Skin Stem Cells: Purification and Characterization

项目摘要 这项研究的总体目标是阐明组织动态平衡和 哺乳动物皮肤的再生，并了解这一过程如何在人类疾病中出错，包括 癌症。实现这一目标的核心是纯化和鉴定不同的干细胞(SC)。 皮肤内的种群及其对组织动态平衡和创伤的相对贡献 修理。过去的AR050452研究导致了毛囊(HF)突起细胞的纯化，并将其确立为自体 更新在毛发循环和伤口修复中起作用的干细胞。这些HF-SC也显示出更强大的自我 比毛囊间表皮干细胞(IFE-SCs)更具更新和更广泛的组织再生潜力。几个 差异可能是这一点的原因。一种是，与人类的IFE-SCs一样，小鼠的HF-SCs以静止状态存在 很长一段时间。另一种是它们独特地表达转录因子，如NFATc1，当 如果不存在，则会导致HF-SCs失去静止状态并持续循环HFs。过去的AR050452研究已经设定了 解决以下关键问题的基础：(1)。HF-SC的静止对他们的长期生存至关重要吗 维护？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？他们能吗？ 伤口愈合更快？(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？什么 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？做词干 细胞后代如何影响这一转变？如果是这样的话？(4)IFE和HF SCs对 伤口修复？这在浅层伤口和深层伤口上有区别吗？幼年小鼠与成年小鼠？(5)IFE和HF如何 SCS因损伤而改变基因表达？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，我们将使用FACS、RNA-SEQ、CHIP-SEQ、条件性基因敲除和RNAi筛选 在体内，并使用这些方法来探索其天然、突变和创伤诱导的皮肤干细胞 环境。

Project Summary 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 differerent 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 does 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 are these changes and what 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.