他们是否会花掉他们的SC？它们能更快地愈合伤口吗？(2)控制HF-SC生态位休眠阶段的分子机制是什么？NFATc1及其相关转录调控因子是如何维持HF-SC静止的？它的直接目标是什么？它们如何调节静止和长期自我更新之间的平衡？(3)什么分子变化定义了茎和过渡放大状态之间的差异？干细胞后代是否影响这一转变？如果是的话，又是如何影响的？(4)IFE和HF SCs对创伤修复的相对贡献是什么？这在浅层伤口和深层伤口上有区别吗？在幼鼠和成年鼠之间。(5)IFE和HF SCs在损伤后如何改变基因表达？这些变化的速度有多快？是什么控制了这个过程？为了回答这些问题，我们将在体内使用FACS、RNA-SEQ、CHIP-SEQ、条件性基因敲除和RNAi筛选，并使用这些方法来探索皮肤干细胞在其天然、突变和创伤诱导的环境中的情况。

DESCRIPTION (provided by applicant): The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the purification and characterization of the different stem cell (SC) populations within skin and determination of their relative contributions to tissue homeostasis and wound- repair. Past AR050452 research led to purification of hair follicle (HF) bulge cells, and established them as self- renewing SCs that function in hair cycling and wound-repair. These HF-SCs also displayed more robust self- renewing and broader tissue regenerative potential than interfollicular epidermal (IFE)-SCs. Several differences might account for this. One is that like human IFE-SCs, mouse HF-SCs exist in a quiescent state for prolonged periods. Another is that they uniquely express transcription factors such as Nfatc1, which when absent, causes HF-SCs to lose quiescence and cycle HFs continuously. Past AR050452 research has set the foundations to tackle the following key questions: (1). Is HF-SC quiescence critical to their long-term maintenance? What happens when HFs cycle continuously? Do they expend their SCs over time? Can they heal wounds faster? (2) What are the molecular mechanisms governing the resting phase of the HF-SC niche? How do Nfatc1 and its associated transcriptional regulators function to maintain HF-SC quiescence? What are its direct targets and how do they regulate the balance between quiescence and long-term self-renewal? (3) What molecular changes define the difference between stemness and the transit amplifying state? Do stem cell progeny influence this transition and if so how? (4) What are the relative contributions of IFE and HF SCs to wound-repair? Does this differ in superficial vs deep wounds? In young vs adult mice. (5) How do IFE and HF SCs alter gene expression in response to injury? How fast these changes and what are controls the process? To answer these questions, we'll use FACS, RNA-seq, ChIP-seq, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, mutant and wound-induced environments.